Sequence Insertion 3 of Escherichia Coli RNA Polymerase Regulates Transcriptional Pausing PDF Download

Author: Yu Bao (Ph.D.)
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Languages : en
Pages : 0

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Book Description
Gene expression in all life forms relies on multi-subunit RNA polymerase (RNAP). In transcription elongation stage, RNAP senses specific DNA signals and enters an offline, pausing state, which has multiple interconverting subtypes and allows versatile regulatory options. This work focuses on understanding the pausing regulatory function of sequence insertion 3 (SI3) in Escherichia coli (E. coli) RNAP. In E. coli, the catalytic trigger loop (TL) is interrupted by surface-exposed, 188-aa sequence insertion 3 (SI3). SI3 occupies different locations in paused transcription complexes, but its dynamics during pausing are undefined. We report design, optimization, and use of a Cys-triplet reporter to measure the positional bias of SI3 in different transcription complexes and to determine the effect of restricting SI3 movement on pausing. RNA hairpin-stabilized pausing biases SI3 toward a swiveled position that inhibits TL folding while the consensus elemental pausing biases SI3 toward a closed position that potentially stabilized a pre-translocated state of RNAP. Our findings establish that SI3 functions by modulating the TL folding to aid transcriptional regulation. To investigate SI3's genomic function in transcriptional pausing, we constructed a viable E. coli strain lacking SI3 enabled by a suppressor TL substitution ([beta]'Ala941-Thr; [delta]SI3*). [delta]SI3* increased transcription rate in vitro relative to [delta]SI3, possibly explaining its viability, but retained both positive and negative effects of [delta]SI3 on pausing. Using NET-seq, we found that [delta]SI3*-enhanced pauses resemble the consensus elemental pause sequence whereas sequences at [delta]SI3*-suppressed pauses, which exhibited greater association with PHs, were more divergent. [delta]SI3*-suppressed pauses also were associated with apparent pausing one nt upstream from the consensus sequence, often generating tandem pause sites. Our results document multiple ways that SI3 modulates pausing in vivo and may explain differences in consensus pause sequences found in some NET-seq studies. In conclusion, our comprehensive studies provide compelling evidence linking the movements of the SI3 domain to the formation and stabilization of diverse transcriptional pause signals. We demonstrate the potential of biochemical crosslink assays in exploring conformational changes within large protein complexes. Additionally, our research emphasizes the necessity for refining current NET-seq protocols to prevent unintended cleavage during library preparation.