Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP) I. Saturation and alanine-scanning mutagenesis

Wei Niu, Yuhong Zhou, Qianping Dong, Yon W. Ebright, Richard H. Ebright

Research output: Contribution to journalArticlepeer-review

70 Scopus citations

Abstract

It has been proposed that the surface loop consisting of amino acid residues 152 to 166 of the catabolite gene activator protein (CAP) of Escherichia coli makes direct protein-protein contact with RNA polymerase at the lac promoter. In this work, we have used targeted saturation mutagenesis of codons 152 to 166 of the gene encoding CAP, followed by a screen, to isolate more than 200 independent mutants of CAP defective in transcription activation but not defective in DNA binding. All isolated single-substitution mutants map to just eight amino acid residues: 156, 157, 158, 159, 160, 162, 163 and 164. We propose that these residues define the full extent of the epitope on CAP for the proposed CAP-RNA polymerase interaction. In addition, we have constructed alanine substitutions at each position from residue 152 to 166 of CAP, and we have analyzed the effects on transcription activation at the lac promoter and on DNA binding. Alanine substitution of Thr 158 results in an approximately eightfold specific defect in transcription activation. In contrast, alanine substitution of no other residue tested results in a more than twofold specific defect in transcription activation. We conclude that, for Thr158, side-chain atom beyond Cβ are essential for transcription activation at the lac promoter, and we propose that Thr158 OHγ makes direct contact with RNA polymerase in the ternary complex for lac promoter. CAP and RNA polymerase. We conclude further that for no residue other than Thr158 are side-chain atoms beyond Cβ essential for transcription activation at the lac promoter.

Original languageEnglish (US)
Pages (from-to)595-602
Number of pages8
JournalJournal of molecular biology
Volume243
Issue number4
DOIs
StatePublished - Nov 4 1994

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Keywords

  • RNA polymerase
  • cAMP receptor protein (CRP)
  • epitope mapping
  • lac promoter
  • transcription activation

Fingerprint Dive into the research topics of 'Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP) I. Saturation and alanine-scanning mutagenesis'. Together they form a unique fingerprint.

Cite this