Jb. Huang et al., Mechanisms by which T7 lysozyme specifically regulates T7 RNA polymerase during different phases of transcription, J MOL BIOL, 293(3), 1999, pp. 457-475
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.