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Mol Cell. 2015 Dec 17;60(6):953-65. doi: 10.1016/j.molcel.2015.10.029. Epub 2015 Nov 25.

Massively Systematic Transcript End Readout, "MASTER": Transcription Start Site Selection, Transcriptional Slippage, and Transcript Yields.

Author information

1
Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA; Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA.
2
Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA; Department of Biomedical and Health Informatics, The Children's Hospital of Philadelphia, Philadelphia, PA 19041, USA.
3
Institute of Biosciences and Bioresources, National Research Council of Italy, Via P. Castellino 111, Naples 80131, Italy.
4
Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA; Department of Biomedical and Health Informatics, The Children's Hospital of Philadelphia, Philadelphia, PA 19041, USA; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104, USA; Department of Obstetrics, Gynecology and Reproductive Sciences, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA.
5
Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA; Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA.
6
Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA; Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA. Electronic address: bnickels@waksman.rutgers.edu.

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) (∼ 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").

PMID:
26626484
PMCID:
PMC4688149
DOI:
10.1016/j.molcel.2015.10.029
[Indexed for MEDLINE]
Free PMC Article

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