T7 RNA polymerase mutants with altered promoter specificities.
The amino acid at position 748 in T7 RNA polymerase (RNAP) functions to discriminate base pairs at positions -10 and -11 in the promoter. We have constructed a series of T7 RNAP mutants having all possible amino acid substitutions at this position. Surprisingly, most (13/19) substitutions result in active RNAPs, and many of these exhibit altered promoter specificities. Identification of mutant RNAPs with altered specificities expands the repertoire of highly specific phage RNAPs that are available for use in phage RNAP-based transcription systems and highlights the complexity of sequence-specific DNA recognition.
Raskin
C A
CA
Morse Institute of Molecular Biology and Genetics, Department of Microbiology and Immunology, State University of New York Health Science Center, Brooklyn 11203-2098.
Diaz
G A
GA
McAllister
W T
WT
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D012321:DNA-Directed RNA Polymerases