Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome

• Published in: mSystems 2018-01, Vol.3 (1)
• Main Authors: Yamamoto, Kaneyoshi, Yamanaka, Yuki, Shimada, Tomohiro, Sarkar, Paramita, Yoshida, Myu, Bhardwaj, Neerupma, Watanabe, Hiroki, Taira, Yuki, Chatterji, Dipankar, Ishihama, Akira
• Format: Article
• Language: English
• Subjects: Binding sites; Chromatin; Defective mutant; Deoxyribonucleic acid; DNA; DNA microarrays; DNA-directed RNA polymerase; E coli; Enzymes; Escherichia coli; Gene regulation; Genes; Genomes; Immunoprecipitation; Molecular Biology and Physiology; omega subunit; Open reading frames; Phenotypes; Polymerase chain reaction; Promoters; prophage; Prophages; Proteins; Regulatory sequences; Research Article; Reverse transcription; RNA polymerase; Stringent response; Subunit structure; Transcription factors; transcription regulation
• ISSN: 2379-5077; 2379-5077
• DOI: 10.1128/msystems.00172-17

The RNA polymerase (RNAP) of K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α ββ'ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the gene product), participates in subunit assembly by supporting the folding of the largest subunit, β', but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and -defective mutant strains. The altered distribution of RpoZ-defective RNAP was identified mostly within open reading frames, in particular, of the genes inside prophages. For the genes that exhibited increased or decreased distribution of RpoZ-defective RNAP, the level of transcripts increased or decreased, respectively, as detected by reverse transcription-quantitative PCR (qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of ligands by exponential enrichment), the distribution of constitutive promoters that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS within prophages. Since all 10 prophages in K-12 carry only a small number of promoters, the altered occupancy of RpoZ-defective RNAP and of transcripts might represent transcription initiated from as-yet-unidentified host promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP are located in the regions of low-level H-NS binding. By using phenotype microarray (PM) assay, alterations of some phenotypes were detected for the -deleted mutant, indicating the involvement of RpoZ in regulation of some genes. Possible mechanisms of altered distribution of RNAP inside prophages are discussed. The 91-amino-acid-residue small-subunit omega (the gene product) of RNA polymerase plays a structural role in the formation of RNA polymerase (RNAP) as a chaperone in folding the largest subunit (β', of 1,407 residues in length), but except for binding of the stringent signal ppGpp, little is known of its role in the control of RNAP function. After analysis of genomewide distribution of wild-type and RpoZ-defective RNAP by the ChIP-chip method, we found alteration of the RpoZ-defective RNAP inside open reading frames, in particular, of the genes within prophages. For a set of the genes that exhibited altered occupancy of the RpoZ-defective RNAP, t