Mechanisms by which T7 lysozyme specifically regulates T7 RNA polymerase during different phases of transcription

Bacteriophage T7 lysozyme binds to T7 RNA polymerase (RNAP) and regulates i ts transcription by differentially repressing initiation from different T7 promoters. This selective repression is due in part to a lysozyme-induced i ncrease in the K-NTP of the initiation complex (IC) and to intrinsically di fferent NTP:concentration requirements for efficient initiation from differ ent T7 promoters. While lysozyme represses initiation, once the enzyme has left the promoter and formed an elongation complex (EC) it is generally res istant to the effects of lysozyme. The mechanism by which the inhibitory ef fects of lysozyme are largely restricted to the initiation phase of transcr iption is not well understood. We find that T7 lysozyme destabilizes initia l transcription complexes (ITCs) and increases the rate of release of trans cripts from these complexes but does not destabilize ECs. However, if the R NA:RNAP interaction proposed to be important for EC stability is disrupted by proteolysis of the RNA-binding domain or use of templates which interfer e with establishment of this RNA:RNAP interaction, the EC becomes sensitive to lysozyme. Comparison of the X-ray structures of T7RNAP and of a T7RNAP: T7 lysozyme complex reveals that lysozyme causes the C terminus of the poly merase to flip out of the active site. Experiments in which carboxypeptidas e;Ais used to probe the lysozyme-induced exposure of the C terminus reveal a large decrease in carboxypeptidase sensitivity following transcription in itiation, suggesting that interactions with the 3'-end of the RNA help stab ilize the active site in a functional (carboxypeptidase protected) conforma tion. Thus, the resistance of the EC to lysozyme appears to be due to the c onsecutive establishment of two sets of RNA:RNAP interactions. The first is made with the 3'-end of the RNA and helps stabilize a functional conformat ion of the active site, thereby suppressing the effects of lysozyme on K-NT P. The second is made with a more upstream element of the RNA and keeps the EC from being destabilized by lysozyme binding. (C) 1999 Academic Press.