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Synergistic action of RNA polymerases in overcoming the nucleosomal barrier

Nature Structural & Molecular Biology volume 17, pages 745–752 (2010)Cite this article

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Abstract

During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet must access the nucleosomal DNA. Previous in vivo evidence has suggested that multiple RNAPs might increase transcription efficiency through nucleosomes. Here we have quantitatively investigated this hypothesis using Escherichia coli RNAP as a model system by directly monitoring its location on the DNA via a single-molecule DNA-unzipping technique. When an RNAP encountered a nucleosome, it paused with a distinctive 10–base pair periodicity and backtracked by ∼10–15 base pairs. When two RNAPs elongate in close proximity, the trailing RNAP apparently assists in the leading RNAP's elongation, reducing its backtracking and enhancing its transcription through a nucleosome by a factor of 5. Taken together, our data indicate that histone-DNA interactions dictate RNAP pausing behavior, and alleviation of nucleosome-induced backtracking by multiple polymerases may prove to be a mechanism for overcoming the nucleosomal barrier in vivo.

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Figure 1: Locating an RNAP during elongation on nucleosomal DNA.
Figure 2: Transcription through a nucleosome shows a distinctive 10-bp periodicity pausing pattern.
Figure 3: Histone-DNA interactions induce RNAP backtracking, and prevention of backtracking facilitates transcription.
Figure 4: The trailing RNAP assists the leading RNAP to exit an arrested state.
Figure 5: Two RNAPs work synergistically to overcome a nucleosomal barrier.
Figure 6: Transcription efficiency comparison and cartoon illustrating the mechanism of transcription through nucleosomal DNA.

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Acknowledgements

We thank members of the Wang laboratory for critical reading of the manuscript, J. Widom (Northwestern University) for the plasmid containing the 601 NPE and the J. Roberts laboratory (Cornell University) for help with the phosphorescence gel scanner. M.D.W. wishes to acknowledge support from the US National Institutes of Health (GM059849), the US National Science Foundation (MCB-0820293), the Keck Foundation Distinguished Young Scholar in Medical Research Award and the Cornell Nanobiotechnology Center.

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  1. Lu Bai & Daniel S Johnson

    Present address: Present address: The Rockefeller University, New York, New York, USA.,

Authors and Affiliations

  1. Department of Physics, Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York, USA

    Jing Jin, Lu Bai, Daniel S Johnson, Robert M Fulbright & Michelle D Wang

  2. Howard Hughes Medical Institute, Cornell University, Ithaca, New York, USA

    Jing Jin & Michelle D Wang

  3. NCI Center for Cancer Research, Frederick, Maryland, USA

    Maria L Kireeva & Mikhail Kashlev

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Contributions

J.J. designed and constructed transcription templates, designed and performed bulk and single-molecule transcription experiments, analyzed the data, participated in all discussions and proposal of the model and also wrote the manuscript; L.B., D.S.J. and M.L.K. offered suggestions and technical advice and helped troubleshoot the experiments; R.M.F. purified E. coli RNAP and histones and revised the manuscript; M.K. helped with the design of the project, offered advice on biochemical studies and contributed significantly to the manuscript revision; M.D.W. supervised the study throughout all stages of the project and wrote the manuscript.

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Correspondence to Michelle D Wang.

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Jin, J., Bai, L., Johnson, D. et al. Synergistic action of RNA polymerases in overcoming the nucleosomal barrier. Nat Struct Mol Biol 17, 745–752 (2010). https://doi.org/10.1038/nsmb.1798

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