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TitleTranscription start site scanning requires the fungi-specific hydrophobic loop of Tfb3.
Journal, issue, pagesNucleic Acids Res, Vol. 52, Issue 19, Page 11602-11611, Year 2024
Publish dateOct 28, 2024
AuthorsChun Yang / Pratik Basnet / Samah Sharmin / Hui Shen / Craig D Kaplan / Kenji Murakami /
PubMed AbstractRNA polymerase II (pol II) initiates transcription from transcription start sites (TSSs) located ∼30-35 bp downstream of the TATA box in metazoans, whereas in the yeast Saccharomyces cerevisiae, ...RNA polymerase II (pol II) initiates transcription from transcription start sites (TSSs) located ∼30-35 bp downstream of the TATA box in metazoans, whereas in the yeast Saccharomyces cerevisiae, pol II scans further downstream TSSs located ∼40-120 bp downstream of the TATA box. Previously, we found that removal of the kinase module TFIIK (Kin28-Ccl1-Tfb3) from TFIIH shifts the TSS in a yeast in vitro system upstream to the location observed in metazoans and that addition of recombinant Tfb3 back to TFIIH-ΔTFIIK restores the downstream TSS usage. Here, we report that this biochemical activity of yeast TFIIK in TSS scanning is attributable to the Tfb3 RING domain at the interface with pol II in the pre-initiation complex (PIC): especially, swapping Tfb3 Pro51-a residue conserved among all fungi-with Ala or Ser as in MAT1, the metazoan homolog of Tfb3, confers an upstream TSS shift in vitro in a similar manner to the removal of TFIIK. Yeast genetic analysis suggests that both Pro51 and Arg64 of Tfb3 are required to maintain the stability of the Tfb3-pol II interface in the PIC. Cryo-electron microscopy analysis of a yeast PIC lacking TFIIK reveals considerable variability in the orientation of TFIIH, which impairs TSS scanning after promoter opening.
External linksNucleic Acids Res / PubMed:39287137 / PubMed Central
MethodsEM (single particle)
Resolution3.7 - 3.8 Å
Structure data

42437

8uoq

EMDB-42437, PDB-8uoq:
Composite map of PIC_delta_TFIIK form2
Method: EM (single particle) / Resolution: 3.8 Å

42438

8uot

EMDB-42438, PDB-8uot:
Composite map of PICdeltaTFIIK form1
Method: EM (single particle) / Resolution: 3.7 Å

Chemicals

ChemComp-ZN:
Unknown entry

ChemComp-MG:
Unknown entry

ChemComp-SF4:
IRON/SULFUR CLUSTER

Source
  • saccharomyces cerevisiae (brewer's yeast)
KeywordsTRANSCRIPTION / pol II / Transcription. POL II

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