Environmental control of the in vivo oligomerization of nucleoid protein H-NS.

The nucleoid-associated transcriptional repressor H-NS forms both dimers and tetramers in vivo. Two types of two-hybrid systems, one capable of detecting protein dimerization and the other protein tetramerization, have been used to determine whether environmental changes could affect the oligomerization capacity of this protein in the cell. Increasing the temperature from 37 degrees C to 48 degrees C and changing the pH between 4.0 and 9.0 did not influence either dimerization or tetramerization, whereas lowering the temperature below 25 degrees C and increasing osmolarity were found to reduce the formation of H-NS tetramers, which are the active form of this protein, without affecting dimerization. These findings provide a rationale to explain the induction of H-NS expression during cold-shock, suggest a mechanism contributing to derepressing osmotic-shock genes transcriptionally regulated by H-NS and indicate that changes of the oligomerization properties of H-NS do not play a role in the H-NS and temperature-dependent control of virulence gene expression.

Mapping the active sites of bacterial translation initiation factor IF3.

IF3C is the C-terminal domain of Escherichia coli translation initiation factor 3 (IF3) and is responsible for all functions of this translation initiation factor but for its ribosomal recycling. To map the number and nature of the active sites of IF3 and to identify the essential Arg residue(s) chemically modified with 2,3-butanedione, the eight arginine residues of IF3C were substituted by Lys, His, Ser and Leu, generating 32 variants that were tested in vitro for all known IF3 activities. The IF3-30S subunit interaction was inhibited strongly by substitutions of Arg99, Arg112, Arg116, Arg147 and Arg168, the positive charges being important at positions 116 and 147. The 70S ribosome dissociation was affected by mutations of Arg112, Arg147 and, to a lesser extent, of Arg99 and Arg116. Pseudo-initiation complex dissociation was impaired by substitution of Arg99 and Arg112 (whose positive charges are important) and, to a lesser extent, of Arg116, Arg129, Arg133 and Arg147, while the dissociation of non-canonical 30S initiation complexes was preserved at wild-type levels in all 32 mutants. Stimulation of mRNA translation was reduced by mutations of Arg116, Arg129 and, to a lesser extent, of Arg99, Arg112 and Arg131 whereas inhibition of non-canonical mRNA translation was affected by substitutions of Arg99, Arg112, Arg168 and, to a lesser extent, Arg116, Arg129 and Arg131. Finally, repositioning the mRNA on the 30S subunit was affected weakly by mutations of Arg133, Arg131, Arg168, Arg147 and Arg129. Overall, the results define two active surfaces in IF3C, and indicate that the different functions of IF3 rely on different molecular mechanisms involving separate active sites.