ISOLATION AND PROPERTIES OF ENHANCER-BYPASS MUTANTS OF SIGMA-54

The N-terminal activation domain of Escherichia coil sigma 54 was rand omly mutated to provide a library of changes that might allow the requ ired enhancer function to be bypassed. Five clones harbouring mutant s igma factors were obtained that exhibited this property in that they e nhanced growth under nitrogen-limiting conditions in cells lacking Ntr C. DNA sequence analysis located all mutations to four leucines in a s mall region between amino acids 25 and 31. No mutant sigma factors ret ained the hydrophobic character of the leucine residues. Mutant sigma factors were shown to transcribe in vitro without the need for enhance r binding activator or ATP hydrolysis, confirming the in vivo phenotyp e. These and other data suggest that a very small set of leucines is c ritical for keeping polymerase function in check, allowing high respon siveness to physiological induction via enhancer proteins such as NtrC .