Article

Template misalignment in multisubunit RNA polymerases and transcription fidelity.

Department of Cell Biology, School of Osteopathic Medicine, University of Medicine and Dentistry of New Jersey, 42 East Laurel Road, Stratford, New Jersey 08084, USA.
Molecular Cell (Impact Factor: 14.46). 11/2006; 24(2):257-66. DOI: 10.1016/j.molcel.2006.10.001
Source: PubMed

ABSTRACT Recent work showed that the single-subunit T7 RNA polymerase (RNAP) can generate misincorporation errors by a mechanism that involves misalignment of the DNA template strand. Here, we show that the same mechanism can produce errors during transcription by the multisubunit yeast RNAP II and bacterial RNAPs. Fluorescence spectroscopy reveals a reorganization of the template strand during this process, and molecular modeling suggests an open space above the polymerase active site that could accommodate a misaligned base. Substrate competition assays indicate that template misalignment, not misincorporation, is the preferred mechanism for substitution errors by cellular RNAPs. Misalignment could account for data previously taken as evidence for additional NTP binding sites downstream of the active site. Analysis of the effects of different template topologies on misincorporation indicates that the duplex DNA immediately downstream of the active site plays an important role in transcription fidelity.

0 Bookmarks
 · 
77 Views
  • Source
  • [Show abstract] [Hide abstract]
    ABSTRACT: Synthetic nucleic acid analogues have profoundly advanced our knowledge of DNA and RNA, as well as the complex biological processes that involve nucleic acids. As a pivotal enzyme, eukaryotic RNA polymerase II (Pol II) is responsible for transcribing DNA into messenger RNA, which serves as a template to direct protein synthesis. Chemically modified nucleic acid analogues have greatly facilitated the structural elucidation of RNA Pol II elongation complex and understanding the key chemical interactions governing RNA Pol II transcriptional fidelity. This review addresses major progress in RNA polymerase II mechanistic studies using modified nucleic acid analogues in recent years.
    Israel Journal of Chemistry 06/2013; 53(6-7). · 2.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We developed a highly sensitive assay to detect transcription errors in vivo. The assay is based on suppression of a missense mutation in the active site tyrosine in the Cre recombinase. Because Cre acts as tetramer, background from translation errors are negligible. Functional Cre resulting from rare transcription errors that restore the tyrosine codon can be detected by Cre-dependent rearrangement of reporter genes. Hence, transient transcription errors are captured as stable genetic changes. We used this Cre-based reporter to screen for mutations of Saccharomyces cerevisiae RPB1 (RPO21) that increase the level of misincorporation during transcription. The mutations are in three domains of Rpb1, the trigger loop, the bridge helix, and in sites involved in binding to TFIIS. Biochemical characterization demonstrates that these variants have elevated misincorporation, and/or ability to extend mispaired bases, or defects in TFIIS mediated editing.
    PLoS Genetics 09/2014; 10(9):e1004532. · 8.17 Impact Factor

Full-text (2 Sources)

Download
41 Downloads
Available from
Jun 2, 2014