Analysis of RovA, a Transcriptional Regulator ofYersinia pseudotuberculosisVirulence That Acts through Antirepression and Direct Transcriptional Activation
Journal of Biological Chemistry
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. FIGURE 6. In vitro run-off transcription from the inv promoter in the presence of the RovA wt protein, RovA L140 * , and/or H-NS. Plasmid pHT102 was incubated with E. coli RNAP and increasing concentrations of RovA, L140 * , and/or H-NS as detailed under "Experimental Procedures." pHT102 carries a 3Ј-terminally truncated inv gene and generates a transcript of 136 nucleotides. The inv and rovA transcripts and the RNA I transcripts (106 and 108 nt) encoded by the vector are indicated by arrows. A radiolabeled DNA marker fragment of 135 nt is indicated on the left. Numbers below show the quantification of the result (relative transcription), the invЈ transcripts were normalized to RNA I transcripts.