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.