title: Functional analysis of the post-transcriptional regulator RsmA reveals a novel RNA-binding site.
abstract: The RsmA family of RNA-binding proteins are global post-transcriptional regulators that mediate extensive changes in gene expression in bacteria. They bind to, and affect the translation rate of target mRNAs, a function that is further modulated by one or more, small, untranslated competitive regulatory RNAs. To gain new insights into the nature of this protein/RNA interaction, we used X-ray crystallography to solve the structure of the Yersinia enterocolitica RsmA homologue. RsmA consists of a dimeric beta barrel from which two alpha helices are projected. From structure-based alignments of the RsmA protein family from diverse bacteria, we identified key amino acid residues likely to be involved in RNA-binding. Site-specific mutagenesis revealed that arginine at position 44, located at the N terminus of the alpha helix is essential for biological activity in vivo and RNA-binding in vitro. Mutation of this site affects swarming motility, exoenzyme and secondary metabolite production in the human pathogen Pseudomonas aeruginosa, carbon metabolism in Escherichia coli, and hydrogen cyanide production in the plant beneficial strain Pseudomonas fluorescens CHA0. R44A mutants are also unable to interact with the small untranslated RNA, RsmZ. Thus, although possessing a motif similar to the KH domain of some eukaryotic RNA-binding proteins, RsmA differs substantially and incorporates a novel class of RNA-binding site.
authors.affiliation: Institute of Infection, Immunity & Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK. stephan.heeb@nottingham.ac.uk
authors.forename: Stephan
authors.initials: S
authors.lastname: Heeb
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Kuehne
authors.forename: Mark
authors.initials: M
authors.lastname: Bycroft
authors.forename: Sorana
authors.initials: S
authors.lastname: Crivii
authors.forename: Mark D
authors.initials: MD
authors.lastname: Allen
authors.forename: Dieter
authors.initials: D
authors.lastname: Haas
authors.forename: Miguel
authors.initials: M
authors.lastname: Cámara
authors.forename: Paul
authors.initials: P
authors.lastname: Williams
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010641:Phenotype; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015008:Yersinia enterocolitica / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D012313:RNA