Genome-wide assessments of transcriptional pauses and errors by nascent transcript sequencing combined with RNase footprinting (RNET-seq)

ABSTRACT: We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with

ORGANISM(S): Escherichia coli

SUBMITTER: Masahiko Imashimizu 

PROVIDER: E-GEOD-62102 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress