The DNA Binding Protein H-NS Binds to and Alters the Stability of RNA in vitro and in vivo

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 fact...

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Bibliographic Details
Published in:Journal of molecular biology 2004-06, Vol.339 (3), p.505-514
Main Authors: Brescia, Cristin C, Kaw, Meenakshi K, Sledjeski, Darren D
Format: Article
Language:English
Subjects:
Bacterial Proteins - metabolism
Base Sequence
DNA Primers
DNA-Binding Proteins - metabolism
Electrophoretic Mobility Shift Assay
Escherichia coli - genetics
Escherichia coli - metabolism
non-coding RNA
Nucleic Acid Conformation
Protein Binding
Reverse Transcriptase Polymerase Chain Reaction
RNA chaperone
RNA, Bacterial - chemistry
RNA, Bacterial - isolation & purification
RpoS
StpA
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Summary: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 μM, whereas binding to rpoS mRNA or DsrA RNA was approximately 3 μM. 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.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2004.03.067