Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II.
Rpb9 is a small non-essential subunit of yeast RNA polymerase II located on the surface on the enzyme. Deletion of the RPB9 gene shows synthetic lethality with the low fidelity rpb1-E1103G mutation localized in the trigger loop, a mobile element of the catalytic Rpb1 subunit, which has been shown to control transcription fidelity. Similar to the rpb1-E1103G mutation, the RPB9 deletion substantially enhances NTP misincorporation and increases the rate of mismatch extension with the next cognate NTP in vitro. Using pre-steady state kinetic analysis, we show that RPB9 deletion promotes sequestration of NTPs in the polymerase active center just prior to the phosphodiester bond formation. We propose a model in which the Rpb9 subunit controls transcription fidelity by delaying the closure of the trigger loop on the incoming NTP via interaction between the C-terminal domain of Rpb9 and the trigger loop. Our findings reveal a mechanism for regulation of transcription fidelity by protein factors located at a large distance from the active center of RNA polymerase II.
NCI Center for Cancer Research, National Institutes of Health, Frederick, Maryland 21702, USA.
Celine
C
Walmacq
Maria L
ML
Kireeva
Jordan
J
Irvin
Yuri
Y
Nedialkov
Lucyna
L
Lubkowska
Francisco
F
Malagon
Jeffrey N
JN
Strathern
Mikhail
M
Kashlev
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D003570:Cytidine Triphosphate / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000378:metabolism
D005656:Fungal Proteins; D009711:Nucleotides; D021122:Protein Subunits; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D012319:RNA Polymerase II; D014544:Uridine Triphosphate