Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation.
To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro. The mutation does not appear to significantly affect translocation state equilibrium of Pol II in a stalled elongation complex. Primarily, it promotes NTP sequestration in the polymerase active center. Furthermore, pre-steady-state analyses revealed that the E1103G mutation shifted the equilibrium between the closed and the open active center conformations toward the closed form. Thus, open conformation of the active center emerges as an intermediate essential for preincorporation fidelity control. Similar mechanisms may control fidelity of DNA-dependent DNA polymerases and RNA-dependent RNA polymerases.
NCI Center for Cancer Research, Frederick, MD 21702, USA.
Maria L
ML
Kireeva
Yuri A
YA
Nedialkov
Gina H
GH
Cremona
Yuri A
YA
Purtov
Lucyna
L
Lubkowska
Francisco
F
Malagon
Zachary F
ZF
Burton
Jeffrey N
JN
Strathern
Mikhail
M
Kashlev
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain; D015966:Gene Expression Regulation, Fungal; D007536:Isomerism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009711:Nucleotides / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D014158:Transcription, Genetic
D009711:Nucleotides; D018626:Retroelements; D029701:Saccharomyces cerevisiae Proteins; D012319:RNA Polymerase II; C511736:RPB1 protein, S cerevisiae