Early steps in the path of nascent ribonucleic acid across the surface of ribonucleic acid polymerase, determined by photoaffinity labeling

Leslie H. DeRiemer, Claude F. Meares

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

The photoaffinity probes β-(4-azidophenyl) adenosine 5′-diphosphate (N3PhppA) and β-(4-azidophenyl) adenylyl-(3′-5′)-uridine 5′-diphosphate (N3PhppApU) were used to determine the RNA polymerase subunit contacts made by the 5′ ends of three nascent RNA chains. Ternary enzyme-poly[d(A-T)]·oligonucleotide complexes were prepared in which the nascent oligonucleotide contained a photoaffinity label at the 5′ end and a 32P radiolabel only at the 3′ end. The length of the RNA was fixed at two, three, or four nucleotides. Photolysis of the ternary complexes was followed by dissociation, polyacrylamide gel electrophoresis, autoradiography, and scintillation counting. With a dinucleotide probe, the enzyme subunits labeled were β′ (71%) and σ (21%). Photolysis of the ternary complex containing trinucleotide RNA also resulted in labeling of the β′ (64%) and σ (35%) subunits. With a tetranucleotide, the β′ subunit was very heavily labeled (88%), and a small amount of labeling of the β (7%) and σ (4%) subunits was observed. The α subunit was not labeled with any of the probes. These results imply that a conformational change, possibly involving dissociation of the σ subunit, occurs in the enzyme as the ribonucleotide is elongated from a tri- to a tetranucleotide.

Original languageEnglish (US)
Pages (from-to)1612-1617
Number of pages6
JournalBiochemistry
Volume20
Issue number6
StatePublished - 1981

ASJC Scopus subject areas

  • Biochemistry

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