The highly conserved glutamic acid 791 of Rpb2 is involved in the binding of NTP and Mg(B) in the active center of human RNA polymerase II.
During transcription by RNA polymerase (RNAP) II, the incoming ribonucleoside triphosphate (NTP) enters the catalytic center in association with an Mg2+ ion, termed metal B [Mg(B)]. When bound to RNAP II, Mg(B) is coordinated by the beta and gamma phosphates of the NTP, Rpb1 residues D481 and D483 and Rpb2 residue D837. Rpb2 residue D837 is highly conserved across species. Notably, its neighboring residue, E836 (E791 in human RNAP II), is also highly conserved. To probe the role of E791 in transcription, we have affinity purified and characterized a human RNAP II mutant in which this residue was substituted for alanine. Our results indicate that the transcription activity of the Rpb2 E791A mutant is impaired at low NTP concentrations both in vitro and in vivo. They also revealed that both its NTP polymerization and transcript cleavage activities are decreased at low Mg concentrations. Because Rpb2 residue E791 appears to be located too far from the NTP-Mg(B) complex to make direct contact at either the entry (E) or addition (A) site, we propose alternative mechanisms by which this highly conserved residue participates in loading NTP-Mg(B) in the active site during transcription.
Langelier
Marie-France
MF
Laboratory of Gene Transcription, Institut de recherches cliniques de Montréal, 110 avenue des Pins Ouest, Montréal, Québec, Canada H2W 1R7.
Baali
Dania
D
Trinh
Vincent
V
Greenblatt
Jack
J
Archambault
Jacques
J
Coulombe
Benoit
B
D019943:Amino Acid Substitution; D001665:Binding Sites; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000378:metabolism
D021122:Protein Subunits; D012333:RNA, Messenger; D012265:Ribonucleotides; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D018698:Glutamic Acid; C500816:POLR2B protein, human; D012319:RNA Polymerase II; D008274:Magnesium