Photoaffinity labeling of RNA polymerase III transcription complexes by nascent RNA

Blaine Bartholomew, Claude F. Meares, Michael E. Dahmus

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


The proteins contacting nascent RNA transcripts in RNA polymerase III transcription complexes have been examined using photoaffinity labeling techniques. The photoaffinity analog 4-S-UTP was incorporated along with [α-32P]CTP into VAI transcripts, using a phosphocellulose fractionated HeLa S-100 extract and DNA containing the adenovirus VAI gene. The photoreactive nascent RNA was cross-linked to proximal proteins in the transcription complex. The photoaffinity labeled proteins were separated by sodium dodecyl sulfate-poly acrylamide gel electrophoresis and detected by autoradiography. The specific photoaffinity labeling of RNA polymerase III was dependent on 4-S-UTP and on DNA containing a class III promoter. Photoaffinity labeling was inhibited by 200 μg/ml α-amanitin. Proteins of 140,160,270, and 310kDa were labeled. These photoaffinity labeled proteins were shown to be stably associated with the DNA template by gel exclusion chromatography. The 160-kDa protein was cross-linked to RNAs approximately 14-18 nucleotides in length, whereas the >250-kDa proteins were cross-linked to RNAs 18-30 nucleotides in length. The 140- and 160-kDa proteins correspond in molecular mass to the two large subunits of RNA polymerase III. The molecular masses of the 270- and 310-kDa proteins, and the length of the RNA cross-linked to them, suggest that these proteins are components of transcription factor (TF) IIIC. These results indicate that the nascent transcript contacts the two largest subunits of RNA polymerase III until the transcription complex reaches the TFIIIC binding site, at which point the nascent transcript contacts TFIIIC.

Original languageEnglish (US)
Pages (from-to)3731-3737
Number of pages7
JournalJournal of Biological Chemistry
Issue number7
StatePublished - Mar 5 1990

ASJC Scopus subject areas

  • Biochemistry


Dive into the research topics of 'Photoaffinity labeling of RNA polymerase III transcription complexes by nascent RNA'. Together they form a unique fingerprint.

Cite this