Template misalignment in multisubunit RNA polymerases and transcription fidelity.
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.
Kashkina
Ekaterina
E
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.
Anikin
Michael
M
Brueckner
Florian
F
Pomerantz
Richard T
RT
McAllister
William T
WT
Cramer
Patrick
P
Temiakov
Dmitry
D
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013824:Thermus / Q000201:enzymology; D013997:Time Factors; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry*
D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases