Dominant lethal mutations near the 5' substrate binding site affect RNA polymerase propagation.
The segment Asp1064-Lys1073 in the beta subunit of Escherichia coli RNA polymerase is evolutionarily conserved and is located near the "5' face" of the nucleotide binding pocket as was shown by affinity labeling with priming substrates (Grachev, M. A., Lukhtamov, E. A., Mustaev, A. A., Zaychikov, E. F., Abdukayumov, M. N., Rabinov, I. V., Richter, V. I., Skoblov, Y. S., and Chistyakov, P. G. (1989) Eur. J. Biochem. 180, 577-585). We engineered single Xaa-->Ala or Ala-->Ser substitutions of eight evolutionarily conserved amino acids in this segment as well as a multiple alanine (KRNK) substitution of four of these residues. The KRNK mutation as well as four of the single substitutions were dominant lethal, two of the single mutations were recessive lethal, and two were viable. RNA polymerase bearing the dominant mutations was prepared for biochemical study by in vitro reconstitution from subunits. All of the mutant enzymes formed stable, specific promoter complexes, capable of initiating RNA synthesis. However, the KRNK polymerase was totally blocked in initiation-to-elongation transition, whereas the four point mutants displayed allele-specific changes in promoter clearance rate. Each of the four mutations changed, in a specific way, both the pattern of short oligomers generated in abortive initiation and the pattern of RNA polymerase pausing during elongation. Thus, the mutations appear to distort but not destroy the active center and to alter, in allele-specific manner, the coupling between the catalytic reaction and RNA polymerase propagation along the template.
Sagitov
V
V
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Nikiforov
V
V
Goldfarb
A
A
D000483:Alleles; D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004279:DNA, Viral / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013329:Structure-Activity Relationship
D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases