title: RapA, Escherichia coli RNA polymerase SWI/SNF subunit-dependent polyadenylation of RNA.
abstract: In this work, we describe RapA-dependent polyadenylation of model RNA substrates and endogenous, RNA polymerase-associated nucleic acid fragments. We demonstrate that the Escherichia coli RNA polymerase obtained through the classic purification procedure carries endogenous RNA oligonucleotides, which, in the presence of ATP, are polyriboadenylated in a RapA-dependent manner by an accessory poly(rA) polymerase. RNA polymerase isolated from poly(A) polymerase- (PAP-) and polynucleotide phosphorylase- (PNP-) deficient E. coli strain lacks accessory (rA)(n)-synthetic activity. Experiments with reconstituted RNA polymerase-PAP and RNA polymerase-PNP mixtures suggest that RapA enables the polyadenylation by PAP of RNA polymerase-associated RNA. Mutations disrupting RapA's ATP-hydrolytic function disrupt RapA-dependent polyadenylation, and the rapA(-)E. coli strain displays a measurable reduction in RNA polyadenylation. RapA-dependent polyadenylation can also be modulated by mutations in the section of RapA's SWI/SNF domain linked to interaction with single-stranded nucleic acid. We have developed enzymatic assays in which model, synthetic RNAs are polyriboadenylated in a RapA-dependent manner. Taken together, our results are consistent with RapA acting as an RNA polymerase-associated, ATP-dependent RNA translocase. Our work further links RapA to RNA remodeling and provides new mechanistic insights into the functional interaction between RNA polymerase and RapA.
authors.affiliation: Department of Chemistry and Biochemistry, Lamar University, Beaumont, Texas 77710, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Richmond
authors.forename: Raghavendra R
authors.initials: RR
authors.lastname: Pasupula
authors.forename: Seema G
authors.initials: SG
authors.lastname: Kansara
authors.forename: Joshua P
authors.initials: JP
authors.lastname: Autery
authors.forename: Brent M
authors.initials: BM
authors.lastname: Monk
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Sukhodolets
mesh_terms: D000595:Amino Acid Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D026723:Polyadenylation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; C074434:RapA protein, E coli; D012313:RNA; D012321:DNA-Directed RNA Polymerases