Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure.
In order to test the proposal that most nucleotide polymerases share a common active site structure and folding topology, we have generated 22 mutations of residues within motifs A, B and C of T7 RNA polymerase (RNAP). Characterization of these T7 RNAP mutants showed the following: (i) most of the mutations resulted in moderate to drastic reductions in T7 RNAP transcriptional activity supporting the idea that motifs A, B and C identify part of the polymerase active site; (ii) the degree of conservation of an amino acid within these motifs correlated with the degree to which mutation of that amino acid reduced transcriptional activity, supporting the predictive ability of this alignment in identifying the most functionally critical residues; (iii) a comparison of DNAP I and T7 RNAP mutants revealed similarities (as well as differences) between corresponding mutant phenotypes; (iv) the Klenow fragment structure is shown to provide a reasonable basis for interpretation of the differential effects of mutating different amino acids within motifs A, B and C in T7 RNAP. These observations support the proposal that these polymerase active sites have similar three-dimensional structures.
Bonner
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G
Department of Biological Sciences, University of Pittsburgh, PA 15260.
Patra
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D
Lafer
E M
EM
Sousa
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R
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D005814:Genes, Viral; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013698:Templates, Genetic; D015678:Viral Structural Proteins / Q000235:genetics
D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D012321:DNA-Directed RNA Polymerases