SPECIFICITY IN TRANSCRIPTIONAL REGULATION IN THE ABSENCE OF SPECIFIC DNA-BINDING SITES - THE CASE OF T7 LYSOZYME

The binding of T7 lysozyme to T7 RNAP increases the apparent K-m for N m during initiation (formation of the first phosphodiester bond). It a lso increases the dissociation constant and dissociation rate of produ ct dinucleotide from the polymerase. Higher Nm concentrations are requ ired for maximal rates of productive initiation from T7 class II versu s class III promoters, though individual promoters display distinct re sponses to changes in NTP concentrations. The greater degree of repres sion of class Il versus class III promoters by T7 lysozyme, which appe ars to be important for the switch to class III gene expression during the phage life cycle, might therefore be a consequence of: (1) T7 lys ozyme generally reducing the affinity of the polymerase for NTPs and i ncreasing the rate of release of transcripts, and (2), intrinsically h igher Nm concentration requirements for productive initiation from cla ss IT promoters. T7 lysozyme is also found to inhibit the addition of untemplated bases to the transcript which can occur when the elongatio n complex reaches the end of a template, and its effects are qualitati vely similar to those reported for mutations in the extreme C terminus of T7 RNAP. Together with the locations of polymerase mutations which cause resistance or hypersensitivity to T7 lysozyme, these observatio ns suggest that the structural mechanism of lysozyme action might incl ude conformational changes in the C-terminal loop (aa. similar to 820- 883) of T7 RNAP. (C) 1998 Academic Press.