title: Suppression of in vivo Rho-dependent transcription termination defects: evidence for kinetically controlled steps.
abstract: The conventional model of Rho-dependent transcription termination in bacteria requires RNA-dependent translocase activity of the termination factor Rho as well as many kinetically controlled steps to execute efficient RNA release from the transcription elongation complex (EC). The involvement of the kinetically controlled steps, such as RNA binding, translocation and RNA release from the EC, means that this termination process must be kinetically coupled to the transcription elongation process. The existence of these steps in vivo has not previously been delineated in detail. Moreover, the requirement for translocase activity in Rho-dependent termination has recently been questioned by a radical view, wherein Rho binds to the elongating RNA polymerase (RNAP) prior to loading onto the mRNA. Using growth assays, microarray analyses and reporter-based transcription termination assays in vivo, we showed that slowing of the transcription elongation rate by using RNAP mutants (rpoB8 and rpoB3445) and growth of the strains in minimal medium suppressed the termination defects of five Rho mutants, three NusG mutants defective for Rho binding and the defects caused by two Rho inhibitors, Psu and bicyclomycin. These results established the existence of kinetically controlled steps in the in vivo Rho-dependent termination process and further reinforced the importance of 'kinetic coupling' between the two molecular motors, Rho and RNAP, and also argue strongly that the Rho translocation model is an accurate representation of the in vivo situation. Finally, these results indicated that one of the major roles of NusG in in vivo Rho-dependent termination is to enhance the speed of RNA release from the EC.
authors.affiliation: Laboratory of Transcription Biology, Center for DNA Fingerprinting and Diagnostics, Tuljaguda complex, 4-1-714 Mozamjahi Road, Nampally, Hyderabad - 500 001, India.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Shashni
authors.affiliation: Laboratory of Transcription Biology, Center for DNA Fingerprinting and Diagnostics, Tuljaguda complex, 4-1-714 Mozamjahi Road, Nampally, Hyderabad - 500 001, India.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Mishra
authors.affiliation: Laboratory of Transcription Biology, Center for DNA Fingerprinting and Diagnostics, Tuljaguda complex, 4-1-714 Mozamjahi Road, Nampally, Hyderabad - 500 001, India.
authors.forename: B Sudha
authors.initials: BS
authors.lastname: Kalayani
authors.affiliation: Laboratory of Transcription Biology, Center for DNA Fingerprinting and Diagnostics, Tuljaguda complex, 4-1-714 Mozamjahi Road, Nampally, Hyderabad - 500 001, India.
authors.forename: Ranjan
authors.initials: R
authors.lastname: Sen
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D012234:Rho Factor / Q000235:genetics / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins; D012234:Rho Factor; D012321:DNA-Directed RNA Polymerases