title: Inactivation of the bacterial RNA polymerase due to acquisition of secondary structure by the ω subunit.
abstract: The widely conserved ω subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of ω is known to be substituted by GroEL in ω-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of ω variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to ω, which is largely unstructured, ω mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, ω6 bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that ω6 binds to β' subunit in vitro with greater affinity than that of ω. The reconstituted RNAP holoenzyme in the presence of ω6 in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant ω results in death of the cell. Furthermore, lethality of ω6 is relieved in cells expressing the rpoC2112 allele encoding β'2112, a variant β' bearing Y457S substitution, immediately adjacent to the β' catalytic center. Our results suggest that the enhanced ω6-β' interaction may perturb the plasticity of the RNAP active center, implicating a role for ω and its flexible state.
authors.affiliation: From the Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Paramita
authors.initials: P
authors.lastname: Sarkar
authors.affiliation: Laboratory of Bacterial Genetics, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500001, India, and.
authors.forename: Abhijit A
authors.initials: AA
authors.lastname: Sardesai
authors.affiliation: Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Katsuhiko S
authors.initials: KS
authors.lastname: Murakami
authors.affiliation: From the Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India,. Electronic address: dipankar@mbu.iisc.ernet.in.
authors.forename: Dipankar
authors.initials: D
authors.lastname: Chatterji
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D061785:Transcription Initiation, Genetic / Q000502:physiology*
chemical_list: D029968:Escherichia coli Proteins; D012321:DNA-Directed RNA Polymerases; C571073:RpoZ protein, E coli
keywords: Bacterial Genetics; Bacterial Transcription; Dominant Lethal; Protein Conformation; Protein-Protein Interactions; RNA Polymerase; Unstructured Protein; ω Subunit of RNA Polymerase