Figure V diagrams some useful terms: coding strand, downstream, promoter, startsite (startpoint), template strand, transcription, upstream.
Figure W (see also Table 26.1 on page 986) shows the subunit composition of RNA polymerase. RNA is synthesized in the 5' to 3' direction (the same direction as DNA is synthesized). The synthesis of RNA does not require a primer, but does require a DNA template strand. The first nucleotide of the RNA chain retains the 5'-triphosphate group, but all subsequent nucleotides that are added to the growing chain only retain the alpha phosphate in the phosphodiester linkage.
The overall reaction is: n rNTP > (rNMP)n + n PPi
The promoter has consensus sequences at the -35 and -10 regions (Figure 26.11). Nucleotide changes in the consensus sequences affect the efficiency of the promoter (Figure 26.12). The spacing between consensus sequences is also important for promoter function. Promoters that resemble the consensus most closely are highly active, wherease those promoters with sequences deviating from the consensus work less well.
Basal, UP element-dependent, and activator-dependent transcription complexes (Figure X).
Sigma
factor makes protein-DNA interactions
with promoter -35 and -10 elements. The alpha subunit carboxy-terminal
domain (alpha CTD) occupies different positions at different promoters.
It is thought that the flexible linker between the alpha CTD and
the rest of the RNA polymerase, together with differences in DNA
bending, permits the alpha CTD to engage in different interactions.
Note: Alpha NTD = the alpha subunit amino-terminal domain.
(A) Simple promoter. Alpha CTD makes no specific interactions.
(B) Promoter containing an UP element (an AT-rich sequence about 20 bp in size located immediately upstream of the -35 region; see page 970). The seven E. coli rrn genes, which encode ribosomal RNA, are unusually strong, accounting for more than 60% of total RNA synthesis in rapidly growing cells. Alpha CTD makes specific protein-DNA contacts with the UP element (either immediately upstream of the -35 element or further upstream).
(C) Promoter containing a site for an activator that interacts with alpha CTD (for example, CRP at the lac promoter). Alpha CTD makes specific protein-protein interactions (closed circle) with the activator. This recruits alpha CTD to DNA (either immediately upstream of the -35 element or further upstream), compensating for the absence of an UP element.
Conclusion: It is likely that promoter strength is a function of all three promoter elements, with very strong promoters, notably the rrn promoters, having near consensus -35, -10, and UP elements and with weaker promoters having one, two, or three non-consensus promoter elements.
Sigma factor does two things: reduces non-specific binding to DNA and increases specific binding to promoter sequences. The initial contacts of RNA polymerase holoenzyme with the promoter span the region from -55 to +20. When sigma is released (during the elongation phase of RNA synthesis), the core RNA polymerase covers about 30-40 bp of DNA.
Figure M1 (see also Figure 26.9b on page 991) shows a current view of the transcription elongation complex. RNAP is NOT an inchworm! The central notion of the discontinuous (aka inchworm) model of elongation was that the advancing RNAP undergoes conformational transitions that are not synchronous with single-step nucleotide additions; that is, the transcription elongation complex (TEC) moves non-monotonically. Mechanistically, this was envisioned as contraction and expansion of the advancing TEC. Originally, it was proposed that inchworming is intrinsic to elongation; that is, it constitutes the very mechanism of TEC advancement. However, currently available data are consistent with a simple "monotonic" model of the translocation step, in which RNAP structure does not change as the enzyme moves along the RNA and DNA chains, thus advancing the whole enzyme 1 base pair and 1 nucleotide for each polymerization event. The exceptions to monotonic advancement are incidental situations when specific sites in DNA induce irregularities in the RNAP "footprint." There is reversible loss of catalytic activity in the TEC at the inchworming sites. In such complexes, RNAP translocated backward with time after halting. The transient backtracking is an alternate explanation for inchworming. The TEC backtracks as the result of the weakness of the 3'- proximal RNA:DNA hybrid. The RNAP molecule that has backtracked appears to be in the same overall conformation as that in the productive elongation-competent complex. Moreover, the RNA:DNA hybrid in the backtracked complex appears to have shifted to an upstream region. Thus, apparent discontinuous advancement reflects a reversible side pathway rather than a succession of elongation intermediates. The irregular DNA and RNA footprints that have been observed will have to be reinterpreted as reflections of mixed populations of the TEC alternating between the productive and backtracked states. Backtracking signals are A-U rich, but the stability of the 3'-terminal heteroduplex is not the whole story. Sequences behind and ahead of the heteroduplex area affect elongation arrest. Backtracking may be resolved through internal RNA cleavage (stimulated by GreA and GreB factors) and resumption of elongation from the newly generated 3' terminus (see Figure 26.10 on page 992). It is easy to imagine that mismatched ribonucleotides that destabilize the 3' proximal hybrid would facilitate backtracking. There are also antitermination factors that prevent pausing or backtracking (but we will not consider them here).
The catalytic cycle of RNA chain elongation minimally involves (i) nucleoside triphosphate (NTP) binding, (ii) nucleophilic displacement of pyrophosphate from the NTP by the RNA 3' hydroxyl, (iii) pyrophosphate release, and (iv) translocation of the new RNA 3' nucleotide to vacate the NTP-binding site.
Overwinding in front of the transcription bubble (which puts in positive supercoils) is removed by the action of gyrase (also known as topoisomerase II, which puts in negative supercoils). Likewise, topoisomerase I (which relaxes negative supercoils) eliminates the underwinding (negative supercoils) behind the transcription bubble.
Factor-independent (also called rho-independent) terminators have the following general structure (Figure 26.15): a stem-loop (where the stem is GC-rich) and a string of U's (templated by a string of A's). Note: We will not discuss rho factor or rho-dependent terminators. Figure M2 outlines events during factor-indepentent termination of transcription. Figure M3 shows a detailed translocation model for termination of transcription.