Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking.
The subunits of Saccharomyces cerevisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3'-extension of deoxycytidines, a "tailed template". We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N(3)RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N(3)RdUTP, which has a photoreactive azido group 9 A from the uridine base, we used the photoaffinity cross-linker 5N(3)dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N(3)RdUTP. With 5N(3)dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex.
Wooddell
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McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Burgess
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D001386:Azides / Q000737:chemistry; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018983:DNA Footprinting; D004271:DNA, Fungal / Q000737:chemistry; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D019920:Photoaffinity Labels / Q000737:chemistry*; D012319:RNA Polymerase II / Q000737:chemistry*; D012331:RNA, Fungal / Q000737:chemistry; D016914:Ribonuclease H / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry
D001386:Azides; D003432:Cross-Linking Reagents; D004271:DNA, Fungal; D003856:Deoxyuracil Nucleotides; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D019920:Photoaffinity Labels; D012331:RNA, Fungal; C048865:5-azido-2'-deoxyuridine 5'-triphosphate; C064622:5-(N-(4-azidobenzoyl)-3-aminoallyl)deoxyuridine 5'-monophosphate; D014542:Uridine Monophosphate; D012319:RNA Polymerase II; D016914:Ribonuclease H