No abstract

The Mesh1 class of hydrolases found in bacteria, metazoans and humans was discovered as able to cleave an intact pyrophosphate residue esterified on the 3 hydroxyl of (p)ppGpp in a Mn 2+ dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from Drosophila melanogaster and human MESH1 also extends to the (p)ppApp purine analogs. More importantly, we developed real time enzymatic assays, coupling ppNpp hydrolysis to NADH oxidation and pppNpp hydrolysis to NADP + reduction, which facilitate estimation of kinetic constants. Furthermore, by using this assay technique we confirmed TLC observations and also revealed that purified small alarmone hydrolase (SAH Mex) from Methylobacterium extorquens displays a strong hydrolase activity toward (p)ppApp but only negligible activity toward (p)ppGpp. In contrast, the substrate specificity of the hydrolase present in catalytically active N-terminal domain of the RSH protein from Streptococcus equisimilis (Rel Seq) includes (p)ppGpp but not (p)ppApp. It is noteworthy that the RSH protein from M. extorquens (RSH Mex) has been recently shown to synthesize both (p)ppApp and (p)ppGpp.

Background:No structure exists of the bacteriophage T4 activator MotA with DNA. Results: Using FeBABE, physical models, and ICM Molsoft, we determined how MotA interacts with DNA within the transcription complex. Conclusion:The unusual "double-wing" motif in MotA CTD sits within the DNA major groove. Significance: FeBABE analyses together with structures can be used to determine protein-DNA architecture within multiprotein complexes.