Dataset Information

RNA polymerase trafficking in Bacillus subtilis cells.

ABSTRACT: To obtain insight into the in vivo dynamics of RNA polymerase (RNAP) on the Bacillus subtilis genome, we analyzed the distribution of the σ(A) and β' subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells using chromatin affinity precipitation coupled with gene chip mapping (ChAP-chip). In contrast to Escherichia coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RΝΑP is evenly distributed from the promoter to the coding sequences. This finding suggests that, in general, B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex and proceeds without intragenic transcription attenuation. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which can be identified as transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior between E. coli and B. subtilis during initiation and elongation steps might result in distinct strategies for postinitiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis.

SUBMITTER: Ishikawa S

PROVIDER: S-EPMC2953687 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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RNA polymerase trafficking in Bacillus subtilis cells.

Ishikawa Shu S Oshima Taku T Kurokawa Ken K Kusuya Yoko Y Ogasawara Naotake N

Journal of bacteriology 20100903 21

To obtain insight into the in vivo dynamics of RNA polymerase (RNAP) on the Bacillus subtilis genome, we analyzed the distribution of the σ(A) and β' subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells using chromatin affinity precipitation coupled with gene chip mapping (ChAP-chip). In contrast to Escherichia coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RΝΑP is evenly distributed from the promoter to the coding sequence ...[more]

PMID: 20817769

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Project description:Transcription elongation is a highly processive process that is punctuated by RNA polymerase (RNAP) pausing. Long-lived pauses can provide time for diverse regulatory events to occur, which play important roles in modulating gene expression. Transcription elongation factors can dramatically affect RNAP pausing in vitro. The genome-wide role of such factors in pausing in vivo has been examined only for NusG in Bacillus subtilis. NusA is another transcription elongation factor known to stimulate pausing of B. subtilis and Escherichia coli RNAP in vitro. Here, we present the first in vivo study to identify the genome-wide role of NusA in RNAP pausing. Using native elongation transcript sequencing followed by RNase digestion (RNET-seq), we analyzed factor-dependent RNAP pausing in B. subtilis and found that NusA has a relatively minor role in RNAP pausing compared to NusG. We demonstrate that NusA has both stimulating and suppressing effects on pausing in vivo. Based on our thresholding criteria on in vivo data, NusA stimulates pausing at 129 pause peaks in 93 different genes or 5' untranslated regions (5' UTRs). Putative pause hairpins were identified for 87 (67%) of the 129 NusA-stimulated pause peaks, suggesting that RNA hairpins are a common component of NusA-stimulated pause signals. However, a consensus sequence was not identified as a NusA-stimulated pause motif. We further demonstrate that NusA stimulates pausing in vitro at some of the pause sites identified in vivo. IMPORTANCE NusA is an essential transcription elongation factor that was assumed to play a major role in RNAP pausing. NusA stimulates pausing in vitro; however, the essential nature of NusA had prevented an assessment of its role in pausing in vivo. Using a NusA depletion strain and RNET-seq, we identified a similar number of NusA-stimulated and NusA-suppressed pause peaks throughout the genome. NusA-stimulated pausing was confirmed at several sites in vitro. However, NusA did not always stimulate pausing at sites identified in vivo, while in other instances NusA stimulated pausing at sites not observed in vivo. We found that NusA has only a minor role in stimulating RNAP pausing in B. subtilis.

Project description:Here we investigated the refolding of Bacillus subtilis 6S-1 RNA and its release from σA-RNA polymerase (σA-RNAP) in vitro using truncated and mutated 6S-1 RNA variants. Truncated 6S-1 RNAs, only consisting of the central bubble (CB) flanked by two short helical arms, can still traverse the mechanistic 6S RNA cycle in vitro despite ~10-fold reduced σA-RNAP affinity. This indicates that the RNA's extended helical arms including the '-35'-like region are not required for basic 6S-1 RNA functionality. The role of the 'central bubble collapse helix' (CBCH) in pRNA-induced refolding and release of 6S-1 RNA from σA-RNAP was studied by stabilizing mutations. This also revealed base identities in the 5'-part of the CB (5'-CB), upstream of the pRNA transcription start site (nt 40), that impact ground state binding of 6S-1 RNA to σA-RNAP. Stabilization of the CBCH by the C44/45 double mutation shifted the pRNA length pattern to shorter pRNAs and, combined with a weakened P2 helix, resulted in more effective release from RNAP. We conclude that formation of the CBCH supports pRNA-induced 6S-1 RNA refolding and release. Our mutational analysis also unveiled that formation of a second short hairpin in the 3'-CB is detrimental to 6S-1 RNA release. Furthermore, an LNA mimic of a pRNA as short as 6 nt, when annealed to 6S-1 RNA, retarded the RNA's gel mobility and interfered with σA-RNAP binding. This effect incrementally increased with pLNA 7- and 8-mers, suggesting that restricted conformational flexibility introduced into the 5'-CB by base pairing with pRNAs prevents 6S-1 RNA from adopting an elongated shape. Accordingly, atomic force microscopy of free 6S-1 RNA versus 6S-1:pLNA 8- and 14-mer complexes revealed that 6S-1:pRNA hybrid structures, on average, adopt a more compact structure than 6S-1 RNA alone. Overall, our findings also illustrate that the wild-type 6S-1 RNA sequence and structure ensures an optimal balance of the different functional aspects involved in the mechanistic cycle of 6S-1 RNA.

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RNA polymerase trafficking in Bacillus subtilis cells.

Publications

RNA polymerase trafficking in Bacillus subtilis cells.

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