Dataset Information

Multiple roles of the RNA polymerase {beta}' SW2 region in transcription initiation, promoter escape, and RNA elongation.

ABSTRACT: Interactions of RNA polymerase (RNAP) with nucleic acids must be tightly controlled to ensure precise and processive RNA synthesis. The RNAP ?'-subunit Switch-2 (SW2) region is part of a protein network that connects the clamp domain with the RNAP body and mediates opening and closing of the active center cleft. SW2 interacts with the template DNA near the RNAP active center and is a target for antibiotics that block DNA melting during initiation. Here, we show that substitutions of a conserved Arg339 residue in the Escherichia coli RNAP SW2 confer diverse effects on transcription that include defects in DNA melting in promoter complexes, decreased stability of RNAP/promoter complexes, increased apparent K(M) for initiating nucleotide substrates (2- to 13-fold for different substitutions), decreased efficiency of promoter escape, and decreased stability of elongation complexes. We propose that interactions of Arg339 with DNA directly stabilize transcription complexes to promote stable closure of the clamp domain around nucleic acids. During initiation, SW2 may cooperate with the ?(3.2) region to stabilize the template DNA strand in the RNAP active site. Together, our data suggest that SW2 may serve as a key regulatory element that affects transcription initiation and RNAP processivity through controlling RNAP/DNA template interactions.

SUBMITTER: Pupov D

PROVIDER: S-EPMC2943606 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Multiple roles of the RNA polymerase {beta}' SW2 region in transcription initiation, promoter escape, and RNA elongation.

Pupov Danil D Miropolskaya Nataliya N Sevostyanova Anastasiya A Bass Irina I Artsimovitch Irina I Kulbachinskiy Andrey A

Nucleic acids research 20100510 17

Interactions of RNA polymerase (RNAP) with nucleic acids must be tightly controlled to ensure precise and processive RNA synthesis. The RNAP β'-subunit Switch-2 (SW2) region is part of a protein network that connects the clamp domain with the RNAP body and mediates opening and closing of the active center cleft. SW2 interacts with the template DNA near the RNAP active center and is a target for antibiotics that block DNA melting during initiation. Here, we show that substitutions of a conserved ...[more]

PMID: 20457751

Similar Datasets

Project description:Influenza viruses encode a viral RNA-dependent RNA polymerase (FluPol), which is responsible for transcribing and replicating the negative-sense viral RNA (vRNA) genome. FluPol transcribes vRNA using a host-capped mRNA primer and replicates it by synthesizing a positive-sense cRNA intermediate, which is copied back into vRNA. To carry out these functions, FluPol interacts with vRNA and cRNA using conserved promoter elements at the 5' and 3' termini. Recent structural studies have identified a new surface binding site for the 3' vRNA and cRNA promoters on FluPol, referred to as the mode B site. However, the role of this binding site in FluPol function is unknown. In this study, we used a combination of cell-based and biochemical assays to show that the mode B site is important for both viral genome transcription and replication in influenza A virus. Furthermore, we show that the mode B site is not needed for initiating transcription in vitro but is required to synthesize a full-length product. This is consistent with a model in which the 3' terminus of the vRNA template binds in the mode B site during elongation. Our data provide the first functional insights into the role of the mode B site on FluPol, which advances our understanding of FluPol function and influenza virus replication.IMPORTANCE Influenza viruses are responsible for up to 650,000 deaths per year through seasonal epidemics, and pandemics have caused tens of millions of deaths in the past. Most current therapeutics suffer from widespread resistance, creating a need for new drug targets against influenza virus. The virus encodes an RNA-dependent RNA polymerase, which replicates and transcribes the vRNA genome. The polymerase interacts with vRNA and the complementary replicative intermediate cRNA using several specific binding sites; however, the functions associated with these binding sites remain unknown. Here, we functionally characterize a binding site for the 3' vRNA and cRNA promoters. Our data offer insight into the mechanism of viral genome transcription by the influenza virus polymerase and may be applicable to other related viruses.

Dataset Information

Multiple roles of the RNA polymerase {beta}' SW2 region in transcription initiation, promoter escape, and RNA elongation.

Publications

Multiple roles of the RNA polymerase {beta}' SW2 region in transcription initiation, promoter escape, and RNA elongation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets