NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I.

Abstract:

: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.

authors

Clarke AM,Engel KL,Giles KE,Petit CM,Schneider DA

doi

10.1073/pnas.1809421115

subject

Has Abstract

pub_date

2018-12-11 00:00:00

pages

E11633-E11641

issue

50

eissn

0027-8424

issn

1091-6490

pii

1809421115

journal_volume

115

pub_type

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