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Bacterial Transcription as a Target for Antibacterial Drug
Cong Ma, Xiao Yang, Peter J. Lewis
School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, Australia
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .139
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .140
RNAP STRUCTURE AND FUNCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .140
Overview of the Transcription Cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .140
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Bacterial RNAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .140
Human RNAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .141
COMPOUNDS THAT INHIBIT TRANSCRIPTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .141
PRIMARY CHANNEL INHIBITORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .142
Rifamycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .142
Sorangicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .144
GE23077 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .145
MOBILE ELEMENTS OF THE PRIMARY CHANNEL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .145
Streptolydigin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .145
Salinamide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .146
CBR Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .147
SECONDARY-CHANNEL INHIBITORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .147
Tagetitoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .147
Microcin J25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .148
SWITCH REGION INHIBITORS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .148
Myxopyronin, Corallopyronin, Ripostatin, and Squaramides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .148
Fidaxomicin and Lipiarmycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149
INHIBITORS WITH UNKNOWN TARGETS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149
TRANSCRIPTION FACTORS AND RNAP BINDING SITES AS TARGETS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149
Transcription Factor Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149
Termination Factor Rho . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149
Bicyclomycin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150
The RNAP-␴ Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150
The GKL series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150
DSHS00507 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151
The SB series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151
Potential Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151
NusA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151
NusB/E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152
NusG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152
NusGSP factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .153
Helicases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .153
CONCLUSIONS AND PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .153
ACKNOWLEDGMENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154
AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .160
SUMMARY scribe the inhibition of the bacterial transcription process with
Transcription, the first step of gene expression, is carried out by respect to structural studies of RNAP, highlight recent progress
the enzyme RNA polymerase (RNAP) and is regulated through toward the discovery of novel transcription inhibitors, and suggest
interaction with a series of protein transcription factors. RNAP additional potential antibacterial targets for rational drug design.
and its associated transcription factors are highly conserved across
the bacterial domain and represent excellent targets for broad-
spectrum antibacterial agent discovery. Despite the numerous
antibiotics on the market, there are only two series currently ap-
Citation Ma C, Yang X, Lewis PJ. 2016. Bacterial transcription as a target for
proved that target transcription. The determination of the three- antibacterial drug development. Microbiol Mol Biol Rev 80:139 –160.
dimensional structures of RNAP and transcription complexes at doi:10.1128/MMBR.00055-15.
high resolution over the last 15 years has led to renewed interest in Address correspondence to Peter J. Lewis, Peter.Lewis@newcastle.edu.au.
targeting this essential process for antibiotic development by uti- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
lizing rational structure-based approaches. In this review, we de-
March 2016 Volume 80 Number 1 Microbiology and Molecular Biology Reviews mmbr.asm.org 139
Ma et al.
INTRODUCTION tial cytotoxicity (20); (iv) numerous high-resolution structures
are available, enabling structure-based drug design (21); and (v)
B acteria are prokaryotic microorganisms representing one of
the three domains of life (1). While most bacteria occupy an
incredibly diverse range of ecological niches, a small proportion
the essential RNAP-associated transcription factors are not con-
served between bacterial and eukaryotic cells, providing the op-
portunity for development of compounds targeting RNAP-tran-
can cause disease (2, 3). Due to the rate at which these pathogens
scription factor interactions (22).
are acquiring resistance to antibiotics, the very real possibility that
we will not be able to effectively treat many infections is fast be-
coming reality (4, 5). A recent report projects that if there is no RNAP STRUCTURE AND FUNCTION
concerted effort to discover and develop new antibiotics, by 2050 Overview of the Transcription Cycle
there will be ⬎10,000,000 deaths per year associated with antibi- There are three main sequential steps in the transcription cycle:
otic-resistant infections, with an associated cost to the global promoter binding/initiation, RNA chain elongation, and termi-
economy of ⬃$1 trillion (6). nation. For transcription initiation to occur, the bacterial RNAP
Prior to the clinical development of sulfonamides and pen- core must associate with an initiation protein factor, ␴, forming a
icillin in the 1930s and 1940s, bacterial infections were the
holoenzyme, which is competent for specific binding to the pro-
primary cause of death for children and working-age adults. moter regions on DNA (23). After promoter recognition, ⬃14 bp
This is rare now, and few people remain who can still recall life of DNA is melted upstream of the transcription start site to form
before access to effective antibiotics. Since the 1960s, antibiotic an open promoter complex (24). The template strand, including
research and development by major pharmaceutical compa- the transcription start site, is directed toward the active site of
nies have dropped precipitously (7), and there has been com- RNAP, where RNA synthesis is initiated (21). Several rounds of
placency within the medical profession as well as the general abortive initiation are likely to occur, generating short transcripts
public based on the assumption that we can effectively control 2 to 9 nucleotides (nt) in length (25).
and treat all microbial infections. The blasé overuse of these Once about 12 nucleotides of RNA have been synthesized, ␴
precious drugs has contributed to the rate at which antibiotic factor is released, while RNAP escapes from the promoter and
resistance has developed within hospital environments as well undergoes a significant conformational change resulting in the
as the community. formation of a stable transcription elongation complex (EC) (21,
Antibiotic resistance represents a serious and growing prob- 26). Actively transcribing EC is stably associated with template
lem in the treatment of bacterial infections (8). The dramatic DNA and RNA, adding one nucleotide (NMP) at a time to the
slowdown in development of new antibacterial agents has co- growing transcript. It is highly processive, transcribing at 30 to 100
incided with an alarming increase in the number of resistant, nt/s along the DNA template in vivo (27). Once an intrinsic or
multiresistant, and even totally antibiotic-resistant infections. factor-dependent transcription termination signal is reached,
The new compounds released to market are almost all deriva- RNAP core is released from the template DNA, allowing initiation
tives of existing classes, and consequently cross-resistance is of another round of transcription (28, 29).
often preexisting within the microbiome (9). Resistance is rap-
idly acquired by spontaneous or induced mutation and hori- Bacterial RNAP
zontal gene transfer from resistant species. Decreased mem- In bacteria there is only one RNAP responsible for the transcrip-
brane permeability, increased efflux capacity, enzymatic tion of all classes of RNA (30), and the ⬃350-kDa bacterial RNAP
inactivation, and direct mutation of the binding sites of drug core enzyme (subunit composition, ␣2␤␤=␻) consists of five sub-
targets are the major causes of antibiotic resistance (10–14). units: two large subunits (␤ and ␤=), two ␣ subunits, and an ac-
Consequently, structure-function-based studies on known and cessory subunit (ε) (Fig. 1A) (31–34). In the firmicutes, there is a
potential targets essential to bacterial viability would help in sixth subunit, ε, of unknown function (33). Due to recent ad-
optimizing current antibiotics and for the rational design of vances in structural biology, high-resolution structures of RNAP
new antibacterial agents. Most clinically approved antibiotics have been solved in different forms (see reference 35 for a com-
target bacterial cell wall growth/integrity, translation, and prehensive review of available structures), including core RNAP
DNA replication/segregation, while transcription appears to be (36), holoenzyme (37, 38), and initiation (39, 40) and elongation
an underutilized target. (41) complexes.
Transcription is the process by which RNA is synthesized from Bacterial RNAP core enzyme has an overall "crab claw"
its template DNA by the enzyme RNA polymerase (RNAP) (15). structure, being 150 Å long, 110 Å wide, and 115 Å tall (36). The
So far there are only two antibiotics targeting bacterial RNAP on two ␣ subunits lie at the back of the enzyme, and the two large
the market: the rifamycin series (16) and fidaxomicin/lipiarmicin subunits ␤ and ␤= interact extensively, each forming one "pin-
(17). A transcription factor Rho inhibitor, bicyclomycin, has also cer" of the claw (36). A number of important structural fea-
been commercialized, mainly for use as a growth promoter in tures of bacterial RNAP can be identified from the core and
animal feedstock (18). Nevertheless, bacterial transcription repre- holoenzyme homology models (Fig. 1). The primary channel
sents an excellent target for novel antibacterial development for formed by the cleft between ␤ and ␤= is about 27 Å wide, which
the following reasons: (i) transcription is an essential process for is adequate to accommodate a double-stranded DNA (dsDNA)
cell viability; (ii) bacterial RNAP and its associated transcription template, allowing RNA synthesis to occur (36). The switch
factors are highly conserved, permitting the potential develop- region is located at the top of the RNAP clamp (Fig. 1C) and
ment of broad-spectrum antitranscriptional antibiotics (19); (iii) which mediates opening and closing of the RNAP clamp to
eukaryotic RNAP is not similar to its bacterial homolog at the allow DNA loading at the active site (15). The downstream face
sequence level (barring the active site), which suggests low poten- of the active center is formed by the bridge helix (BH) (also
140 mmbr.asm.org Microbiology and Molecular Biology Reviews March 2016 Volume 80 Number 1
Targeting Transcription
FIG 1 RNAP structure and functional motifs. In all panels the structure of the Thermus thermophilus RNAP elongation complex was used. (A and B) Side and
front views, respectively. ␣ subunit, dark gray; ␤ subunit, light gray; ␤= subunit, medium gray; ␻ subunit, black. The DNA primary channel and secondary
channel for NTP entry are highlighted with yellow circles. Template-strand DNA, green; nontemplate-strand DNA, orange; RNA, red. The active-site magnesium
ion is shown as a cyan sphere. (C and D) Enlarged views from panels A and B, respectively, with functional regions colored and labeled. CH region, pale green;
rudder, cyan; switch, red; active-site residues (NADFDGD), purple; ␤ link, orange; F loop, magenta, fork loop II, yellow; bridge helix (BH), blue; trigger loop
(TL), green; lid, bronze; zipper, brown; ␤ flap tip, salmon. The approximate path of RNA through the RNA exit channel is indicated by the arrow in panel D.
Structure images were prepared using PDB files 1IW7 and 2O5I in PyMol v1.7.4 (Schrödinger, LLC).
referred to as the F helix) and the trigger loop (TL) (or G loop). ␣-amanitin, an anticancer drug that traps the bridge helix and
The BH extends directly across the active channel and splits it trigger loop in RNAP II, preventing nucleotide addition, is also a
into two separate channels, the downstream DNA (active) bactericidal agent (48, 49). Consequently, cytotoxicity could be a
channel and the secondary channel (Fig. 1A) (36, 42). The 10- considerable issue when adopting a strategy of drug design based
to 12-Å-wide secondary channel is not large enough for dou- on the conserved active-site region.
ble-stranded nucleic acids and is proposed to be involved in
nucleoside triphosphate (NTP) entry (36). On the upstream COMPOUNDS THAT INHIBIT TRANSCRIPTION
side of RNAP, the ␤ G region (␤-flap) and the rest of the up- Considerable effort has been spent in the discovery of inhibitors of
stream boundary of the active site together form an RNA exit bacterial transcription since the isolation of rifampin (RIF) (17),
channel, through which the newly synthesized RNA transcript and several reviews, listed in Table 1, have summarized the re-
is threaded (Fig. 1B) (43). The ␣-helical motif at the top of the search results from different perspectives. Representative struc-
␤-flap, called the ␤-flap tip, is functionally important, as it is tures for compounds with specific activities outlined in this review
the binding site for ␴ region 4 and the essential elongation are shown in Fig. 2 and will be referred to in the appropriate
factor NusA (Fig. 1D) (44). Although there are many contacts sections below. These molecules bind to a diverse range of sites on
between core RNAP and ␴ factor, a solvent-exposed clamp- RNAP and inhibit transcription through binding to sites in and
helix (CH) region of the RNAP ␤= subunit (Fig. 1C) represents around the primary channel, as well as targeting the secondary
the major site for ␴ interaction (38). channel and switch regions. The activity or interaction with RNAP
of transcription factors is also a source of established (bicyclomy-
Human RNAP cin) (Fig. 2) or emerging lead compounds.
In the nuclei of eukaryotic cells there are three distinct multisub- Since the mid-20th century, most of the inhibitors of bacterial
unit RNAPs: RNAP I transcribes rRNA precursors, RNAP II is transcription have been isolated from microorganisms, except for
responsible for the synthesis of mRNA and certain small nuclear synthetic squaramides, ureidothiophene, CBR703, and the SB se-
RNAs, and RNAP III performs the synthesis of 5S rRNA and tRNA ries of compounds (Fig. 2) (50, 51). So far, however, only rifamy-
(45). The overall architecture of RNAP is similar and the sequence cins and fidaxomicin/lipiarmycin (Fig. 2) have been approved for
of the active site highly conserved in all living organisms, so drugs clinical use, and no new transcription inhibitor compounds are in
targeting this region may have cross activity (30, 46, 47). Indeed, clinical trials. The high rate of acquisition of bacterial resistance
TABLE 1 Past reviews on inhibitors of bacterial transcription
Author(s) Yr Content summary (reference)
Darst 2004 Described four inhibitors, i.e., two classic molecules, rifampin and streptolydigin, and two new inhibitors, microcin J25 and
CBR703; their binding sites on bacterial RNAP structures were also examined (229)
Villain-Guillot et al. 2007 Summarized all of the inhibitors known to date; this complete review illustrated the chemical structures of the inhibitors and
their binding sites on RNAP (230)
Mariani and Maffioli 2009 A comprehensive review of all the inhibitors known to date; the progress of chemical modification of inhibitors and their
activity against bacteria, as well as clinical applications, were detailed (50)
Ho et al. 2009 Focused on the structure of RNAP and inhibitor complexes; four cocrystal structures of Thermus aquaticus RNAP with
rifampin and sorangicin and of Thermus thermophilus RNAP with streptolydigin, and myxopyronin were presented; the
authors also constructed a homology model of Mycobacterium tuberculosis RNAP in complex with myxopyronin (231)
Srivastava et al. 2011 A comprehensive review of the inhibitors myxopyronin, corallopyronin, ripostatin, and lipiarmycin, which target the switch
region; the sequence of this region is highly conserved in bacteria but not in eukaryotes, providing a new avenue for the
rational development of a potent new class of broad-spectrum antibiotics (136)
prevented the further development of many compounds, while nel formed by the ␤ subunit flap domain, as well as the ␤= lid and
myxopyronins were sequestered by serum albumin, preventing zipper domains (Fig. 1C and D).
them from being free to access their target, CBR703 was proven to
be unattractive due to cytotoxicity, and the SB series were found to Rifamycins
have a nonspecific mode of action (52–54). Rifamycins (Fig. 2) were the first group of antimicrobials targeting
In the following sections, we describe binding sites and modes RNAP, discovered in the last century from metabolites of Amyco-
of action of known inhibitors and envision some potential targets latopsis mediterranei (60). The first drug of this class was rifamycin
for structure-based drug design. SV, which was used only intravenously and topically in clinics
(61). Rifampin (RIF), synthesized from rifamycin SV, is orally
PRIMARY CHANNEL INHIBITORS effective and has broad-spectrum activity against Gram-positive,
The primary channel is a large cleft formed by the two large sub- Gram-negative, and especially mycobacterial pathogens (62). As it
units ␤and ␤= and is highly conserved among RNAPs (31, 55). The is the key element in combinatorial antitubercular chemotherapy,
cleft has an overall positive charge, while the surface of RNAP is rigorous studies on the chemical derivation of RIF led to the de-
mostly negatively charged. Specifically, ␤ regions H and I interact velopment of two analogues, rifabutin (RBT) and rifapentine
with ␤= region D, which positions the absolutely conserved -NAD (RPT), possessing improved pharmacological characteristics (63,
FDGD- ␤= D motif at the base of the cleft (Fig. 1C). The Mg2⫹ ion 64). Rifaximin was also derived from RIF and was approved for
is chelated by the aspartate (D) residues from the NADFDGD the treatment of gastrointestinal disorders and hepatic encepha-
motif at the base of the channel, catalyzes nucleotide addition to lopathy (64, 65).
the growing RNA transcript, and may also be involved in DNA Cocrystal structures of different rifampins with Thermus
melting (36, 56). aquaticus RNAP (66) and Thermus thermophilus holoenzyme (67)
During elongation, a transcription bubble is formed when 10 indicated that they bind within the cleft close to the active center of
to 12 bp of downstream duplex DNA yet to be transcribed enters RNAP, which sterically hinders growth of the RNA product rather
the active channel (21). The duplex DNA is then separated within than DNA binding or RNA synthesis sites (Fig. 3A and B). Bio-
the active channel, and the newly synthesized RNA forms a 9- to chemical experiments also showed that RIF did not inhibit the
10-bp hybrid with the template DNA strand, with the 3= end of the formation of the RNAP-promoter open complex but could stall
RNA positioned within the active site near the center of the en- the formation of RNA products that are ⬎3 nt (66–68). Structur-
zyme (Fig. 1B). The nontemplate DNA strand is rewound with the ally, the rifamycin naphthyl group interacts with Escherichia coli ␤
template DNA on the upstream side, and the length of the RNA- subunit residues 146, 511, 513, 529, 531, 533, 534, 568, and 572,
DNA hybrid and DNA bubble stays approximately constant as and a recent cocrystal structure of E. coli RNAP holoenzyme and
transcription proceeds (57). The four-step nucleotide addition RIF showed that it also interacts with ␤ fork loop II, especially
cycle starts with the binding of an NTP complementary to the residue 540 (69), while the remaining functional groups bind to
template DNA at the insertion site (i⫹1) in the active center. Ini- residues 143, 510, 511, 512, 514, 516, 525, 526, 564, and 761 (Fig.
tially, the TL is in its unfolded conformation (Fig. 1C and D), and 3B). Artsimovitch et al. demonstrated that RIF and RPT bind to
the NTP enters via the secondary channel to form a preinsertion RNAP in a similar fashion despite their different side chains,
complex. Upon folding of the TL (see below), an insertion com- whereas RBT can additionally interact with the ␴ subunit (67)
plex forms. Catalyzed by the active-site-bound Mg2⫹ ions, the (Fig. 3C).
3=-OH group of the growing RNA chain attacks the ␣-phosphate The antimicrobial activity of RIF proved that the region adja-
of the incoming NTP, resulting in one nucleotide addition to the cent to the active center of bacterial RNAP was a valid target for
3= end of the RNA transcript and release of pyrophosphate (PPi). discovery of drugs acting against transcription. However, the ease
The newly added RNA nucleotide is then translocated to the prod- with which resistance to RIF is gained by mutation at multiple sites
uct site (i site), exposing the next template base in the i⫹1 site for illustrates that there are significant problems with this site for the
the incoming NTP (39, 58, 59). The newly synthesized RNA tran- development of antimicrobials with long therapeutic lives. While
script moves away from the active site through the RNA exit chan- alteration of the RIF binding site does have implications for cell
FIG 2 Compounds that inhibit bacterial transcription. The compounds featured in this review are shown as chemical structures, illustrating the diversity in size
and complexity of RNAP inhibitors. The compounds are arranged with respect to their targets. The naphthyl group of rifampin in the primary-channel inhibitors
is circled, with the equivalent region of sorangicin also circled. Numbered circles: streptolol (1) and monosaccharide (2) moieties of streptolydigin, squarate ring
(3), benzylamine ring (4) and piperidine (5) groups of squaramides, and steroid-like carboxylic acid (6) and indolone (7) groups of DSHS0057. See the text for
further details.
FIG 3 Antibiotics that bind close to the active site. (A) Space-filled structure of T. thermophilus RNAP holoenzyme with core subunits (␣, ␤, ␤=, and ␻) in gray
and initiation factor ␴ shown as a slate blue cartoon. The catalytic Mg2⫹ ion is shown as a cyan sphere, and its location is indicated by the red arrow and
star. The rifamycin/sorangicin binding site is shown in green, and the area used in panels B and C is boxed. The approximate rotations for regions shown
in panels B and C are indicated with arrows. In all panels the catalytic Mg2⫹ ion is shown as a cyan sphere, and in panels B and C an initiating RNA
dinucleotide (orange) is shown adjacent to the active site. (B) Rifampin (RIF) (red) and sorangicin (SOR) (yellow) have overlapping binding sites. (C)
Rifabutin (RBT) (magenta) has improved binding to holoenzyme through interaction with D513 (red stick) located on a region of the ␴ factor called the
␴3 loop. When benzoxazinorifamycin 2b (BZR) (blue) is bound, the C-3= tail causes a distortion in the ␴3 loop (teal), preventing it interacting with the
template strand of DNA. (D) GE23077 (orange) binds in the i and i⫹1 sites, inhibiting RNA synthesis. The binding site is shown in pale green. ATP
entering RNAP via the secondary channel is shown in red. Rifamycin SV (RifSV) (orange) binds near to GE23077, and the two molecules can be covalently
linked to form a compound with activity against rifampin-resistant RNAP. Structure images were prepared using PDB files 1IW7, 1YNJ, 1YNN, 2A68,
2O5J, 4G7O, 4KN4, and 4OIR in PyMol v1.7.4 (Schrödinger, LLC).
fitness, a whole-genome sequencing study of RIF-resistant Myco- vent ␴3 loop interaction with the ⫺3 and ⫺4 bases of the template
bacterium tuberculosis strains showed that alterations of residues ␣ DNA, which is required for binding the initiating NTP at the i⫹1
187 and ␤= 434, 483, 485, 491, and 698 were compensatory (70). A site (69). Residue D513 of the ␴3 loop is able to interact with the
separate study with Staphylococcus aureus also showed that alter- ansa ring of RBT (Fig. 3C, dotted line) (67), whereas benzoxazin-
ations at ␤ residues 471 and 481 gave intermediate levels of resis- orifamycin 2b (BZR) causes a distortion of the ␴3 loop that pre-
tance to RIF with no loss of cell fitness (71). Efforts to minimize vents template DNA interaction (Fig. 3C, teal ribbon) (69). The
RIF drug resistance include combinatorial therapy (with isonia- improved properties of the benzoxazinorifamycins has led to one
zid, ethambutol, and pyrazinamide for tuberculosis), short-term derivative, Rifalazil, entering, but failing, phase II trials for the
use in clinical practice, and the development of new delivery treatment of Chlamydia infection (77, 78).
mechanisms such as nanoparticles (72) and metal conjugation
(73). Furthermore, chemical derivatives have also been developed, Sorangicin
such as the benzoxazinorifamycins, which demonstrate superior Sorangicin is a macrocyclic antibiotic isolated from Sorangium
affinity to RIF-resistant M. tuberculosis strains and reduced ad- cellulosum (79). A cocrystal structure of sorangicin with T. aquati-
verse side effects caused by cytochrome P450 induction (69, 74– cus RNAP demonstrated that this molecule bound to exactly the
76). Cocrystal structures showed that the interactions of benzox- same site as RIF adjacent to the active center and shared similar
azinorifamycins with the RNAP ␤ fork loop II and the ␴3 loop may interactions with the corresponding residues (Fig. 3B) (80). Thus,
occur and may explain the improved biological activity (67, 69). the same mechanism of action, biological activity, and cross-resis-
Careful inspection of the structure of E. coli RNAP in complex tance with RIF mutants can be expected (81).
with benzoxazinorifamycins suggests that the C-3= tail may pre- However, despite being structurally related to rifamycins,
sorangicin comprises a more flexible skeleton than RIF, which subunit of RNAP spans the DNA binding cleft downstream of the
contains a naphthyl moiety (equivalent regions are circled in catalytic center and makes tight contacts with the mobile ␤= trig-
Fig. 2), suggesting that the former might be able to adapt better ger loop (TL) (Fig. 1C and 4A and B). The TL and BH are involved
than RIF to conformational change and mutations in RNAP (80). in the regulation of substrate loading and nucleotide addition by
Studies on RIF and sorangicin against resistant mutant M. tuber- switching the DNA binding cleft between open and closed confor-
culosis strains revealed that a narrower range of mutant strains mations. The open conformation (Fig. 4A) allows the transloca-
were resistant to sorangicin than to RIF and proved that RIF was tion of the 3= end of the RNA transcript and release of the pyro-
more sensitive to the change of shape of the binding pocket due to phosphate by-product (89). Bending of the BH toward the DNA/
its rigid structure, while sorangicin displayed more conforma- RNA hybrid has been proposed to facilitate RNA translocation
tional flexibility, as demonstrated by molecular dynamics simula- (90, 91). The closed conformation (Fig. 4B) involves refolding of
tions (80, 82). the TL into a three-helix bundle with the BH into the catalytic
Extensive studies have been performed on the structure-activity conformation (59). The three-helix bundle aligns the NTP sub-
relationship of rifampin and sorangicin, which could help us to strate with the active site, as well as shifting the position of the 3=
understand how the region around the active site of RNAP could end of the RNA and catalytic Mg2⫹ ions (92). Residues in the
amino-terminal extension of the BH, the F-loop, and the ␤-link
be exploited as a valid drug target (50, 66, 67). Using information
based on the RIF-RNAP cocrystal structure, five amino acids were (Fig. 1C) contact the TL directly and stabilize its refolding (93).
identified, corresponding to T. thermophilus RNAP ␤ subunit res-
idues Q390, F394, R405, Q567, and Q633, and used in fragment- Streptolydigin
based drug design. Two compounds containing four functional Streptolydigin (STL) was isolated from Streptomyces lydicus cul-
groups designed for binding to residues Q390, F394, R405, and tures in the last century, even earlier than the discovery of rifamy-
Q567 displayed good in vitro activities of 70 and 62 ␮M, respec- cins (94–96). This molecule is a derivative of tetramic acid con-
tively (83). However, R405 is not conserved in E. coli (T525), and taining an acyl side chain (streptolol) and a monosaccharide (Fig.
these compounds were subsequently also shown to inhibit E. coli 2, circles 1 and 2, respectively), and it displayed broad-spectrum
␤-galactosidase but not other E. coli or mammalian enzymes (84). antibacterial activity by inhibiting bacterial transcription initia-
tion, elongation, and pyrophosphorolysis (97). Extensive studies
GE23077 on STL have been performed since its isolation, and it was dem-
GE23077 (Fig. 2) was isolated from an Actinomadura sp. in 2004 onstrated that STL could stabilize the translocation state by pre-
and is a relatively new macrocyclic heptapeptide antibiotic (85). venting the conformational changes in the BH and TL during the
The cocrystal structure of T. thermophilus RNAP with GE23077 nucleotide addition cycle, thus decreasing the rate of nucleotide
has recently been published, which illustrated that the compound addition (98, 99).
bound to the i and i⫹1 sites of the active center, adjacent to the The cocrystal structure of STL with T. thermophilus RNAP
catalytic Mg2⫹ ion and close to the RIF binding site (Fig. 3D) (86). showed that STL bound to the region adjacent to the active site,
More precisely, it was demonstrated using saturation mutagenesis which is functionally involved in the nucleotide addition cycle (59,
that E. coli ␤ subunit residues P564, E565, G566, N568, R678, 98, 99). More specifically, the streptolol side chain of STL interacts
M681, N684, M685, Q688, K1065, K1073, and H1237 and ␤= res- with the BH and the "STL pocket," which is formed by E. coli ␤
idues D462, T786, and A787 were involved in binding with residues 538 to 552 and 557 to 576 (98), while the tetramic acid
GE23077. moiety binds to the TL, causing its displacement (Fig. 4C, dashed
GE23077 alone is not a particularly good antibiotic, as it has arrow). In fact, deletion of the TL can increase the binding affinity
poor membrane permeativity due to its hydrophilic nature and of T. thermophilus RNAP to STL (98). The sugar moiety substi-
shows good antibacterial activity only when used against ⌬tolC tuted on the N-tetramic acid was not thought to interact directly
strains or in combination with a membrane-perturbing agent (85, with RNAP, but the cocrystal structure with an RNAP-DNA com-
87). Chemical derivation does not result in a great improvement plex revealed that it forms a hydrogen bond with the downstream
of antibiotic activity (88). However, the target-dependent resis- DNA and hydrophobic interactions with the DNA and TL (59).
tance spectrum for GE23077 was much smaller than those for RIF Analysis of the same cocrystal structure revealed that the down-
and other RNAP inhibitors, with resistant substitutions being ob- stream DNA also interacts with the streptolol side chain and the
tained at only four residues (E565, G566, M681, and N684) in the tetramic acid of STL.
␤ subunit (86), suggesting that the i and i⫹1 sites at the active Although STL was shown to bind multiple sites within RNAP,
center might be a good target for drug discovery. most of the interactions were hydrophobic van der Waals forces
By combining GE23077 and rifamycin SV, a bipartite molecule (59, 98, 99), except for the interaction of the streptolol ring with ␤
which bound to adjacent sites near the active site was created (Fig. fork loop II residue R548 and the interactions of the acetamide
3D) (86). This compound demonstrated excellent activity against substitution of tetramic acid with ␤= BH residues K789 and N792
GE23077 or RIF-resistant RNAP, showing that the RIF binding (59). It was demonstrated that E. coli RNAP with a ␤ subunit
pocket and GE23077 binding i and i⫹1 sites could be considered R548A substitution was resistant to STL, and enzyme with an
for use as a combined target for drug design. N792D substitution in the ␤= subunit was 75 times more sensitive
than the wild type to STL due to enhancement of the polar inter-
MOBILE ELEMENTS OF THE PRIMARY CHANNEL action with acetamide (98).
The activity of RNAP is highly regulated during all stages of the Bacterial resistance to STL was first observed many years ago,
transcription cycle, and a great proportion of the regulation is and a saturation mutagenesis study isolated 72 independent resis-
mediated via mobile structural elements adjacent to the active site. tant mutations on 26 residues, suggesting that it may not be a good
The conserved 37-amino-acid (aa) bridge helix (BH) in the ␤= antibiotic for further development, despite its specificity for bac-
FIG 4 Inhibitors that target the bridge helix and trigger loop. (A and B) Bridge helix (BH) (blue) and trigger loop (TL) (green) in alternative conformations. The
BH and TL colors are conserved in all panels in this figure, and active-site Mg2⫹ ions are shown as cyan spheres. Bending of the BH in panel A, indicated by the
dashed straight arrow, aids translocation of the transcript to help incorporation of the incoming NTP, shown in red behind the BH. The TL is in the "open"
conformation, allowing NTP entry into the active site. In panel B, the BH is straight and the TL has formed extended ␣ helices behind the BH, forming a "closed"
conformation that helps position the incoming NTP (red) in the active site. The movement of the TL from an open to a closed conformation is indicated with
the curved dashed arrow in panel A. (C) Streptolydigin (STL) bound in a space-filled model of RNAP. Amino acids involved in interaction with STL are colored
yellow. The STL pocket formed by the BH and ␤ fork loop II is circled. The TL in the open conformation (shown as a cartoon) clashes with the tetramic moiety
of STL, providing an understanding of how its deletion can stabilize STL binding. (D) Salinamide (SAL) bound within the secondary channel adjacent to the TL
and CBR703 bound adjacent to the F loop (magenta). The binding site for SAL is shown in orange and that for CBR703 in yellow. RNAP surface and cartoon
elements are shown in semitransparent form so that CBR703 within its binding site can be seen. Conformational change of the TL from the open to the closed
form (transparent cartoons) will be sterically blocked by SAL. Structure images were prepared using PDB files 1IW7, 1ZYR, 2O5J, 4G7O, 4MEX, 4OIP, 4XSX, and
4ZH2 in PyMol v1.7.4 (Schrödinger, LLC).
terial RNAP (59, 100, 101). However, the information obtained not conserved in eukaryotic RNAP, accounting for the specificity
from studies of STL binding to RNAP may help with the of this antibiotic against bacteria (105). A mutagenesis study to
development of RNAP inhibitors targeting the nucleotide addi- identify amino acid substitutions conferring salinamide A resis-
tion cycle. tance correlated well with the information obtained from the co-
crystal structure. Substitutions in ␤= BH residues R738, A779, and
Salinamide
G782 and ␤ residues D675 and N677 result in resistance to salin-
Salinamides A to E are a series of bicyclic polypeptidic molecules amide A.
isolated from marine and soil Streptomyces spp. (102–104). Salin- During the nucleotide addition cycle, salinamide A may also
amides A and B (Fig. 2) displayed good inhibition of transcription
interact with the TL due to conformational changes in RNAP
in both Gram-positive and -negative organisms and with moder-
(105). Although the binding site of salinamide A was different
ate antibacterial activity, which was probably due to the hydro-
from that of STL and did not show cross-resistance with other
philic peptidic structure having poor membrane-crossing proper-
ties (104). The cocrystal structure of salinamide A with E. coli RNAP inhibitors, functionally salinamide A was similar to STL, as
RNAP holoenzyme demonstrated that it bound to a region be- it did not prevent the formation of a transcription open complex
tween the BH and secondary channel (Fig. 4D) and involved in- but was able to inhibit transcription initiation and elongation,
teraction with ␤= residues R738, R744, L746, M747, A748, S775, probably by prevention of TL refolding due to steric interference
and K781 and ␤ residues D675 and N677. These amino acids are (Fig. 4D, dashed line).
Although salinamide A is not suitable for further clinical devel- ponents may have affected assays that suggest that CBR703 has no
opment due to poor membrane permeativity, the binding site effect on human tissue culture cell lines at concentrations as high
close to the secondary channel and nucleotide addition site repre- as 100 ␮M (53, 110). The inhibitory effect on biofilm formation
sents an interesting validated target for new drug design. may also be an artifact, as it occurs at high concentrations (2 to 400
␮M) when CBR703 starts to precipitate at 100 ␮M, and the hy-
CBR Compounds drophobic aggregates may have prevented biofilm formation (53).
The CBR series (Fig. 2) consists of small molecules comprising Nevertheless, the information gained from the CBR compounds
two linked aromatic rings, isolated by screening a chemical com- may provide approaches for development of allosteric inhibitors
pound library to inhibit E. coli transcription (106). CBR703, to- of transcription that bind the same site. The close proximity (⬃8 Å
gether with CBR9379 and CBR9393, demonstrated the ability to between the closest atoms) of the CBR703 and salinamide binding
inhibit transcription elongation by stabilizing elongation complex sites may also permit fragment-based development of bipartite
isomerization and slowing translocation (107). The inhibitory ac- inhibitors.
tivity of these compounds is proposed to be due to an allosteric
effect that prevents TL folding, mediated via the F-loop, and the SECONDARY-CHANNEL INHIBITORS
inhibition of BH movement at its N-terminal hinge (108). An During transcription, NTP substrates need to be made available
interesting aspect of their antibacterial activity is their ability to for the growing RNA chain, but the active site is buried deep inside
inhibit biofilm formation, which is especially important in a clin- RNAP at the apex of the primary channel (Fig. 1A). The funnel-
ical setting as biofilms are a major source of nosocomial infection shaped secondary channel is formed by the ␤= BH spanning the
(109). DNA binding clamp immediately downstream of the active site
Recent X-ray crystal structures show that CBR703 and several (36). The secondary channel is about ⬃12 Å in diameter, which is
derivatives bind to E. coli RNAP between ␤ lobes 1 and 2 to regions too narrow for double-stranded nucleic acids and allows the pas-
linking the ␤ flap loop II and ␤DII motifs, the ␤= F-loop, and the sage of only one NTP at a time, and the negative electrostatic
N-terminal portion of the ␤= BH (Fig. 4D) (108, 110). Specifically, potential that predominates in the secondary channel imposes a
CBR703 forms interactions with ␤ residues V550, H551, P552, further constraint on NTP diffusion (55). Several transcription
Y555, R637, G640, E641, and S642 and ␤= residues K749, P750, factors regulate RNAP activity through interaction with the sec-
I755 (part of the F-loop), P770, F773, I744, and H777 (BH N ondary channel, such as the Gre factors DksA and Rnk (111–114).
terminus) (108). Most of these interactions are hydrophobic, with For example, in a backtracked elongation complex, the 3= end of
only ␤ S642 making a polar interaction with the amidoxime moi- the RNA transcript slides into the secondary channel, and the
ety of CBR7903 (108, 110). The structural information agrees well acidic tips of the Gre factors penetrate into the enzyme close to the
with data inferring the binding site from analysis of resistance active site and facilitate an intrinsic RNA cleavage activity of
mutants (at ␤ P560, R637 and S642) (106), and this was further RNAP (115). The backtracked RNA transcript is then released
elaborated by saturation mutagenesis experiments that identified from the secondary channel, allowing the stalled transcription
a further 11 resistance alleles (110). Despite resistance arising at 14 elongation complex to be rescued (116).
separate loci, the rate of resistance due to spontaneous mutation
in one tested strain was extremely low at ⬍1 ⫻ 10⫺12, which is 102- Tagetitoxin
to 104-fold lower than those for other antibiotics, suggesting that Tagetitoxin (Fig. 2) was isolated from a culture of the plant patho-
the CBR binding site may have potential as a useful resistance- gen Pseudomonas syringae pv. tagetis and was expected to have
refractory binding site (110). Interestingly, two mutations (␤= broad-spectrum activity on RNAP (117). A series of studies
P750L and ␤= F773V) make cells dependent on CBR compounds showed that it targeted chloroplast RNAP and RNAP III (but not
for growth as well as conferring resistance, although the mecha- RNAPs I and II) and could also target bacterial RNAP by inhibi-
nism for this is currently unknown (108). The predicted structure tion of transcription elongation (118, 119). Despite this in vitro
of CBR703 bound to RNAP determined by in silico docking was activity, tagetitoxin has no antibacterial activity against live cells
very similar to what was found in the X-ray crystal structures, due to low cell permeativity (120).
except it was inserted into the two hydrophobic pockets the wrong The structure of tagetitoxin in complex with T. thermophilus
way around (107, 108, 110). This is not surprising since CBR703 is RNAP (Fig. 5A) shows that it binds in the secondary channel
highly symmetrical and brominated derivatives were needed to mainly via polar interactions due to its high oxygen content,
unambiguously determine the orientation of the compound in the which also results in poor membrane permeativity of the com-
structural experiments (108, 110). pound (120). ␤= residues N458, Q504, S733, and Q736 are in-
Despite the promise of the CBR family, significant hurdles still volved in binding to the bicyclic ring of tagetitoxin, while ␤ resi-
need to be overcome before they can be considered bona fide dues R678 and R1106 and ␤= residue R731 interact with the
antibiotic leads. Although these compounds have good activity phosphate group, which also binds to a noncatalytic Mg2⫹ ion
against RNAPs from problem Gram-negative organisms, includ- (121). It has also been proposed that tagetitoxin is capable of in-
ing Klebsiella pneumoniae, and are reported to have activity teraction with the TL region (Fig. 5A, closed form, green cartoon),
against Gram-positive Staphylococcus aureus, despite the presence which could account for its inhibitory effect on transcription, but
of N rather than S at the ␤642 position that is essential for binding this is a currently unresolved issue (121–123). While tagetitoxin is
and specificity, they have antibacterial activity only against strains not an antibiotic, further studies to resolve its mechanism of in-
in which the TolC outer membrane drug efflux pump has been hibition may provide important information for the development
deleted (53, 108, 110). In addition, despite not inhibiting human of new leads that have a similar binding site. The structure of
RNAP II activity in vitro, the compounds are cytotoxic (53). The tagetitoxin has also been revised recently (124) and is the structure
fact that they are hydrophobic compounds and bind serum com- presented in Fig. 2, which may help with the development of new
FIG 5 Secondary-channel binding compounds. (A) Tagetitoxin bound inside the secondary channel. The binding site is shown in yellow with the TL (green) in
the closed conformation as a cartoon to aid visualization of tagetitoxin, and the BH is shown in blue. (B) Binding site for microcin J25 in yellow, with the TL
surface rendered in the open conformation. No structure of microcin J25 in complex with RNAP is available, but modeling indicates that it would block the
secondary channel, preventing NTP entry (rendered as a semitransparent red surface). Structure images were prepared using PDB files 1PP5 and 2BE5 in PyMol
v1.7.4 (Schrödinger, LLC).
leads through reexamination of electron density maps of the DNA loading/unwinding and clamp closure during initiation and
RNAP-tagetitoxin structure along with structure-activity rela- elongation (41).
tionship analysis.
Myxopyronin, Corallopyronin, Ripostatin, and Squaramides
Microcin J25
Myxopyronins (Fig. 2) were isolated from cultures of Myxococcus
Microcin J25 (Fig. 2) is a 21-mer peptide produced by E. coli AY25 fulvus and displayed broad-spectrum antibacterial activity against
that is active against Gram-negative bacteria (125, 126). It has a Gram-positive and -negative bacteria by inhibiting transcription
very unusual lassoed tail structure comprising a ring with the C- initiation (133, 134). Mechanistic studies showed that myxopy-
terminal end passing through the center (127–129). It inhibits ronins bind to the switch region and lock the clamp and switch
abortive initiation and elongation by competitively preventing regions in a partly closed/fully closed conformation. This prevents
NTP uptake or binding (130). Saturation mutagenesis identified the clamp from opening and RNAP from binding double-
substitutions on 51 residues in the regions of the ␤ and ␤= subunits stranded DNA during the transition from a closed to an open
that form the secondary channel, which result in resistance to complex during transcription initiation (132, 135).
microcin J25. The binding site was proposed to comprise ␤= resi- The binding site of myxopyronin A and 8-desmethyl myxopy-
dues 498 to 504, 732 to 733, 922 to 926, and 1244 to 1248 (Fig. 5B) ronin B on T. thermophilus RNAP has been determined and
(130). The inhibitory constant (Ki) of microcin J25 was also mea- shown to comprise a hydrophobic pocket formed by "switch 1,"
sured to be approximately 20 ␮M (131), but due to the relatively "switch 2," and adjacent segments, including ␤ residues 1270 to
low potency, narrow spectrum, and very large number of substi- 1292 and 1318 to 1328 and ␤= residues 330 to 347, 1319 to 1328,
tutions that result in resistance to the compound, it is not partic- and 1346 to 1357 (132, 135). Although the switch region is con-
ularly amenable for further development. served across prokaryotic and eukaryotic RNAPs, the adjacent
segments are conserved only in bacterial RNAPs, which may ex-
SWITCH REGION INHIBITORS plain the specificity of myxopyronins against bacteria (132, 133).
The overall architecture of the RNAP crab claw undergoes a series It was shown that ␤ E1272, E1279, and S1322 and ␤= G344, K345,
of conformational changes to allow template DNA to access the and K1348 were involved in polar interactions with both myxopy-
catalytic center. One of the pincers of the claw formed by part of ronin A and 8-desmethyl myxopyronin B. Mutagenesis studies
the ␤= subunit (termed the clamp region) has been shown to ex- demonstrated that amino acid substitutions at ␤ residues E1279
hibit significant structural flexibility (26, 74) and is hinged, which and S1322 and ␤= residue K345 generated resistance against 8-des-
enables widening of the claw, helping to load DNA into the active methyl myxopyronin B, while changes at ␤ V1275 and L1291 and
site. The clamp then swings back to close the active channel in ␤= K345 resulted in resistance to myxopyronin A (132, 135).
order to retain the DNA template in the transcription bubble dur- It is worth noting that corallopyronin, which is structurally sim-
ing transcription elongation. The swing motion of the clamp is ilar to the myxopyronins, as well as the structurally distinct ri-
regulated by the switch region, which is located at the base of the postatin, demonstrated the same inhibitory activity on transcrip-
clamp domain (15, 76) (Fig. 1C and D). There are five segments of tion initiation, and the amino acid substitutions that resulted in
the switch region, termed "switch 1" to "switch 5." In particular, resistance to these molecules also overlapped (136–138). These
switches 1 and 2 undergo relatively large conformational changes results indicated that corallopyronin and ripostatin also bind to
to mediate clamp movement (132). Important residues of the the switch region (132).
switch region form direct interactions with the DNA template as Squaramides (Fig. 2) are a series of synthetic small molecules
well as the nascent RNA transcript, serving regulatory roles in identified in a high-throughput screen using a bacterial transcrip-
tion-coupled translation assay with an in vitro 50% inhibitory INHIBITORS WITH UNKNOWN TARGETS
concentration (IC50) as low as 0.3 ␮M (51). It was then demon- Ureidothiophene was discovered though chemical library screen-
strated that these compounds were inactive against the translation ing against S. aureus RNAP holoenzyme and demonstrated good
process by substituting mRNA for plasmid DNA. The compounds antibacterial activity against Gram-positive bacteria, including S.
had no activity against representatives of the ESKAPE pathogen aureus and S. epidermidis (147). However, mammalian albumin
group (139) but did show weak activity (MIC values of 100 to 200 binds tightly to ureidothiophene, eliminating its in vivo activity.
␮M) in wild-type Haemophilus influenzae that could be improved Thiolutin and holomycin are types of pyrrothine natural prod-
on deletion of the acrB gene, which encodes a component of the ucts isolated from Streptomyces luteosporeus and Streptomyces cla-
drug efflux system. vuligerus, respectively (148, 149). Both compounds demonstrated
These squaramide derivatives comprise an isoxazole containing broad activity against bacterial and fungal RNAPs, and functional
a sulfonamide substitution on a four-member squarate ring, as analysis showed that that they could inhibit bacterial transcription
well as a benzylamine moiety and a piperidine as its linker to the elongation and yeast transcription initiation (149–151).
squarate (Fig. 2, circles 3, 4, and 5, respectively). Cocrystal struc- Etnangien is a natural antibiotic isolated from the Gram-neg-
tures of two squaramides with E. coli RNAP holoenzyme have ative soil bacterium Sorangium cellulosum, a member of the myxo-
recently been published, which showed that their major binding bacteria with a particularly large (⬎13-Mb) genome that produces
site was broadly similar to that of the myxopyronins within the many secondary metabolites with a wide range of potential med-
switch 1 and 2 regions, with less substantial interaction with ical applications. Etnangien displayed activity against Gram-pos-
switch regions 3 and 4 (140). The cocrystal structures also dem- itive bacteria with MICs as low as 0.06 ␮g/ml but with no effect
onstrated that the binding of squaramide compounds to RNAP against Gram-negative organisms (152). Although E. coli was
pushed switch 2 into the DNA binding main channel of RNAP, shown to be resistant to etnangien, in vitro inhibitory activity
which would prevent the correct positioning of the melted tem- against E. coli DNA polymerase and RNAP and low cytotoxicity
plate DNA. Mutagenesis studies using H. influenzae RNAP con- against a mammalian cell line was observed.
firmed that ␤= residues A1323, L1332, and K1348 and ␤ residue
L1326 (E. coli numbering) interacted directly with squaramides, TRANSCRIPTION FACTORS AND RNAP BINDING SITES AS
while mutations at ␤ residues Q1257, E1279, D1296, and P1320 TARGETS
may also affect squaramide binding (51). Squaramides have po- Transcription Factor Overview
tential for drug development, as they are small synthetic molecules
Each step of the transcription cycle is highly regulated by tran-
and have plenty of options for structural elaboration and because
scription factors during normal cell growth and in response to
the cocrystal structures with RNAP showed that they did not com- environmental signals. To start transcription at the appropriate
pletely occupy the area encompassing the switch region. site, RNAP must be associated with an initiation factor. Most bac-
teria have one primary ␴ factor for the transcription of housekeep-
Fidaxomicin and Lipiarmycin ing genes (␴70 in Gram-negative bacteria and ␴A in Gram-positive
bacteria) and one or more alternative ␴ factors for transcription of
Fidaxomicin (tiacumicin B) (Fig. 2) is a derivative from fermen-
specific subsets of genes in response to environmental stimuli
tation products of Dactylosporangium aurantiacum and is struc-
(153, 154). In the elongation phase, the ␴ factor dissociates from
turally very similar to lipiarmycin, a metabolite from Actinoplanes
RNAP while elongation factors become associated (155). Elonga-
deccanensis (141–143). Both of these compounds target bacterial
tion factors represent a diverse range of proteins that regulate
RNAP and exhibit antibacterial activity selectively against Gram-
transcription activity, such as modulating transcription rates, the
positive bacteria, and fidaxomicin is now used for the treatment of
response to pause and termination signals, rescue of stalled/back-
Clostridium difficile-associated diarrhea (CDAD), with efficacy tracked complexes, and removal of protein roadblocks (156). In
similar to but relapse rates lower than those for vancomycin (144). addition, the compositions of transcription complexes (TCs) in-
There is currently no published structure of fidaxomicin or li- volved in mRNA and rRNA synthesis are quite different (157,
piarmycin in complex with RNAP, but biochemical experiments 158). TCs involved in rRNA synthesis are resistant to pausing
indicated that both of these molecules bind to the switch region signals and transcribe at higher rates than those involved in
(136, 145, 146). Lipiarmycin did not prevent promoter binding mRNA synthesis. The difference in responses of RNAP to pause
but could inhibit promoter DNA melting and ␴-dependent tran- signals, etc., during mRNA and rRNA synthesis is because of its
scription, as well as template strand DNA binding to RNAP. A association with different suites of elongation factors (157, 159,
mutagenesis study demonstrated that deletion of ␴70 subunit re- 160). Many of these transcription factors are essential for bacterial
gion 3.2 residues 513 to 519 of E. coli RNAP and mutation at viability and are not highly conserved in eukaryotic cells. There-
switch 2 residue R337 resulted in resistance to lipiarmycin, sug- fore, the factors as well as their binding sites on RNAP represent
gesting that it might target both the ␴ factor and the switch region potential novel drug targets (Fig. 6).
to prevent formation of a transcription open complex (145).
However, despite having analogous structures and the fact that Termination Factor Rho
amino acid substitutions at appropriate sites result in cross-resis- The bacterial transcription termination factor Rho is a ring-
tance between lipiarmycin and fidaxomicin, fidaxomicin is prob- shaped homohexameric RNA translocase. Each Rho protomer has
ably a more promising compound, as deletion of the ␴3 loop does two distinct functional domains: the N-terminal oligonucleotide/
not result in resistance to the drug (146). These results indicate oligosaccharide binding (OB) domain, which constitutes Rho's
that the way in which these molecules function to inhibit tran- primary RNA binding site, and the C-terminal domain with a
scription is yet to be fully elucidated. secondary RNA binding and ATP binding site (29, 161). The Rho
FIG 6 Transcription factor interaction network. The space-filled RNAP elongation complex is shown in gray, with template-strand DNA in green and
nontemplate-strand DNA in orange. Arrows indicate known interactions, although the binding sites for Rho and NusE on RNAP are not known. Subscript
N and C indicate N- and C-terminal protein domains. NusGN binds to the CH region of RNAP and across the active channel and has an overlapping
binding site with region 2.2 of initiation factor ␴. NusGC can partner switch between Rho and NusE as appropriate. NusE forms a heterodimer with NusB
and binds RNAP. NusAN and region 4 of ␴ have overlapping binding sites on the ␤ flap tip helix at the top of the RNA exit channel. All of these interactions
could potentially be exploited in the development of new antimicrobial compounds. Structure images were prepared using PDB file 2O5I in PyMol v1.7.4,
Schrödinger, LLC.
hexamer exists in two forms, as an open or a closed ring. Open- cin indicated that the binding site was between two C-terminal
form Rho binds to a specific Rho utilization site (rut site) on the domains of Rho protomers, close to the ATP binding site (Fig. 7A)
RNA transcript via its N-terminal primary RNA binding site, and (172). The bicyclomycin binding pocket is formed by residues
the newly synthesized RNA is then guided through the primary E211, R212, D265, S266, and R269 of one protomer and G337 of
binding site into the central hole of the hexamer, where the ring the adjacent protomer, while bicyclomycin also interacts with the
closes and RNA binding triggers ATP hydrolysis (29). It is still associated Mg2⫹ ion that is required for ATP hydrolysis (Fig. 7B).
unclear whether Rho binds to RNAP throughout/for extended
periods of the transcription cycle or only during the final stages of The RNAP-␴ Interaction
termination, but direct interaction with RNAP appears to be re- ␴ factors are required for initiation of transcription. In all bacteria,
quired to induce allosteric changes in the active site of RNAP to the general housekeeping ␴ factor (␴70 in Gram-negative bacteria
terminate transcription (162). and ␴A in Gram-positive bacteria) is absolutely essential for cell
Bicyclomycin. Bicyclomycin (Fig. 2) was isolated from Strepto- viability (173, 174). ␴ factors are flexible elongated proteins that
myces sapporonensis and is active predominately against Gram- make multiple contacts with RNAP when forming a transcription
negative bacteria (163), although it is reported to be more effective initiation complex. However, only one region of the ␴ factor,
against problematic nosocomial infections such as Klebsiella called ␴2.2, is absolutely necessary for initiation complex forma-
pneumoniae and Acinetobacter baumannii when used in combina- tion, and it binds to a region of the ␤= subunit of RNAP called the
tion with inhibitors of translation (164). While Rho is essential for ␤= clamp helix (CH) region (175–177) (Fig. 1B). The ␴2.2-CH
viability in many Gram-negative organisms, Gram-positive or- interaction involves ␤= residues R275, R278, R281, L282, I291,
ganisms appear to be less dependent on it, although it is important N294, and M298 and ␴70 residues Q403, Q406, E407, I410, and
in preventing transcription of noncoding RNA in the latter and M413 (37, 38, 176, 177), but this is a nontraditional drug target as
has activity against Micrococcus luteus (165, 166). Bicyclomycin is it lacks any deep clefts, which are considered desirable binding
the only known antibiotic to target Rho, and it does not affect sites for small molecules. Nevertheless, a peptide-based study
RNA or ATP binding to Rho but does inhibit Rho-dependent showed that peptides mimicking ␴2.2 could inhibit RNAP-␴ bind-
transcription termination and ATP hydrolysis (167–169). Substi- ing to prevent in vitro transcription initiation, and prevention of ␴
tutions in E. coli Rho residues L208, M219, S266, and G337 con- production by antisense peptide nucleic acids could inhibit the
ferred resistance to bicyclomycin, suggesting that Rho was the growth of clinically relevant multidrug-resistant S. aureus (178,
binding target of bicyclomycin (170, 171). 179).
A cocrystal structure of E. coli open-form Rho and bicyclomy- The GKL series. The GKL series (Fig. 2) is a class of bis-indole-
FIG 7 Inhibition of transcription termination factor Rho activity. (A) The Rho hexamer in its open-ring conformation. Four subunits are shown in slate blue
with the remaining subunits in gray and white, sandwiching the Rho inhibitor bicyclomycin (red sticks). The bicyclomycin binding site is boxed. (B) Expanded
view of bicyclomycin binding site area, with protein shown as ribbons. The bicyclomycin binding site is highlighted in yellow, with contributing amino acids
shown as sticks. ATP is shown in blue in stick format and Mg2⫹ as a cyan sphere. Structure images were prepared using PDB file 1XPO in PyMol v1.7.4
(Schrödinger, LLC).
derived compounds targeting bacterial transcription based on the against eukaryotic RNAP in in vitro binding assays or in cell
strategy of structure-based drug design (177). Detailed binding culture, suggesting that this compound will be a potentially
studies enabled the characterization of the precise contributions useful lead for future development.
of individual amino acids to the RNAP-␴ interaction (176, 177). The SB series. The SB series (Fig. 2) was discovered by com-
This resulted in the construction of a pharmacophore model pound library screening against E. coli RNAP holoenzyme forma-
which was used to screen an in-house compound library. Careful tion using an enzyme-linked immunosorbent assay (ELISA)-
selection of potential hit compounds resulted in ⬃1/3 having po- based assay (185). These compounds could inhibit E. coli ␴70
tent in vitro inhibitory activity against the formation of RNAP-␴ binding to RNAP with an IC50 of 2 to 15 ␮M, although their
complexes (176, 177, 180). binding site was unknown. The ␴-independent transcription in-
GKL003 was shown to specifically bind to the ␤= subunit CH hibition activity of the SB series suggested that the compound
region and had a Ki of ⬃6 nM as determined from in vitro tran- binding site was on RNAP rather than ␴ (186).
scription assays (177). Compared to fidaxomicin, which functions A modified SB series of furanyl rhodanine compounds dem-
only when added prior to RNAP-␴ interaction, GKL003 is a com- onstrated activity against Gram-positive bacteria and inhibited
petitive inhibitor which can also inhibit transcription initiation formation of biofilm (109). However, it was shown that the SB
after the holoenzyme has formed (177). Despite having excellent series could target multiple targets on the membrane and
activity in vitro, GKL003 had poor activity against live cultures of within bacteria instead of acting as a specific inhibitor of tran-
bacteria, probably due to its low solubility in aqueous media and scription (52), making any further development complicated
inability to cross bacterial membranes. Nevertheless, it did exhibit until comprehensive structure-activity relationship studies are
broad-spectrum antibacterial activity at high concentrations and performed.
has been extensively derivatized to establish structure-activity re-
lationships (181–183). Determination of the structures of GKL Potential Targets
compounds in complex with RNAP will be important for their NusA. As an essential transcription factor, NusA is tightly associ-
continued development. ated with RNAP throughout the elongation phase of transcription
DSHS00507. Using structure-based drug design, an in silico (187) and is important for the regulation of several aspects of
screen of a publically available drug-like compound library led transcription elongation, including pausing, termination, and the
to seven hits, with one, DSHS00507 (Fig. 2), showing excellent formation of termination-resistant (antitermination) complexes
inhibitory activity against RNAP holoenzyme formation (184). essential for the production of rRNA (157, 188). Recent biochem-
This compound, comprising a steroid-like ABC tricyclic car- ical work found that NusA is involved in the "immune system" in
boxylic acid and an indolone (Fig. 2, circles 6 and 7, respec- E. coli host cells to suppress the toxic activity of foreign genes
tively), binds to the ␤= subunit CH region of RNAP with great (189). NusA has also been shown to interact with translesion DNA
specificity and inhibits in vitro transcription by preventing ␴A synthesis polymerases and to play a role along with the UvrD
binding. Antibacterial assays indicated that the compound was helicase in nucleotide excision repair (190, 191). Residues F59,
effective against Gram-positive bacteria, including communi- R61, E94, and Q96 of the N-terminal domain of NusA are re-
ty-acquired methicillin-resistant Staphylococcus aureus, and mi- quired for interaction with L895, E899, K900, R903, and E908 on
croscopic analysis gave results consistent with RNAP being the a conserved region of RNAP called the ␤-flap tip helix (Fig. 1D),
target for this compound in live cells. Although eukaryotic which forms one side of the RNA exit channel, although the struc-
RNAPs also contain a CH structure, it shows no sequence sim- ture of the complex at high resolution is yet to be elucidated (44,
ilarity to the bacterial CH, and DSHS00507 showed no activity 192, 193). NusA is highly conserved among the eubacteria, is
FIG 8 NusE is an "interaction hub" that links transcription and translation. (A) Nonoverlapping interactions that NusE makes with NusB (red), NusGC (yellow),
boxA RNA from an rRNA operon leader (green), and double-stranded RNA (dsRNA) (cyan), representative of the boxB hairpin from an rRNA operon leader.
(B) Location of NusE (gray) (also known as ribosomal protein S10) in the context of a translating ribosome. The 30S subunit is shown in green and the 50S
subunit in cyan. NusGC (yellow) would not be sterically inhibited from interacting with NusE integrated into the 30S ribosomal subunit and so could be
important in linking transcription with translation. Structure images were prepared using PDB files 2JVV, 2KVQ, 3CXC, 3D3B, 3R2C, and 3R2D in PyMol v1.7.4
unique to this kingdom, and is essential for viability in all organ- a wide range of proteins and nucleic acids, with very little over-
isms tested (173, 174, 194, 195), indicating its importance in tran- lap between the binding sites (Fig. 8A). Indeed, the context of
scription and making it an ideal target for the development of new NusE/S10 within the small ribosomal subunit leaves the NusGC
antitranscription compounds that prevent its interaction with binding site available for interaction and has been suggested to
RNAP. be important in linking transcription and translation in bacte-
NusB/E. In bacteria, rRNA is the most abundant RNA species ria (Fig. 8B) (203).
in the cell and the major component of the ribosomes, and its NusG. As the only conserved transcription factor across all
rate of synthesis is growth rate limiting. NusB and NusE are three domains of life (204), bacterial NusG exerts diverse, or
small transcription factors required for the formation of anti- even opposite, functions in the regulation of gene expression.
termination complexes that carry out rRNA synthesis (158, NusG is an essential protein in Gram-negative bacteria (173,
188, 196). NusE is ribosomal protein S10 and so has dual func- 195) and is involved in enhancing factor-dependent termina-
tions in the cell as both a transcription factor and a component tion by termination factors such as Rho and bacteriophage
of the small ribosomal subunit. Antitermination complex as- HK022 Nun (205, 206). Together with Rho, NusG is responsi-
sembly initiates when NusB binds to a newly transcribed con- ble for limiting the expression of toxic foreign genes on cryptic
served RNA sequence in the rRNA operon leader sequence phages integrated in the E. coli chromosome (189). On the other
called boxA. Next, a NusB-NusE heterodimer is formed to hand, NusG has been shown to increase the transcription elonga-
strengthen the interaction with boxA and facilitate binding of tion rate both in vitro and in vivo by preventing transcription
other regulatory factors, and the complex interacts with RNAP pauses incurred by RNAP backtracking (207–209). In conjunc-
via NusE (188, 197). Therefore, formation of the NusB-NusE- tion with other Nus factors (NusA and NusB/E; see above), NusG
boxA complex represents a regulatory step in antitermination participates in the formation of antitermination complexes re-
complex assembly (198). The NusB-NusE heterodimer is also quired for the rapid and efficient transcription of rRNA operons
able to bind double-stranded RNA (199) and in vivo may bind (188). NusG has also been suggested to have a role in coupling
the RNA hairpin boxB, which is also part of the leader sequence transcription and translation (Fig. 8B) (203). NusG consists of an
of rRNA operons, although it does not appear to be important amino-terminal domain (NusGN) joined by a flexible linker to the
for viability (200). Since rapid cell proliferation is a character- carboxyl-terminal domain (NusGC) (210). As illustrated in Fig. 6,
istic of bacterial infection, small molecules that disrupt NusB- NusGN competes with ␴ factors for RNAP binding (211), while
NusE heterodimer formation or interaction with boxA or that NusGC binds mutually exclusively to either Rho or NusE factors
inhibit NusB-NusE-boxA interaction with RNAP have poten- (203). By analogy to the interaction between the Spt4/5 in com-
tial as lead compounds for novel antibiotics (Fig. 6). While the plex with the CH region from the archaeon Pyrococcus furiosus
structure and interactions between NusB, NusE, and RNA (212), the tip of the ␤= CH region of RNAP binds to a hydrophobic
(both boxA and dsRNA) have been determined (199, 201) and cavity on NusGN comprising F65, P66, Y68, L70, I93, I103, E107,
NusE is known to bind to the ␤ subunit of RNAP (202), its V108, and I111. Analysis of a NusB-NusE-NusGC tripartite com-
precise binding site on RNAP remains to be determined. NusE plex by nuclear magnetic resonance (NMR) indicates that residues
also interacts with the C-terminal domain of NusG (NusGC) G139, P140, F141, S161, V162, I164, F165, G166, R167, A168,
(see below). This small protein is therefore able to interact with T169, E172, and D174 are involved in interaction with NusE
(203), whereas mutagenic studies imply a role for G146, V148, subtilis has also been shown to interact with its cognate RNAP
L158, V160, and I164 in interaction with Rho (213). While the (222–224). Once the elongation complex is stalled at DNA lesion
residues involved in interaction with NusE are all solvent exposed, sites, UvrD is able to pull RNAP backwards (in the direction op-
those implicated in binding to Rho are buried within the hydro- posite to that of transcription), exposing the damaged DNA for
phobic core of NusGC. NusG is closely related to another special- repair (191). E. coli RapA (HepA), a Swi2/Snf2 helicase (225), has
ized transcription factor, RfaH, whose C-terminal domain under- been shown to be involved in RNAP recycling and RNA polyade-
goes drastic rearrangement from a ␤-barrel form to a NusGC-like nylation (226, 227), and in B. subtilis, HelD is required for efficient
conformation (214), and it is possible that NusGC undergoes con- recycling of RNAP, which is achieved synergistically with the ␦
formational change upon binding to Rho, although this seems subunit (228). As our understanding of the roles of helicases in
unlikely as circular dichroism spectra of wild-type and mutant transcription increases, these important enzymes could also rep-
NusG are all similar (213). The NusGC binding site on Rho is resent potential targets for the design of transcription inhibitors.
solvent exposed and is within two small depressions on the outer
surface of the hexameric ring comprising hydrophobic residues CONCLUSIONS AND PERSPECTIVES
203 to 208 (VLMVLL), V227 and V228 (213). Small molecules
The need for new antibiotics is pressing, and it is essential that new
targeting the NusGN-RNAP interaction (with the possibility of
also inhibiting ␴-RNAP binding), as well as the NusGC-Rho/NusE targets are identified, validated, and developed to ensure that al-
binding surface, would have the potential to inhibit the cellular ternative compounds are available to combat organisms that are
function of the essential factor NusG in bacteria, although the resistant to currently available antibiotics. Transcription repre-
apparent hydrophobic nature of the regions involved in the RNAP sents an excellent target for the continued development of impor-
␤= CH-NusGN and Rho-NusGC interactions may prove difficult tant drugs, as it can be targeted through direct and allosteric
to target with specificity. inhibition of enzymatic activity or through inhibition of protein-
NusGSP factors. While essentially all microbes contain a gene protein/protein-nucleic acid interactions that are essential for vi-
encoding a NusG transcription elongation factor, many also con- ability. Historically, high-throughput screens of natural product
tain paralogous NusG-like proteins that control transcription of libraries have been used in the discovery of potent antitranscrip-
small subsets of genes, and these have been called specialized tion inhibitors, and this approach is still yielding compounds that
NusGs (NusGSPs) (215). The best characterized NusGSP is RfaH, have great promise as stand-alone compounds or in combination
which is involved in the control of transcription of genes required with existing inhibitors as novel chimeric antibiotic leads (see,
for extracytoplasmic functions such as lipopolysaccharide (LPS) e.g., reference 86).
synthesis, toxin production, and phage sensitivity (216). NusGSPs More directed approaches are now also possible due to the
bind to the same region of RNAP as NusG via their structurally rapid increase in high-resolution structural information on RNAP
conserved N-terminal domains (217), and their C-terminal do- and transcription complexes that is becoming available. Chemical
main (as determined for RfaH) undergoes a dramatic structural space can be explored for the design and development of new
transformation into a structure analogous to the C-terminal do- inhibitor compounds using more rational approaches that require
main of NusG, which permits interaction with NusE/S10, ensur- the careful construction of templates (pharmacophores) around
ing efficient translation (214). NusGSPs are not essential, but since which inhibitors are constructed. Such approaches can be used in
they are required for the expression of genes associated with viru- the design/modification of compounds that target the enzymatic
lence (e.g., LPS and toxin production), compounds that target activity of RNAP as well as protein-protein interactions.
their interaction with RNAP and/or NusE may be useful as anti- There are advantages and disadvantages to both approaches.
infective agents. However, given the similarity of NusGSP binding Through the process of natural selection, most natural products
sites with NusG, the same problems with producing compounds tend to have very good activity (low MICs) but are also subject to
of sufficient specificity may be encountered. the coevolution of resistance mechanisms that can result in a short
Helicases. Helicases are enzymes that catalyze the unwinding of effective lifetime in the clinic. Furthermore, since current screens
DNA · DNA, RNA · RNA, and RNA · DNA hybrid molecules into have tended to focus on discovery of compounds isolated from
their component single strands using the energy produced by the soil samples, which are dominated by Gram-positive organisms,
hydrolysis of a nucleoside 5=-triphosphate (218). Helicases are re- few new compounds that target the Gram-negative organisms that
quired for all aspects of nucleic acid metabolism/genome mainte- are starting to dominate antibiotic-resistant infections have been
nance in both prokaryotic and eukaryotic cells, including transcrip- identified. This is exacerbated by the fact that many high-through-
tion, RNA splicing, and translation initiation, as well as DNA put screens use strains of Gram-negative bacteria carrying tolC
replication, repair, and recombination (219). Although the role of deletions (TolC is a component of Gram-negative efflux systems)
helicases in eukaryotic transcription has been studied for many years or mutations that result in defective outer membranes, thus allow-
(reviewed in reference 219), other than Rho, transcription in bacteria ing easier access of compounds to the cytoplasm. Screening natu-
is not a process traditionally thought to require the activity of heli- ral product libraries from other environments may well provide
cases to unwind or rewind DNA/RNA or DNA-RNA hybrids. In E. leads effective against both Gram-positive and -negative bacteria.
coli, UvrD is a superfamily 1 (SF 1) helicase involved primarily in Compounds that are rationally designed, screened, and iden-
nucleotide excision repair, a process whereby various DNA lesions tified based on the production of a pharmacophore have the ad-
are located and removed (220, 221). More recently, it has been vantage that the target site is known, enabling the rapid develop-
shown in E. coli that UvrD is able to bind directly to transcribing ment of compounds with high specificity. While such inhibitors
RNAP to a level comparable to that for common transcription often have excellent properties when used in vitro, they may re-
factors (such as NusA [191]). This is likely to be a highly conserved quire substantial modification in order to be developed into useful
interaction, as the essential UvrD ortholog PcrA from Bacillus clinical compounds; the medicinal chemist is required to perform
in months/years the tasks that evolution has carried out on natural protein synthesis. Nature 207:417– 418. http://dx.doi.org/10.1038
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ACKNOWLEDGMENTS 21. Murakami KS, Darst SA. 2003. Bacterial RNA polymerases: the wholo
We thank Richard Ebright and Yu Feng for supply of PDB files of RNAP in story. Curr Opin Struct Biol 13:31–39. http://dx.doi.org/10.1016/S0959
complex with CBR compounds prior to their public release. We also -440X(02)00005-2.
thank other members of the Lewis laboratory for helpful comments dur- 22. Seshasayee AS, Sivaraman K, Luscombe NM. 2011. An overview of
prokaryotic transcription factors: a summary of function and occurrence
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We declare that we have no conflict of interest with any institutions or .1007/978-90-481-9069-0_2.
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The funders had no role in determining the content of the paper or in coli RNA polymerase facilitates investigation of closed complexes with
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Cong Ma was awarded a Ph.D. in synthetic Peter J. Lewis was awarded a Ph.D. in biochem-
chemistry of natural products by the Ecole istry by the University of Sydney in 1991. He
Polytechnique, France, in 2009 and is a regis- then moved to the University of Oxford as an
tered pharmacist. After obtaining his Ph.D., SERC/NATO postdoctoral fellow to work on
he worked with Gregory C. Fu at the Massa- the control of differential gene expression and
chusetts Institute of Technology on catalytic chromosome segregation with Jeff Errington.
chemistry. In 2011 Dr. Ma moved to the Lewis In 2000 he took up an academic position at the
laboratory at the University of Newcastle, es- University of Newcastle, where his research fo-
tablishing a range of skills in molecular biol- cuses on how transcription factors modulate
ogy, biochemistry, and microbiology. His re- the activity of RNA polymerase and whether the
search focus is on combining his chemical essential protein-protein interactions between
and biological skills for the rational design of compounds that inhibit the these factors and RNA polymerase can be exploited in the development of
interaction of transcription factors with RNA polymerase. new antibiotic leads.
Xiao Yang undertook her undergraduate and
postgraduate studies at the University of New-
castle and was awarded a Ph.D. in molecular
microbiology in 2010. She was subsequently
awarded an Australian Research Council Aus-
tralian Postdoctoral Fellowship to continue her
work on structure-function studies of tran-
scription elongation factors in complex with
RNA polymerase in collaboration with Peter
Lewis.
Promoter (Genetics)
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