Molecular genetics of translational accuracy

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The process of protein synthesis on the ribosome is among the oldest of biological mechanisms, having evolved probably in the RNA world before the first cells, The ribosome has evolved an elaborate structure, the essential parts conserved in all domains of life, Understanding the mechanism by which the ribosome faithfully translates mRNAs into proteins has become both more important and more tractable with the availability of multiple molecular structures of both subunits of bacterial ribosomes with various ligands, Our laboratory for many years has been interested in the problem of how mRNA sequences interact with the ribosome to cause programmed errors, We have mainly studied the mechanism of programmed translational frameshifting in which particular sites in mRNAs stimulate translational errors at rates several orders of magnitude greater than at random positions in mRNAs, Our work and that of others revealed that programmed frameshift sites manipulate the decoding center of the ribosome to exacerbate errors. More recently, we have begun to study the mechanism of missense errors, Our study has shown that errors vary widely by type of missense error (first, second or wobble position mispairing) and according to the tRNA isoacceptors involved in decoding the codon that is the site of the error. The major conclusion of our work is that wobble errors commonly occur, tllOugh they are not universal, and that other types of errors occur when the competing cognate tRNA is in insufficient supply to preclude decoding by the errant tRNA, We propose to further characterize the phenomenology of missense errors by creating a set of error-reporter constructs based on the E, coli lacZ and the Photinus (firefly) luciferase genes. Venki Ramakrishnan has proposed that a major part of the accuracy mechanism requiring the disruption ofa protein.protein interaction between ribosomal proteins S4 (rpS4) and rpS5, which allows the ribosome to shift into a "closed" conformation that traps the aminoacyl-tRNA in the A site and allows exit of EF-l A.GDP, The model is based on the existence of mutants targeting the interface that are proposed to destabilize it and cause increased errors, Our preliminary data are inconsistent with this hypothesis, We propose to further test the hypothesis by identifying novel mutations that alter accuracy, either in the rRNA or ribosomal proteins, The process of protein synthesis on the ribosome is among the oldest of biological mechanisms, having evolved probably in the RNA world before the first cells, The ribosome has evolved an elaborate structure, the essential parts conserved in all domains of life, Understanding the mechanism by which the ribosome faithfully translates mRNAs into proteins has become both more important and more tractable with the availability of multiple molecular structures of both subunits of bacterial ribosomes with various ligands, Our laboratory for many years has been interested in the problem of how mRNA sequences interact with the ribosome to cause programmed errors, We have mainly studied the mechanism of programmed translational frameshifting in which particular sites in mRNAs stimulate translational errors at rates several orders of magnitude greater than at random positions in mRNAs, Our work and that of others revealed that programmed frameshift sites manipulate the decoding center of the ribosome to exacerbate errors. More recently, we have begun to study the mechanism of missense errors, Our study has shown that errors vary widely by type of missense error (first, second or wobble position mispairing) and according to the tRNA isoacceptors involved in decoding the codon that is the site of the error. The conclusion of our work is that wobble errors commonly occur, tllOugh they are not universal, and that other types of errors occur when the competing