The expression of genes in prokaryotes is a complex process that involves several steps. One of the first steps is transcription of the gene by an RNA polymerase (RNAP). Transcription is initiated after an RNA polymerase recognizes and binds to a region of nucleotides upstream of the nucleotides that are transcribed. This region is typically referred to as a promoter. Transcription factor binding sites are often located near promoters. In contrast to promoters, transcription factor binding sites are nucleotide sequences to which transcription factors bind. A bound transcription factor then interacts with the RNA polymerase to increase the affinity of the RNA polymerase for the promoter, or to increase the rates of later steps in the mechanism of transcription.
Escherichia coli promoters recognized by the major form of RNA polymerase (RNAP E"sgr"70) typically contain at least two nucleotide sequences that the RNA polymerase recognizes. The first is a hexamer centered approximately 10 base pairs upstream of the transcription start site, and is referred to as the xe2x88x9210 sequence. The second is also a hexamer, and is referred to as the xe2x88x9235 sequence. Typically, about 16 to about 18 base pairs separate the xe2x88x9235 sequence and the xe2x88x9210 sequence in a promoter; however, as few as about 15 base pairs and as many as about 20 base pairs have been observed to separate the xe2x88x9235 sequence and the xe2x88x9210 sequence. The xe2x88x9235 sequence and the xe2x88x9210 sequence are recognized by the a subunit of RNAP.
A third recognition element, referred to in the art as the UP element, has been identified upstream of the xe2x88x9235 sequence (Ross, W. et al. (1993) Science 262, 1407-1413). The most extensively characterized UP element is an adenosine and thymidine rich sequence located between about nucleotide xe2x88x9240 and about nucleotide xe2x88x9260 in the rrnB P1 promoter (i.e., about 40 to about 60 nucleotides 5xe2x80x2 or upstream of the transcription initiation site). The UP element is believed to stimulate promoter activity by increasing the initial equilibrium constant (KB) and possibly later step(s) in the transcription initiation pathway (kf) (Ross, W. et al. (1993) Science 262, 1407-1413; Rao, L. et al. (1994) J. Mol. Biol. 235, 1421-1435).
RNAP E"sgr"70 is composed of 2 xcex1 subunits, a xcex2 subunit, a xcex2xe2x80x2 subunit, and a "sgr" subunit. Each RNAP xcex1 subunit consists of two domains, a 28 kDa N-terminal domain and an 8 kDa C-terminal domain (xcex1CTD) connected by a long unstructured and/or flexible linker (Blatter, E. E. et al. (1994) Cell 78, 889-896). The UP element is recognized by the xcex1CTD of RNAP. The seven xcex1CTD residues most crucial for DNA binding (L262, R265, N268, C269, G296, K298, S299) are highly conserved in bacteria (Gaal, T. et al. (1996) Genes Develop. 10, 16-26). Despite the importance of the xcex1CTD-DNA interaction for bacterial transcription, the details of DNA recognition by a remain to be elucidated.
UP elements have also been described in other promoters and can function with holoenzymes containing different a factors (Ross, W. et al. (1993) Science 262, 1407-1413; Newlands, J. T. et al. (1993) J. Bacteriol. 175, 661-668; Fredrick, K. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2582-2586; Giladi, H. et al. (1996) J. Mol. Biol. 260, 484-491; van Ulsen, P. et al. (1997) J. Bacteriol. 179, 530-537). It appears that UP element-like sequences occur frequently in promoters from gram positive bacteria (Helman, J. D. (1995) Nucl. Acids Res. 23, 2351-2360; Graves, M. C. et al. (1986) J. Biol. Chem. 261, 11409-11415). Consensus sequences derived previously from E. coli promoters contain highly conserved xe2x88x9210 and xe2x88x9235 hexamers; however, no highly conserved upstream sequences have been identified.
In the recent past there has been a significant increase in the prevalence of antibiotic resistant bacteria. However, the identification of new anti-bacterial compounds has not kept pace with the occurrence of these bacteria. Accordingly, there has been a significant increase in human and animal morbidity and mortality due to infectious diseases. The present invention represents a significant advance in the art of identifying compounds that inhibit the transcription of certain promoters, preferably bacterial promoters. Such compounds can be used to inhibit the expression of transcription units and are expected to inhibit the growth of bacteria
Prior to the present invention, there were not enough accessory promoter elements (of the type known to the art as UP elements) characterized to derive a consensus sequence accessory promoter element. Therefore, an in vitro selection was developed and followed by an in vivo screen to identify accessory promoter elements from a random DNA population that greatly increased promoter activity. Accessory promoter elements were identified that conferred larger increases on rrnB P1 core promoter activity than any previously identified accessory promoter elements, and a consensus accessory promoter element was derived. The same type of in vitro selection and in vivo screen was also used to identify two portions of the consensus accessory promoter element. These two portions, referred to herein as the distal subsite and the proximal subsite, each conferred increases on rrnB P1 core promoter activity. An advantage of a preferred aspect of the present invention is the increased sensitivity to compounds that inhibit transcription: the higher levels of transcription by promoters of the invention allow changes in transcription to be more easily measured.
Accordingly, the present invention provides a method for detecting whether a compound alters transcription of a transcription unit. The method includes providing in a reaction mixture a first polynucleotide that includes a promoter operably linked to a transcription unit, adding an amount of the compound to be tested to the reaction mixture under conditions effective to cause transcription, detecting an amount of a transcription product, and comparing the amount of the transcription product in the presence of the compound to an amount of the transcription product in the absence of the compound under the same conditions. The promoter includes an accessory promoter element and a core promoter element, and the reaction mixture includes at least one RNA polymerase. Optionally, the compound does not alter the amount of a transcription product from a second polynucleotide, where the second polynucleotide includes a second promoter operably linked to the same transcription unit that is operably linked to the first promoter, where the second promoter includes the same core promoter element of the first promoter and not an accessory promoter element. Typically, transcription of the transcription unit is decreased; The polynucleotide can be present on a plasmid vector.
Optionally, the reaction mixture can include an rNTP that includes a detectable label. The detectable label can be a radioactive label, a fluorescent label, or an enzymatic label, or a combination. Optionally, the transcription unit can also include a coding region that optionally encodes a detectable marker. The detectable marker can be xcex2-galactosidase, green fluorescent protein, luciferase, or chloramphenicol acetyl transferase. The reaction mixture can include a detectably labeled amino acid. The detectable label can be a radioactive label, a fluorescent label, an enzymatic label, and a combination thereof.
The accessory promoter element can be a distal accessory promoter element, a proximal accessory promoter element, or a combined accessory promoter element. The distal accessory promoter element can include the nucleotide sequence of SEQ ID NOs:4, 6-24, 27, or complements thereof. When a series of sequence identification numbers are disclosed herein, it should be understood that each sequence can be used separately. For instance, stating that an accessory promoter element can include the nucleotide sequence of SEQ ID NOs:4, 6-24, or 27 means the nucleotide sequence can be SEQ ID NO:4, or SEQ ID NO:6, or SEQ ID NO:7, etc. The proximal accessory promoter element can include the nucleotide sequence of SEQ ID NOs:3, 29-36, or complements thereof. The combined accessory promoter element can include a nucleotide sequence of SEQ ID NOs:1-2, 40-70, or complements thereof.
The present invention also provides a kit for testing the ability of a compound to alter transcription of a transcription unit. The kit includes a first polynucleotide that includes a first promoter operably linked to a transcription unit. The first promoter includes an accessory promoter element and a core promoter element. Optionally, the kit includes a second polynucleotide that includes a second promoter operably linked to the same transcription unit that is operably linked to the first promoter. The second promoter includes the same core promoter element of the first promoter and not an accessory promoter element. The polynucleotide can be present on a plasmid vector.
Optionally, the kit can include at least one rNTP that includes a detectable label. The detectable label can be a radioactive label, a fluorescent label, an enzymatic label, or a combination thereof Optionally, the transcription unit can also include a coding region that optionally encodes a detectable marker. The detectable marker can be xcex2-galactosidase, green fluorescent protein, luciferase, or chloramphenicol acetyl transferase. The kit can include a detectably labeled amino acid. The detectable label can be a radioactive label, a fluorescent label, an enzymatic label, and a combination thereof.
For the kits of the present invention, the accessory promoter element can be a distal accessory promoter element, a proximal accessory promoter element, or combined accessory promoter element. The distal accessory promoter element can include the nucleotide sequence of SEQ ID NOs:4, 6-24, 27, or complements thereof. The proximal accessory promoter element can include the nucleotide sequence of SEQ ID NOs:3, 29-36, or complements thereof. The combined accessory promoter element can include a nucleotide sequence of SEQ ID NOs: 1-2, 40-70, or complements thereof.
Promoters are used in recombinant DNA techniques to express RNA polynucleotides or polypeptides in bacterial strains. The RNA polynucleotides or polypeptides can be naturally present in a bacterial strain, or can be from a different type of cell, for instance a different bacterial cell or an animal cell. Isolated RNA polynucleotides or polypeptides are useful in, for example, the development of antibodies, vaccines, and drugs to modify the activity of an RNA polynucleotide or polypeptide, but the ability to isolate RNA polynucleotides or polypeptides is often hampered by low levels of expression in a bacterial strain. Thus, there is a continuing need for the development of methods to increase expression of RNA polynucleotides and/or polypeptides by bacterial cells. The present invention also represents an advance in the art of producing RNA polynucleotides and/or polypeptides by providing promoters that increase transcription and the resulting synthesis of RNA polynucleotides and/or polypeptides.
Prior to the present invention, transcription factor binding sites were often used to increase transcription of a promoter. The use of transcription factors to increase transcription requires not only the use of bacterial cells that encode the correct transcription factors, but growth of the cells under conditions such that the transcription factors are expressed. The promoters of the present invention obviate the need for transcription factor binding sites and transcription factors to increase transcription. Accordingly, the present invention also provides a method of producing an RNA polynucleotide. The method includes introducing to a bacterium a polynucleotide that includes a promoter operably linked to a transcription unit, incubating the bacterium under conditions that promote expression of the transcription unit such that an RNA polynucleotide encoded by the transcription unit is produced, and isolating the RNA polynucleotide. The promoter includes an accessory promoter element and a core promoter element. The RNA polynucleotide can be a structural RNA, an antisense RNA, or a catalytic RNA.
The present invention further provides a method of producing a polypeptide. The method includes introducing to a bacterium a polynucleotide that includes a promoter operably linked to a transcription unit including a coding region, incubating the bacterium under conditions that promote expression of the coding region such that a polypeptide encoded by the coding region is produced, and isolating the polypeptide. The promoter includes an accessory promoter element and a core promoter element. The polynucleotide can be present on a plasmid vector.
For the methods of the present invention, the accessory promoter element can be a distal accessory promoter element, a proximal accessory promoter element, or a combined accessory promoter element. The distal accessory promoter element can include the nucleotide sequence of SEQ ID NOs:4, 6-24, 27, or complements thereof. The proximal accessory promoter element can include the nucleotide sequence of SEQ ID NOs:3, 29-36, or complements thereof. The combined accessory promoter element can include a nucleotide sequence of SEQ ID NOs: 1-2, 40-70, or complements thereof.
The present invention further provides kits for expressing an RNA polynucleotide or a polypeptide. The kits include a polynucleotide that includes a promoter and at least one restriction endonuclease site. The promoter includes an accessory promoter element and a core promoter element, and the restriction endonuclease site is present 3xe2x80x2 of the promoter and provides an insertion site for a transcription unit encoding an RNA polynucleotide, or a coding region encoding a polypeptide.
Also provided by the present invention are polynucleotides. The polynucleotides include an accessory promoter element having a nucleotide sequence of SEQ ID NOs: 1-4, 6-24, 27, 29-36, 40-70, or complements thereof.