Mapping the contacts of yeast TFIIIB and RNA polymerase III at various distances from the major groove of DNA by DNA photoaffinity labeling.
The structure of the Saccharomyces cerevisiae RNA polymerase III transcription complex on the SUP4 tRNATyr gene was probed at distances of approximately 10 to approximately 23 A from the C-5 methyl of thymidine in the major groove of DNA using photoreactive aryl azides attached to deoxyuridine by variable chain lengths. The nucleotide analogs contained an azidobenzoyl group attached with chain lengths that were incrementally increased by approximately 4. 3 A by inserting 1-3 glycine residues into the chain. Another photoreactive deoxyuridine analog was made that contained a butyl chain (ABU-dUMP) to assess the effect of the chain's hydrophobicity on its ability to photoaffinity label the transcription complex. These nucleotide analogs were incorporated at base pairs (bp) -26/-21, -17, or -3/-2 on the nontranscribed strand of the SUP4 tRNATyr gene along with an [alpha-32P]dNMP by primer extension using an immobilized single-stranded DNA template annealed to specific oligonucleotides. The 27-kDa subunit of TFIIIB or the TATA box binding protein was photoaffinity labeled at bp -26/-21 with nucleotide analogs containing a approximately 19- or approximately 23-A chain and not with shorter chains of approximately 10 to approximately 15 A in length. The B" subunit of TFIIIB (Mr = 90 kDa) was photoaffinity labeled at bps -26/-21 with DNA containing a approximately 14-A chain and not with shorter or longer chains. Cross-linking of the B" subunit was inhibited by binding of RNA polymerase III (Pol III) to the TFIIIB-DNA complex and suggested that Pol III binding causes a conformational change in the TFIIIB-DNA complex resulting in the displacement of the 90-kDa subunit at bps -26/-21. Next, the chain length dependence of photoaffinity labeling the 34-kDa subunit of Pol III at bps -17 and -3/-2 indicated that the 34-kDa subunit of Pol III is slightly removed from the major groove at bp -17 in the initiation complex and makes closer contact at bps -3/-2 in a stalled elongation complex.
Persinger
J
J
Department of Medical Biochemistry, School of Medicine, Southern Illinois University at Carbondale, Carbondale, Illinois 62901-4413, USA.
Bartholomew
B
B
D000345:Affinity Labels; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003852:Deoxyribonucleoproteins / Q000737:chemistry / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012320:RNA Polymerase III / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000737:chemistry*; D014158:Transcription, Genetic
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D004247:DNA; D012320:RNA Polymerase III