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Proc Natl Acad Sci U S A. 2016 May 24;113(21):E2899-905. doi: 10.1073/pnas.1603271113. Epub 2016 May 9.

Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection.

Author information

1
Department of Genetics, Rutgers University, Piscataway, NJ 08854; Waksman Institute, Rutgers University, Piscataway, NJ 08854;
2
Waksman Institute, Rutgers University, Piscataway, NJ 08854; Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854;
3
Department of Genetics, Rutgers University, Piscataway, NJ 08854; Department of Biomedical and Health Informatics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104;
4
Department of Genetics, Rutgers University, Piscataway, NJ 08854; Department of Biomedical and Health Informatics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; Department of Obstetrics, Gynecology and Reproductive Sciences, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08901.
5
Waksman Institute, Rutgers University, Piscataway, NJ 08854; Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854; ebright@waksman.rutgers.edu bnickels@waksman.rutgers.edu.
6
Department of Genetics, Rutgers University, Piscataway, NJ 08854; Waksman Institute, Rutgers University, Piscataway, NJ 08854; ebright@waksman.rutgers.edu bnickels@waksman.rutgers.edu.

Abstract

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein-DNA interactions with the downstream part of the nontemplate strand of the transcription bubble ("core recognition element," CRE). Here, we investigated whether sequence-specific RNAP-CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP-CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP-CRE interactions on TSS selection in vitro and in vivo for a library of 4(7) (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP-CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5' merodiploid native-elongating-transcript sequencing, 5' mNET-seq, we assessed effects of RNAP-CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP-CRE interactions determine TSS selection. Our findings establish RNAP-CRE interactions are a functional determinant of TSS selection. We propose that RNAP-CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

KEYWORDS:

RNA polymerase; promoter; transcription bubble; transcription initiation; transcription start site selection

PMID:
27162333
PMCID:
PMC4889395
DOI:
10.1073/pnas.1603271113
[Indexed for MEDLINE]
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