Mechanism of sequence-specific pausing of bacterial RNA polymerase.

Sequence-specific pausing of multisubunit RNA polymerases (RNAPs) represents a rate-limiting step during transcription elongation. Pausing occurs on average every 100 bases of DNA. Several models have been proposed to explain pausing, including backtracking of the ternary elongation complex, delay of translocation of the enzyme along DNA, or a conformational change in the active site preventing formation of the phosphodiester bond. Here, we performed biochemical characterization of previously-reported pauses of Escherichia coli RNAP and found that they are not associated with backtracking or a translocation delay. Instead, the paused complex contains the 3' end of the transcript in the active center and is capable of binding the next cognate NTP. However, bond formation occurs much slower in the paused complex compared with its fully-active counterpart. The pausing is dramatically decreased by a substitution of the base encoding the next incoming NTP and the base encoding the 3' end of the nascent RNA, suggesting that (mis)-alignment of the 3' end of the RNA and the incoming NTP in the active site is crucial for pausing. These pause sites are conserved between E. coli and Thermus thermophilus RNAPs, but are not recognized by Saccharomyces cerevisiae RNAP II, indicating that prokaryotic RNAPs might be more sensitive to the changes in the alignment of the nascent transcript and the substrate NTP in the active site.