PROBING THE PROTEIN-DNA CONTACTS OF A YEAST RNA-POLYMERASE-III TRANSCRIPTION COMPLEX IN A CRUDE EXTRACT - SOLID-PHASE SYNTHESIS OF DNA PHOTOAFFINITY PROBES CONTAINING A NOVEL PHOTOREACTIVE DEOXYCYTIDINE ANALOG

A novel photoreactive deoxycytidine analog, 4-[N-(p-azidobenzoyl)-2-am inoethyl]-dCTP (AB-dCTP), has been synthesized and incorporated at spe cific sites within the SUP4 tRNA(Tyr) gene. Immobilized single-strande d DNA was annealed to specific oligonucleotides and AB-dCMP incorporat ed into DNA by primer extension. DNA photoaffinity labeling with AB-dC MP was used to survey protein-DNA contacts in initiation and elongatio n complexes of RNA polymerase III (pol III), and compared to DNA photo affinity labeling using the previously described photoreactive deoxyur idine analog, 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP (AB-dUMP) [Bart holomew et al. (1993) Mel. Cell.Biol. 13, 942-952]. In contrast to pre vious studies, we have used a crude protein fraction rather than highl y purified preparations of Pol III and transcription factors TFIIIC an d TFIIIB to examine if some component of the transcription complex is lost upon purification. Eleven nucleotide positions from bp -17 to bp +17 (+1 being the start site of transcription) on the nontranscribed s trand were modified and shown to have little or no effect on transcrip tion complex formation, initiation, or elongation as determined by mul tiple-round transcription assays. Efficient photoaffinity labeling by DNA containing AB-dCMP gave results comparable to that with AB-dUMP at proximal nucleotide positions and provided new evidence for the place ment of the 160 and 31 kDa subunits of Pol III near the 5' end of the transcriptional bubble in an elongation complex. A novel 40 kDa protei n was cross-linked at bps -17, -9, and -8 in a TFIIIC-dependent manner that had not been previously detected.