PROBING CLOSE DNA CONTACTS OF RNA-POLYMERASE-III TRANSCRIPTION COMPLEXES WITH THE PHOTOACTIVE NUCLEOSIDE 4-THIODEOXYTHYMIDINE

Photoactive 4-thiodeoxythymidine 5'-triphosphate (4-S-dTTP) has been s ynthesized and used to enzymatically incorporate the corresponding nuc leotide, 4-thiodeoxythymidine 5'-phosphate (4-S-dTMP), at specific pos itions of the Saccharomyces cerevisiae 5 S rRNA and SUP4 tRNA(Tyr) gen es. RNA polymerase III transcription complexes have been assembled on this DNA and analyzed by photocrosslinking for proteins making close c ontact with DNA. Comparison DNA probes with a long-tether photoactive nucleotide 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP (N(3)RdUMP)(1) inc orporated at the same positions have also been analyzed, in order to c ompare the properties of these two crosslinking reagents. At least 10 of the 16 different S. cerevisiae polymerase III subunits make direct contact with DNA. The 120-kDa subunit of transcription factor (TF)IIIC , which is thought to play the key role in positioning TFIIIB upstream of the transcriptional start site, also contacts DNA near the transcr iptional start site in TFIII(C+B) complexes with a SUP4 tRNA(Tyr) gene . The photocrosslinking patterns generated by 4-S-dTMP and N(3)RdUMP a re distinctive, implying that these two reagents can yield complementa ry information about the structures of complex protein assemblies on D NA. Surprisingly, some subunits of the S. cerevisiae RNA polymerase II I are crosslinked by 4-S-dTMP but not by N(3)RdUMP.