Yeast RNA polymerase I. Derivatization of the 190 and 135 subunits by 4-thiouridine monophosphate positioned uniquely at the 3' terminus of an enzyme-bound 32P-containing transcript initiated by a triribonucleotide primer on synthetic single-stranded DNA.
Specific transcription complexes were formed with yeast RNA polymerase I using a cognate oligoribotri-nucleotide primer (GCG) to initiate transcription on short synthetic single-stranded DNA templates. The templates were designed to limit the incorporation of a photoprobe, 4-thiouridine triphosphate, to a single unique position at the 3' terminus of the product RNA (position 12, 13, 14, or 15). The resulting transcription complexes were photolyzed to cross-link the bound transcript (radiolabeled with [alpha-32P]CTP) to the protein with the probe located at the catalytic site. Separation of the protein subunit components by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analysis by autoradiography and silver staining revealed that the two largest subunits (A190 and A135) were radiolabeled. The ratio of subunit labeling (A190/A135) decreased as the RNA transcript increased from 12 to 15 nucleotides in length. This decrease in ratio resulted from a progressive reduction of A190 subunit labeling while the A135 subunit derivatization remained essentially constant. It was also observed that the DNA template was radiolabeled.
Kelly
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Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Sheng
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Dennis
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D000345:Affinity Labels; D001345:Autoradiography; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010777:Photochemistry; D012318:RNA Polymerase I / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*
D000345:Affinity Labels; D004277:DNA, Single-Stranded; D009843:Oligoribonucleotides; C059149:4-thiouridine triphosphate; D012318:RNA Polymerase I; D010727:Phosphoric Diester Hydrolases; D014544:Uridine Triphosphate