Massively Systematic Transcript End Readout, "MASTER": Transcription Start Site Selection, Transcriptional Slippage, and Transcript Yields.
Mol Cell
; 60(6): 953-65, 2015 Dec 17.
Article
em En
| MEDLINE
| ID: mdl-26626484
ABSTRACT
We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 4(7) (â¼ 16,000) barcoded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields (massively systematic transcript end readout, "MASTER"). Using MASTER, we define 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") and transcription-bubble contraction ("anti-scrunching").
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sítio de Iniciação de Transcrição
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Escherichia coli
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Sequenciamento de Nucleotídeos em Larga Escala
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article