Systematic mutagenesis identifies p52/Tfb2 residues required for XPB/Ssl2 function within TFIIH and genetic interactions with TFB6

ABSTRACT: TFIIH is an evolutionarily conserved complex that plays central roles in both RNA polymerase II (pol II) transcription and DNA repair. As an integral component of the pol II Pre-Initiation Complex (PIC), TFIIH regulates the activity of the pol II enzyme in numerous ways. The TFIIH subunit XPB/Ssl2 is an ATP-dependent DNA translocase that stimulates promoter opening prior to transcription initiation. Crosslinking-mass spectrometry and cryo-EM results have shown a conserved interaction network involving XPB/Ssl2 and the C-terminal Hub region of the TFIIH p52/Tfb2 subunit. Here, we systematically mutagenized the HubA region of Tfb2 and screened for growth phenotypes in a TFB6 deletion background in Saccharomyces cerevisiae. We identified six lethal and twelve conditional mutants. Slow growth phenotypes of all but three conditional mutants were relieved in the presence of TFB6, thus identifying a functional interaction between Tfb2 HubA mutants and Tfb6, a protein that dissociates Ssl2 from TFIIH. Biochemical analysis of Tfb2 mutants with severe growth phenotypes revealed defects in Ssl2 association, with similar results in human cells. Further characterization of these tfb2 mutant cells revealed defects in GAL gene induction, and reduced occupancy of TFIIH and pol II at GAL gene promoters, suggesting that functionally competent TFIIH is required for proper pol II recruitment to PICs in vivo. Consistent with recent structural models of TFIIH, our results identify key residues in the p52/Tfb2 HubA domain that are required for stable incorporation of XPB/Ssl2 into TFIIH, and for pol II transcription.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Jacob Bassett  

LAB HEAD: Jeffrey Alan Ranish

PROVIDER: PXD035576 | Pride | 2023-03-11

REPOSITORIES: Pride