Isolation and properties of enhancer-bypass mutants of sigma 54.
The N-terminal activation domain of Escherichia coli sigma 54 was randomly mutated to provide a library of changes that might allow the required enhancer function to be bypassed. Five clones harbouring mutant sigma factors were obtained that exhibited this property in that they enhanced growth under nitrogen-limiting conditions in cells lacking NtrC. DNA sequence analysis located all mutations to four leucines in a small region between amino acids 25 and 31. No mutant sigma factors retained the hydrophobic character of the leucine residues. Mutant sigma factors were shown to transcribe in vitro without the need for enhancer binding activator or ATP hydrolysis, confirming the in vivo phenotype. These and other data suggest that a very small set of leucines is critical for keeping polymerase function in check, allowing high responsiveness to physiological induction via enhancer proteins such as NtrC.
Syed
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Department of Chemistry and Biochemistry, University of California, Los Angeles 90095-1569, USA.
Gralla
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JD
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D006493:Heparin / Q000494:pharmacology; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009584:Nitrogen / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D017422:Sequence Analysis, DNA; D012808:Sigma Factor / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D012808:Sigma Factor; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli; C497751:rpoN protein, E coli; D000255:Adenosine Triphosphate; D006493:Heparin; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D007930:Leucine; D009584:Nitrogen