Determinants of RNA polymerase α subunit for interaction with β, β′, and σ subunits: Hydroxyl-radical protein footprinting

Tomasz Heyduk, Ewa Heyduk, Konstantin Severinov, Hong Tang, Richard H. Ebright

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78 Citations (Scopus)

Abstract

Escherichia coli RNA polymerase (RNAP) α subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2α → α2 → α2β → α2ββ′ → α2ββ′ σ. In this work, we have used hydroxyl-radical protein footprinting to define determinants of α for interaction with β, β′, and σ. Our results indicate that amino acids 30-75 of α are protected from hydroxyl-radical-mediated proteolysis upon interaction with β (i.e., in α2β, α2ββ′, and α2ββ′ σ), and amino acids 175-210 of α are protected from hydroxyl-radical-mediated proteolysis upon interaction with β′ (i.e., in α2ββ′ and α2ββ′ σ). The protected regions are conserved in the α homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of α for direct functional interaction with β and β′. The observed maximal magnitude of protection upon interaction with β and the observed maximal magnitude of protection upon interaction with β′ both correspond to the expected value for complete protection of one of the two α protomers of RNAP (i.e., 50% protection). We propose that only one of the two α protomers of RNAP interacts with β and that only one of the two α protomers of RNAP interacts with β′.

Original languageEnglish
Pages (from-to)10162-10166
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number19
DOIs
Publication statusPublished - 17 Sep 1996
Externally publishedYes

Keywords

  • Epitope mapping
  • Protein-ligand interaction
  • Protein-protein interaction
  • Transcription

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