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J. Biol. Chem., Vol. 265, Issue 14, 7787-7792, 05, 1990

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

S Kelly, N Sheng and D Dennis
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.

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.
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