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 trans cription complex on the SUP4 tRNA(Tyr) gene was probed at distances of similar to 10 to similar to 23 Angstrom from the C-5 methyl of thymid ine in the major groove of DNA using photoreactive aryl azides attache d to deoxyuridine by variable chain lengths. The nucleotide analogs co ntained an azidobenzoyl group attached with chain lengths that were in crementally increased by similar to 4.3 Angstrom by inserting 1-3 glyc ine 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 t he transcription complex. These nucleotide analogs were incorporated a t base pairs (bp) -26/-21, -17, or -3/-2 on the nontranscribed strand of the SUP4 tRNA(Tyr) gene along with an [alpha-P-32]dNMP by primer ex tension using an immobilized single-stranded DNA template annealed to specific oligonucleotides. The 27-kDa subunit of TFIIIB or the TATA bo x binding protein was photoaffinity labeled at bp -26/-21 with nucleot ide analogs containing a similar to 19- or similar to 23-Angstrom chai n and not with shorter chains of similar to 10 to similar to 15 Angstr om in length. The B '' subunit of TFIIIB (M(r) 90 kDa) was photoaffini ty labeled at bps -26/-21 with DNA containing a similar to 14-Angstrom 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 con formational change in the TFIIIB-DNA complex resulting in the displace ment of the 90-kDa subunit at bps -26/-21. Next, the chain length depe ndence 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.