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Structural and mechanistic basis of RNA extension in reiterative transcription initiation: RNA slipping without DNA scrunching

Yu Liu, Jared T. Winkelman, Libing Yu, Chirangini Pukhrambam, Yu Zhang, View ORCID ProfileBryce E. Nickels, View ORCID ProfileRichard H. Ebright
doi: https://doi.org/10.1101/2021.05.08.443277
Yu Liu
1Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA
2Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854, USA
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Jared T. Winkelman
1Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA
2Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854, USA
3Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA
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Libing Yu
1Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA
2Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854, USA
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Chirangini Pukhrambam
2Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854, USA
3Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA
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Yu Zhang
4Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China, 200032
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Bryce E. Nickels
2Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854, USA
3Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA
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  • ORCID record for Bryce E. Nickels
  • For correspondence: ebright@waksman.rutgers.edu
Richard H. Ebright
1Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA
2Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854, USA
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  • ORCID record for Richard H. Ebright
  • For correspondence: ebright@waksman.rutgers.edu
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Abstract

In standard transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA, selects a transcription start site, and--using a “scrunching” mechanism, in which RNAP remains bound to the promoter, unwinds additional DNA, and pulls the additional unwound DNA past its active center, synthesizing an RNA product having a 5’ sequence complementary to the DNA template. In an alternative pathway of transcription initiation, termed “reiterative transcription initiation,” primarily observed at promoters containing homopolymeric sequences at or near the transcription start site, RNAP binds to promoter DNA, unwinds promoter DNA, selects a transcription start site, and--using a mechanism that has not previously been defined--generates an RNA product having a 5’ sequence that contains a variable number of nucleotides not complementary to the DNA template. Here, using x-ray crystallography to define structures, using protein-DNA-photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and using single-molecule DNA nanomanipulation to assess RNAP-dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation (1) occurs without DNA scrunching, (2) involves a short, 2 bp (post-translocated state) to 3 bp (pre-translocated state) RNA-DNA hybrid, (3) and can involve an RNA product positioned as in standard transcription initiation and a DNA template strand positioned differently from standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with sliding of RNA relative to DNA within a short, 2-3 bp, RNA-DNA hybrid.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵* equal contribution

  • Impact statement: X-ray crystallography, protein-DNA-photocrosslinking, and single-molecule DNA nanomanipulation show that reiterative transcription initiation proceeds through RNA slipping, with sliding of RNA relative to DNA within a short, 2-3 bp, RNA-DNA hybrid.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted May 09, 2021.
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Structural and mechanistic basis of RNA extension in reiterative transcription initiation: RNA slipping without DNA scrunching
Yu Liu, Jared T. Winkelman, Libing Yu, Chirangini Pukhrambam, Yu Zhang, Bryce E. Nickels, Richard H. Ebright
bioRxiv 2021.05.08.443277; doi: https://doi.org/10.1101/2021.05.08.443277
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Structural and mechanistic basis of RNA extension in reiterative transcription initiation: RNA slipping without DNA scrunching
Yu Liu, Jared T. Winkelman, Libing Yu, Chirangini Pukhrambam, Yu Zhang, Bryce E. Nickels, Richard H. Ebright
bioRxiv 2021.05.08.443277; doi: https://doi.org/10.1101/2021.05.08.443277

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