Antimicrobials, Multiple Drug Resistance & Antibiotics Resistance

Protein synthesis has four steps, initiation, elongation of peptide chain, termination and the recycling of the spent ribosomes, mRNA and tRNA. Th e last step was discovered by our laboratories and catalyzed by a unique protein called ribosome recycling factor (RRF). Th e recycling step consists of three reactions, release of mRNA, tRNA and splitting of ribosomes. With the use of fl uorescent labeled mRNA similar to the natural mRNA and labeled tRNA, we demonstrate in vitro that the major action of RRF is to release of mRNA and not the splitting of ribosomes into subunits. Th is corrects the general misconception that the major action of RRF is to split the ribosomes into subunits. Th e order of events with the naturally occurring substrate of RRF, the chain of events is release of tRNA, mRNA followed by the splitting of ribosomes. Th e release of mRNA is not dependent on the splitting of ribosomes. Th e in vitro results are supported by in vivo experiments where we used the translational coupling followed by the reporter gene expression (beta galactosidase expression). Using the basic reaction of RRF, release of ribosomes from mRNA, we developed a new screening system for the inhibitor of RRF. In this system, the inhibition of RRF reaction, makes ribosome stay on the mRNA at the termination codon, but start translating downstream which is linked to GFP. We show that this screening method functions by the use of known specifi c inhibitor of RRF, low concentration of fusidic acid. Th e assay method is simple and can be performed in 96 hole plate overnight. We look forward to finding collaborators who has access to the collection of possible inhibitors.