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PMID:10801345

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Citation

Schröder, O and Wagner, R (2000) The bacterial DNA-binding protein H-NS represses ribosomal RNA transcription by trapping RNA polymerase in the initiation complex. J. Mol. Biol. 298:737-48

Abstract

The interaction of the bacterial regulatory protein H-NS with RNA polymerase and the ribosomal RNA P1 promoter was analyzed to better understand the mechanism of H-NS-dependent transcriptional repression. We could show that initial binding of RNA polymerase to the promoter was not inhibited by the simultaneous interaction of H-NS, although H-NS binding sites extend into the core promoter region. Binding of sigma(70)-saturated RNA polymerase and H-NS to the promoter DNA occurs cooperatively and results in a stable complex of slower gel electrophoretic mobility as compared to complexes formed with the single proteins. The presence of the upstream curved H-NS binding site contributes strongly to the cooperative RNA polymerase-promoter interaction. By KMnO(4) modification of single-stranded template nucleotides we could show that open complex formation at the rrnB P1 promoter was not inhibited by H-NS binding. An increased KMnO(4) reactivity of several positions within the open complex rather supports the view that open complex formation is stimulated in presence of H-NS. Moreover, subtle changes in the modification pattern indicate that the open complex formed in the presence of H-NS are structurally distinct from the H-NS-free complex. In vitro transcriptional analysis of the abortive and productive yields revealed that the formation of transcription products longer than three nucleotides is dramatically reduced in the presence of H-NS, while the amount of shorter abortive products remained unaffected. Together the results demonstrate that H-NS inhibits transcription at the rrnB P1 promoter not by interfering with initial RNA polymerase binding but by blocking chain elongation steps subsequent to the first (two) phosphodiester bond formations. The mechanism of H-NS dependent repression at rRNA promoters can thus be explained as a trap which inhibits substrate NTP incorporation beyond template position +3 into the initial transcribing complex.

Links

PubMed Online version:10.1006/jmbi.2000.3708

Keywords

Allosteric Site; Bacterial Proteins/metabolism; Carrier Proteins/metabolism; DNA Footprinting; DNA, Bacterial/chemistry; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; DNA-Binding Proteins/metabolism; DNA-Directed RNA Polymerases/antagonists & inhibitors; DNA-Directed RNA Polymerases/metabolism; Escherichia coli/genetics; Escherichia coli Proteins; Gene Expression Regulation, Bacterial/genetics; Holoenzymes/metabolism; Integration Host Factors; Nucleic Acid Conformation; Potassium Permanganate/metabolism; Promoter Regions, Genetic/genetics; Protein Binding; RNA, Bacterial/biosynthesis; RNA, Bacterial/genetics; RNA, Ribosomal/biosynthesis; RNA, Ribosomal/genetics; Repressor Proteins/metabolism; Sequence Deletion/genetics; Sigma Factor/metabolism; Transcription, Genetic/genetics; rRNA Operon/genetics

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:HNS

GO:0045943: positive regulation of transcription from RNA polymerase I promoter

ECO:0000314:

P

See Fig 5

complete


See also

References

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