Template Misalignment in Multisubunit RNA Polymerases and Transcription Fidelity

Department of Microbiology and Immunology, State University of New York Downstate Medical Center, Brooklyn, New York, United States
Molecular Cell (Impact Factor: 14.02). 11/2006; 24(2):257-66. DOI: 10.1016/j.molcel.2006.10.001
Source: PubMed


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.

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Available from: Florian Brückner
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    • "Fidelity analysis for the entire transcriptome has been limited by a lack of a reliable methodology. In the past decade, extensive in vitro analyses of transcription fidelity revealed several error-avoidance and error-correcting mechanisms based on biochemical assays for misincorporation of a unique NMP (12,13,15–20) and single-molecule assays using optical trapping techniques (11,21). Typically, these experiments included limited or unbalanced substrate concentrations to detect misincorporation. "
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    • "The Syn TEC18 showed higher fidelity than the E. coli TEC irrespective of the coexisting divalent cations, also for misincorporating UMP and GMP (Figure 5(c)). Because this misincorporation may be interpreted as the misincorporation at +19 with the substrate at +20, this misincorporation could be due to the misalignment mechanism [37] [38]. "
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    • "The scaffolds T, G, C, and A differed in the +1 nucleotide opposite the NTP site (Figure 1A). The +1 and +2 nucleotides were identical to prevent misincorporation by template misalignment (Kashkina et al., 2006). To compare the efficiency of all 16 incorporation events (four correct incorporations and 12 misincorporations ), the four scaffolds were assembled with Pol II into ECs that were incubated with 0.1 mM of each NTP. "
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