New insights into the des pathway: DesR activation mechanism and dissection of the des promoter.

NAJLE SR; CYBULSKI LE; CRAIG PO; MENDOZA D

Rosario, Santa Fe, Argentina

Congreso; XLII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

The Des pathway of Bacillus subtilis regulates the synthesis of the ∆5-fatty acid desaturase, encoded by the des gene. Upon a decrease in the membrane lipid fluidity, the histidine kinase DesK phosphorylates DesR. Phosphorylated DesR binds to the des promoter, recruiting the RNA polymerase. The molecular mechanism by which DesR is activated by phosphorylation, as well as the dynamics of its interaction with DNA, remain unknown. To unveil the mechanism by which phosphorylation activates DesR, we constructed a truncated DesR protein, consisting only on its DNA-binding C-terminal domain (DesRC). Electromobility shift assays have shown that DesRC is able to bind to the des promoter regardless of phosphorylation, but incapable of activating des transcription. These results show that DesR activation implies the remotion of inhibitory effects of Nterminal domain over the DNA-binding capacity of the C-terminal domain. In order to comprehend the dynamics of the interaction of DesR-P with Pdes, the two sites, RA and RB, where dissociated through mutagenesis. Mutant versions of Pdes promoter, with different deletions of RB site, have been constructed. EMSA as well as in vivo transcriptional fusion experiments indicate that the RA site, in face with -35 and -10 elements, is enough to allow des transcription. A possible explanation for the existence of the low affinity RB site is proposed.