We report the development of a next-generation sequencing-basedtechnologythat entailsconstruction of a DNA library comprising up to at least 47 (16,000) barcoded sequences, production of RNA transcripts,and analysis of transcript ends and transcript yields (massively systematic transcript end readout,''MASTER''). UsingMASTER,wedefine full inventories of transcription start sites (''TSSomes'') of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo;we define the TSS-region DNA sequence determinants for TSS selection, reiterative initiation (''slippage synthesis''), and transcript yield; and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion('' scrunching'')andtranscription-ubblecontraction (''anti-scrunching'').
Massively Systematic Transcript End Readout,''MASTER'': Transcription Start Site Selection,Transcriptional Slippage, and Transcript Yields
Anna Valenti;
2015
Abstract
We report the development of a next-generation sequencing-basedtechnologythat entailsconstruction of a DNA library comprising up to at least 47 (16,000) barcoded sequences, production of RNA transcripts,and analysis of transcript ends and transcript yields (massively systematic transcript end readout,''MASTER''). UsingMASTER,wedefine full inventories of transcription start sites (''TSSomes'') of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo;we define the TSS-region DNA sequence determinants for TSS selection, reiterative initiation (''slippage synthesis''), and transcript yield; and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion('' scrunching'')andtranscription-ubblecontraction (''anti-scrunching'').I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
