title: The DNA binding protein H-NS binds to and alters the stability of RNA in vitro and in vivo.
abstract: H-NS is an abundant prokaryotic transcription factor that preferentially binds to intrinsically bent DNA. Although H-NS has been shown to reduce the transcription of over 100 genes, evidence suggests that H-NS can also affect the translation of some genes. One such gene, rpoS, specifies a sigma factor, RpoS. The ability of H-NS to bind to the rpoS mRNA and the non-coding RNA regulator, DsrA, was tested. Electrophoretic mobility-shift assays yielded an apparent binding affinity of H-NS binding to curved DNA of approximately 1 microM, whereas binding to rpoS mRNA or DsrA RNA was approximately 3 microM. This RNA binding was not prevented by an excess of competitor yeast RNA, suggesting that H-NS specifically bound these RNAs. Footprint analysis with a single strand-specific ribonuclease was used to identify the H-NS binding site(s) on DsrA and rpoS mRNA. Surprisingly, H-NS appeared to enhance the cleavage of DsrA and rpoS mRNA. The enhanced cleavage was at sites that were predicted to be single-stranded and did not result from contaminating nucleases in the H-NS protein preparation or non-specific effects of the nuclease. Quantitative RT-PCR of RNA isolated from wild-type and hns- strains revealed that H-NS also affects the stability of DsrA in vivo. Thus H-NS appears to modulate RNA stability in vivo and in vitro.
authors.affiliation: Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA.
authors.forename: Cristin C
authors.initials: CC
authors.lastname: Brescia
authors.forename: Meenakshi K
authors.initials: MK
authors.lastname: Kaw
authors.forename: Darren D
authors.initials: DD
authors.lastname: Sledjeski
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012329:RNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D012329:RNA, Bacterial