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    huang Steffi
    Structural studies of RNA polymerase II backtracking were discussed. RNA polymerase II can pause or arrest during elongation by backtracking, where the 3' end of the RNA disengages from the active site. This inhibits further elongation. Backtracking is stimulated by DNA sequences or protein obstacles and can result in the RNA polymerase binding to and being locked by the backtracked transcript. Crystallization of a backtracked polymerase complex revealed density for 9 nucleotides of backtracked RNA. Interactions between the polymerase and backtracked RNA help lock it in the backtracked conformation. Reactivation requires forward translocation or RNA cleavage to restore elongation.

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    Workshop Presentation

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    huang Steffi
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    Structural studies of RNA polymerase II backtracking were discussed. RNA polymerase II can pause or arrest during elongation by backtracking, where the 3' end of the RNA disengages from the active site. This inhibits further elongation. Backtracking is stimulated by DNA sequences or protein obstacles and can result in the RNA polymerase binding to and being locked by the backtracked transcript. Crystallization of a backtracked polymerase complex revealed density for 9 nucleotides of backtracked RNA. Interactions between the polymerase and backtracked RNA help lock it in the backtracked conformation. Reactivation requires forward translocation or RNA cleavage to restore elongation.

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    Structural studies of RNA polymerase II backtracking were discussed. RNA polymerase II can pause or arrest during elongation by backtracking, where the 3' end of the RNA disengages from the active site. This inhibits further elongation. Backtracking is stimulated by DNA sequences or protein obstacles and can result in the RNA polymerase binding to and being locked by the backtracked transcript. Crystallization of a backtracked polymerase complex revealed density for 9 nucleotides of backtracked RNA. Interactions between the polymerase and backtracked RNA help lock it in the backtracked conformation. Reactivation requires forward translocation or RNA cleavage to restore elongation.

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    61 views24 pages

    Workshop Presentation

    Uploaded by

    huang Steffi
    Structural studies of RNA polymerase II backtracking were discussed. RNA polymerase II can pause or arrest during elongation by backtracking, where the 3' end of the RNA disengages from the active site. This inhibits further elongation. Backtracking is stimulated by DNA sequences or protein obstacles and can result in the RNA polymerase binding to and being locked by the backtracked transcript. Crystallization of a backtracked polymerase complex revealed density for 9 nucleotides of backtracked RNA. Interactions between the polymerase and backtracked RNA help lock it in the backtracked conformation. Reactivation requires forward translocation or RNA cleavage to restore elongation.

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    Structural Studies of RNA

    Polymerase II Backtracking

    UCL BIOC 0007 Workshop


    24th October 2019
    Eukaryotic RNA Polymerases

    Pol I rRNA

    RNAP
    Pol II mRNA Ribosome
    Protein
    DNA RNA

    Pol III tRNA

    mtRNAP
    DNA RNA Protein
    Initiation

    Termination Elongation
    Initiation Template
    Promoter Promoter Initial RNA
    complex strand
    recognition opening catalysis
    assembly positioning

    RNA
    Elongation Processive
    separation
    factor binding elongation
    from template

    Backtracking Cleavage

    RNAP –
    "Poly-A Tail
    RNA cleavage template
    formation"
    dissociation
    Rpb1 Rpb2 Rpb3 Rpb4 Rpb5 Rpb6
    Rpb7 Rpb8 Rpb9 Rpb10 Rpb11 Rpb12

    RNA Template DNA Non-template DNA


    “Off-cycle processes”
    l Priming
    NTP l Mismatch recognition and
    proofreading
    Post
    Translocation UV Damaged DNA
    translocated l DNA Damage recognition

    Pre Substrate l Translesion synthesis


    translocated bound
    l Transcript slippage

    PPi l Template misalignment


    RNA-DNA Product Catalysis
    mismatch bound l Abortive Initiation
    l Pausing/Backtracking
    l Cleavage
    RNA Polymerase II
    Pausing and Backtracking
    Wang (1995) Cell Galburt (2007) Nature

    Paused vs. arrested RNAP


    • Paused RNAPs eventually recover and continue transcribing
    • Arrested RNAPs cannot continue elongation
    • Arrested RNAPs have backtracked ~ 10nt.
    • Backtracking is stimulated by
    DNA sequence, or by protein
    obstacles on the DNA.

    • 3’OH disengages from active


    site, inhibiting elongation

    • Requires fwd translocation or


    RNA cleavage to restore
    elongation

    Svestrup (2007) TIBS


    The importance of backtracking

    Nudler (2012) Cell


    Crystallising an backtracked Pol II complex

    • Initiation factor
    independent transcription + CTP
    with CTP generated a + Pol II
    Polycytosine RNA
    – Electron density for 9nt of
    backtracked RNA observed
    Backtracked transcript
    binding locks translocation

    Literature :
    - Tailed template transcription arrests after synthesis of 13-17nt
    - Arrested complexes release 7-14nt RNA upon TFIIS treatment
    - Arrested complexes protect an extra 9nt of RNA compared to the elongation complex
    Pol II - backtracked RNA interactions

    1. Conserved, 2. mostly non-base specific 3. TL interactions, 4. Gating Tyr, 5. Pocket, 6. Py preference


    Catalytic residues
    D290, E291

    Pol II binding domain II

    Catalytic domain III


    Linker helix

    DE to AA lethal
    A Reactivation Complex : Backtracked Pol II-TFIIS
    Acknowledgements
    UCL and Birkbeck College Rowan University, NJ, USA
    Luis Miguel Diaz Santin Dmitry Temiakov
    Emir Aciyan
    NCI, Frederick, MD, USA
    Andrew Wu Mikhail Kashlev
    Lisa Watson Celine Walmacq
    Daniel Saunders
    Andy McGee LMU Munich/MPI Goettingen

    George Cameron Patrick Cramer


    Emma Kennedy Christoph Engel
    Sarah Sainsbury

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