In vitro Mutagenesis of E. coli Ribosomal Proteins

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In vitro mutagenesis has been used extensively in probing the structure and function of ribosomal RNA from the bacterium E. coli Only recently has this powerful technique been applied toward the study of ribosomal proteins. The objective of this planning grant is the modification of a two plasmid, inducible expression system based upon T7 RNA polymerase that was adapted for an investigation of E. coli ribosomal protein L2. This system has made it possible to quickly identify dominant-negative mutations by replica plating transformants at two different temperatures. The refinements being carried out in this study is the coupled expression of a mutant ribosomal protein gene and an rRNA operon that codes for an antibiotic resistance. Upon induction of the T7 RNA polymerase expression system, the vast majority of de novo ribosomes will include of an antibiotic resistance marker as well as the targeted mutant ribosomal protein during subunit assembly. Wild type ribosomes assembled prior to induction will be functionally eliminated by the inclusion of the appropriate antibiotic during invitro activity assays. This will allow for mutant ribosomal subunits assembled in vivo to be characterized in vitro for their activity/inactivity in the partial reactions of protein synthesis, eliminating the need to physically separate the two ribosome populations. %%% The planning grant is testing the applicability of a new technique to the study of ribosomal RNA-protein interactions. Completion of the work will help us better understand the mechanism by which proteins are synthesized in all cells.