TY - JOUR
T1 - Mutant bacteriophage T7 RNA polymerases with altered termination properties
AU - Lyakhov, Dmitry L.
AU - He, Biao
AU - Zhang, Xing
AU - Studier, F. W.
AU - Dunn, John J.
AU - McAllister, William T.
N1 - Funding Information:
Work at SUNY Health Science Center at Brooklyn was supported by NIH grant GM38147. Work at Brookhaven National Laboratories was supported by the Office of Health and Environmental Research of the US Department of Energy. We are grateful to Mr Ray Castagna for technical assistance, and to Ms Rita Gould and Roseann Lingeza for secretarial assistance. We thank Drs Sergei Borokhov and Rui Sousa for thoughtful comments on the manuscript.
PY - 1997/5/30
Y1 - 1997/5/30
N2 - We have identified mutants of bacteriophage T7 RNA polymerase (RNAP) that are altered in their ability to pause or terminate at a variety of signals. These signals include a terminator found fortuitously in the human preproparathyroid hormone (PTH) gene, a pause site found in the concatamer junction (CJ) of replicating T7 DNA, and termination signals that are also utilized by Escherichia coli RNAP (e.g. rrnB T1 and T2). Whereas the mutant enzymes terminate normally at the late terminator in T7 DNA (TΦ) and rrnB T2, they fail to terminate at one of the termination sites of rrnB T1, and also fail to recognize the PTH and CJ signals. The mutant enzymes exhibit normal processivity on linear templates, but show a slightly reduced processivity on supercoiled templates and terminate more efficiently when synthesizing poly(U) tracts. The mutant enzymes also show a decreased tendency to produce aberrant transcription products from DNA templates having protruding 3' ends. T7 lysozyme (an inhibitor of T7 RNAP) has been shown to exert its action by preventing the transition of the RNAP from an unstable initiation complex (IC) to a stable elongation complex (EC). We have found that T7 lysozyme enhances recognition of CJ by wild-type T7 RNAP, and that mutant T7 RNAPs that show increased sensitivity to lysozyme show enhanced recognition of this signal, even in the absence of lysozyme. These results, together with the observation that the mutations that result in the termination-deficient phenotype affect a region of the RNAP that has been implicated in RNA binding and upstream promoter contacts, support the hypothesis that, in some cases, termination represents a reversal of the events that occur during initiation.
AB - We have identified mutants of bacteriophage T7 RNA polymerase (RNAP) that are altered in their ability to pause or terminate at a variety of signals. These signals include a terminator found fortuitously in the human preproparathyroid hormone (PTH) gene, a pause site found in the concatamer junction (CJ) of replicating T7 DNA, and termination signals that are also utilized by Escherichia coli RNAP (e.g. rrnB T1 and T2). Whereas the mutant enzymes terminate normally at the late terminator in T7 DNA (TΦ) and rrnB T2, they fail to terminate at one of the termination sites of rrnB T1, and also fail to recognize the PTH and CJ signals. The mutant enzymes exhibit normal processivity on linear templates, but show a slightly reduced processivity on supercoiled templates and terminate more efficiently when synthesizing poly(U) tracts. The mutant enzymes also show a decreased tendency to produce aberrant transcription products from DNA templates having protruding 3' ends. T7 lysozyme (an inhibitor of T7 RNAP) has been shown to exert its action by preventing the transition of the RNAP from an unstable initiation complex (IC) to a stable elongation complex (EC). We have found that T7 lysozyme enhances recognition of CJ by wild-type T7 RNAP, and that mutant T7 RNAPs that show increased sensitivity to lysozyme show enhanced recognition of this signal, even in the absence of lysozyme. These results, together with the observation that the mutations that result in the termination-deficient phenotype affect a region of the RNAP that has been implicated in RNA binding and upstream promoter contacts, support the hypothesis that, in some cases, termination represents a reversal of the events that occur during initiation.
KW - Concatamer junction
KW - Packaging
KW - Phage RNA polymerase
KW - RNA binding
KW - T7 lysozyme
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U2 - 10.1006/jmbi.1997.1015
DO - 10.1006/jmbi.1997.1015
M3 - Article
C2 - 9192998
AN - SCOPUS:0031591385
SN - 0022-2836
VL - 269
SP - 28
EP - 40
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -