–35 Sequence: A consensus sequence (5′-TTGACA-3′), centered about 35 bases to the left of the transcription start site (see Fig. 31.7), is the initial point of contact for the holoenzyme, and a closed complex is formed. [Note: By convention, the regulatory sequences that control transcription are designated by the 5′→3′ nucleotide sequence on the coding strand. A base in the promoter region is assigned a negative number if it occurs prior to (to the left of, toward the 5′-end of, or “upstream” of) the transcription start site. Therefore, the TTGACA sequence is centered at approximately base −35. The first base at the transcription start site is assigned a position of +1. There is no base designated “0”.] b.
Pribnow box: The holoenzyme moves and covers a second consensus sequence (5′-TATAAT-3′), centered at about −10 (see Fig. 31.7), which is the site of melting (unwinding) of a short stretch (~14 base pairs) of DNA. This initial melting converts the closed initiation complex to an open complex known as a transcription bubble. [Note: A mutation in either the −10 or the −35 sequence can affect the transcription of the gene controlled by the mutant promoter.] 2.
Elongation: Once the promoter has been recognized and bound by the holoenzyme, local unwinding of the DNA helix continues (Fig. 31.8), mediated by the polymerase. [Note: Unwinding generates supercoils in the DNA that can be relieved by DNA topoisomerases (see p. 417).] RNA pol begins to synthesize a transcript of the DNA sequence, and several short pieces of RNA are made and discarded. The elongation phase begins when the transcript (typically starting with a purine) exceeds 10 nucleotides in length. Sigma is then released, and the core enzyme is able to leave (clear) the promoter and move along the template strand in a processive manner, serving as its own sliding clamp. During transcription, a short DNA–RNA hybrid helix is formed (see Fig. 31.8). Like DNA pol, RNA pol uses nucleoside triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5′→3′ direction. In contrast to DNA pol, RNA pol does not require a primer and does not have a 3′→5′ exonuclease domain for proofreading. [Note: Misincorporation of a ribonucleotide causes RNA pol to pause, backtrack, cleave the transcript, and restart. Nonetheless, transcription has a higher error rate than does replication.] 3.
Termination: The elongation of the single-stranded RNA chain continues until a termination signal is reached. Termination can be intrinsic (occur without additional proteins) or dependent upon the participation of a protein known as the ρ (rho) factor.
a. ρ-Independent: Seen with most prokaryotic genes, this requires that a sequence in the DNA template generates a sequence in the nascent (newly made) RNA that is self-complementary (Fig. 31.9). This allows the RNA to fold back on itself, forming a GC-rich stem (stabilized by hydrogen bonds) plus a loop. This structure is known as a “hairpin.” Additionally, just beyond the hairpin, the RNA transcript contains a string of Us at the 3′-end. The bonding of these Us to the complementary As of the DNA template is weak. This facilitates the separation of the newly synthesized RNA from its DNA template, as the double helix “zips up” behind the RNA pol.