Analysis of RovA, a transcriptional regulator of Yersinia pseudotuberculosis virulence that acts through antirepression and direct transcriptional activation.
The transcription factor RovA of Yersinia pseudotuberculosis and analogous proteins in other Enterobacteriaceae activate the expression of virulence genes that play a crucial role in stress adaptation and pathogenesis. In this study, we demonstrate that the RovA protein forms dimers independent of DNA binding, stimulates RNA polymerase, most likely via its C-terminal domain, and counteracts transcriptional repression by the histone-like protein H-NS. As the molecular function of the RovA family is largely uncharacterized, random mutagenesis and terminal deletions were used to identify functionally important domains. Our analysis showed that a winged-helix motif in the center of the molecule is essential and directly involved in DNA binding. Terminal deletions and amino acid changes within both termini also abrogate RovA activation and DNA-binding functions, most likely due to their implication in dimer formation. Finally, we show that the last four amino acids of RovA are crucial for activation of gene transcription. Successive deletions of these residues result in a continuous loss of RovA activity. Their removal reduced the capacity of RovA to activate RNA polymerase and abolished transcription of RovA-activated promoters in the presence of H-NS, although dimerization and DNA binding functions were retained. Our structural model implies that the final amino acids of RovA play a role in protein-protein interactions, adjusting RovA activity.
Tran
Hien J
HJ
Junior Research Group 6, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany.
Heroven
Ann Kathrin
AK
Winkler
Lars
L
Spreter
Thomas
T
Beatrix
Birgitta
B
Dersch
Petra
P
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology*; D014774:Virulence / Q000502:physiology; D015011:Yersinia pseudotuberculosis / Q000235:genetics / Q000472:pathogenicity*
D001426:Bacterial Proteins; D017931:DNA Primers; C405626:RovA protein, Yersinia; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases