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RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit

Nature Structural & Molecular Biology volume 13, pages 4954 (2006) | Download Citation

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Abstract

Structural studies of RNA polymerase II have suggested two possible exit paths for the nascent RNA: groove 1, which points toward the subcomplex of subunits Rpb4 and Rpb7, and groove 2, which points toward Rpb8. These alternatives could not be distinguished previously because less than 10 nucleotides (nt) of transcript were resolved in the structures. We have approached this question by UV cross-linking nascent RNA to components of the transcription complex through uridine analogs located within the first six nucleotides of the RNA. We find that the emerging transcript cross-links to the Rpb7 subunit of RNA polymerase II in various complexes containing 26- to 32-nt transcripts. This interaction is greatly reduced in complexes with 41- or 43-nt RNAs and absent when the transcript is 125 nt. Our results are consistent with groove 1 being the exit path for nascent RNA.

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Acknowledgements

We thank F. Werner for human Rpb4 and Rpb7 and the expression constructs to prepare these proteins, M. Gama-Carvalho (University of Lisbon) for the antibody to U2AF65 (MC3), R. Padgett (Cleveland Clinic Foundation) both for an initial gift of the MC3 antibody and for the U2AF-expressing baculovirus, and M. Pal for establishing the protocol for releasing transcription complexes from magnetic beads. We also thank R. Padgett and M. Pal for advice during the course of these experiments. HeLa cells for preparation of nuclear extracts were provided by the US National Cell Culture Center. This work was supported by grant GM 29487 from the US National Institutes of Health.

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  1. Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    • Andrea Újvári
    •  & Donal S Luse

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The authors declare no competing financial interests.

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Correspondence to Donal S Luse.

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DOI

https://doi.org/10.1038/nsmb1026

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