Mutant bacteriophage T7 RNA polymerases with altered termination properties.
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 (T(phi)) 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.
Lyakhov
D L
DL
Department of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York, Health Science Center at Brooklyn, 11203-2098, USA.
He
B
B
Zhang
X
X
Studier
F W
FW
Dunn
J J
JJ
McAllister
W T
WT
D000595:Amino Acid Sequence; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism* / Q000494:pharmacology; D009154:Mutation; D010281:Parathyroid Hormone / Q000235:genetics / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012323:RNA Processing, Post-Transcriptional; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins; D012407:rRNA Operon / Q000235:genetics
D010281:Parathyroid Hormone; D011498:Protein Precursors; D014764:Viral Proteins; C019056:preproparathormone; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D009113:Muramidase