DNA template sequence control of bacterial RNA polymerase escape from the promoter

Ewa Heyduk; Tomasz Heyduk

doi:10.1093/nar/gky172

Influence of DNA Template Choice on Transcription and Inhibition of Escherichia coli RNA Polymerase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00198-12 ◽

2012 ◽

Vol 56 (8) ◽

pp. 4536-4539 ◽

Cited By ~ 9

Author(s):

Joerg Haupenthal ◽

Kristina Hüsecken ◽

Matthias Negri ◽

Christine K. Maurer ◽

Rolf W. Hartmann

Keyword(s):

Escherichia Coli ◽

Drug Discovery ◽

Rna Polymerase ◽

Sigma Factor ◽

Inhibitory Potency ◽

Experimental Conditions ◽

Early Stages ◽

Bacterial Rna ◽

Transcription Efficiency ◽

Dna Template

ABSTRACTIn recent decades, quantitative transcription assays using bacterial RNA polymerase (RNAP) have been performed under widely diverse experimental conditions. We demonstrate that the template choice can influence the inhibitory potency of RNAP inhibitors. Furthermore, we illustrate that the sigma factor (σ70) surprisingly increases the transcription efficiency of templates with nonphysiological nonprokaryotic promoters. Our results might be a useful guideline in the early stages of using RNAP for drug discovery.

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NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809421115 ◽

2018 ◽

Vol 115 (50) ◽

pp. E11633-E11641 ◽

Cited By ~ 7

Author(s):

Andrew M. Clarke ◽

Krysta L. Engel ◽

Keith E. Giles ◽

Chad M. Petit ◽

David A. Schneider

Keyword(s):

Rna Polymerase ◽

Transcription Elongation ◽

Sequence Motifs ◽

Template Sequence ◽

Pol I ◽

Nucleotide Incorporation ◽

Polymerase I ◽

Dna Sequence Motifs ◽

Dna Template

DNA sequence motifs that affect RNA polymerase transcription elongation are well studied in prokaryotic organisms and contribute directly to regulation of gene expression. Despite significant work on the regulation of eukaryotic transcription, the effect of DNA template sequence on RNA polymerase I (Pol I) transcription elongation remains unknown. In this study, we examined the effects of DNA sequence motifs on Pol I transcription elongation kinetics in vitro and in vivo. Specifically, we characterized how the spy rho-independent terminator motif from Escherichia coli directly affects Saccharomyces cerevisiae Pol I activity, demonstrating evolutionary conservation of sequence-specific effects on transcription. The insight gained from this analysis led to the identification of a homologous sequence in the ribosomal DNA of S. cerevisiae. We then used native elongating transcript sequencing (NETSeq) to determine whether Pol I encounters pause-inducing sequences in vivo. We found hundreds of positions within the ribosomal DNA (rDNA) that reproducibly induce pausing in vivo. We also observed significantly lower Pol I occupancy at G residues in the rDNA, independent of other sequence context, indicating differential nucleotide incorporation rates for Pol I in vivo. These data demonstrate that DNA template sequence elements directly influence Pol I transcription elongation. Furthermore, we have developed the necessary experimental and analytical methods to investigate these perturbations in living cells going forward.

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