title: Dissection of two hallmarks of the open promoter complex by mutation in an RNA polymerase core subunit.
abstract: Deletion of 10 evolutionarily conserved amino acids from the beta subunit of Escherichia coli RNA polymerase leads to a mutant enzyme that is unable to efficiently hold onto DNA. Open promoter complexes formed by the mutant enzyme are in rapid equilibrium with closed complexes and, unlike the wild-type complexes, are highly sensitive to the DNA competitor heparin (Martin, E., Sagitov, V., Burova, E., Nikiforov, V., and Goldfarb, A. (1992) J. Biol. Chem. 267, 20175-20180). Here we show that despite this instability, the mutant enzyme forms partially open complexes at temperatures as low as 0 degrees C when the wild-type complex is fully closed. Thus, the two hallmarks of the open promoter complex, the stability toward a challenge with DNA competitors and the sensitivity toward low temperature, can be uncoupled by mutation and may be independent in the wild-type complex. We use the high resolution structure of Thermus aquaticus RNA polymerase core to build a functional model of promoter complex formation that accounts for the observed defects of the E. coli RNA polymerase mutants.
authors.affiliation: Waksman Institute and Department of Genetics, Rutgers, The State University, Piscataway, NJ 08854, USA. severik@waksman.rutgers.edu
authors.forename: S
authors.initials: S
authors.lastname: Nechaev
authors.forename: M
authors.initials: M
authors.lastname: Chlenov
authors.forename: K
authors.initials: K
authors.lastname: Severinov
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D020134:Catalytic Domain; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013824:Thermus / Q000201:enzymology; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D012321:DNA-Directed RNA Polymerases