Mapping the contacts of yeast TFIIIB and RNA polymerase III at various distances from the major groove of DNA by DNA photoaffinity labeling

Abstract

The structure of the Saccharomyces cerevisiae RNA polymerase III transcription complex on the SUP4 tRNA(Tyr) gene was probed at distances of ~10 to ~23 Å from the C-5 methyl of thymidine in the major groove of BNA 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 ~4.3 Å 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 tRNA(Tyr) gene along with an [α-32P]dNMP by primer extension using an immobilized single-stranded DNA template annealed to specific oligonucleotides. The 27-kBa subunit of TFIIIB or the TATA box binding protein was photoaffinity labeled at bp -26/-21 with nucleotide analogs containing a ~19- or ~23-Å chain and not with shorter chains of ~10 to ~15 Å in length. The B' subunit of TFIIIB (M(r) = 90 kDa) was photoaffinity labeled at bps -26/-21 with DNA containing a ~14-Å 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.