Translational accuracy: Ribosomal protein-protein interactions and stop codon recognition by variant-code release factors

Maintenance of accuracy during translation is important for optimal fitness of the cell. The protein synthetic machinery of the cell, including the ribosome, ensures fidelity during decoding. Structural components of the ribosome play an important role in maintaining fidelity. Decoding is also affected by trans- acting factors such as tRNAs, elongation factors and peptide release factors. The concentration of substrates probing the A site also influences the rate of decoding. Slow decoding at the A site not only increases misreading but also promotes the occurrence of alternate recoding events like frameshifting. To understand how different factors affect translational accuracy, I have studied the roles of ribosomal protein- protein interactions near the decoding center and that of variant code release factors in decoding. The binding of a cognate tRNA at the A site induces a closed conformation of the ribosomal small subunit. The closure of the small subunit was suggested to disrupt the interface between two accuracy modulating proteins rpS4 and rpS5. Mutations in either protein confer an error- prone or ram phenotype. Mutations in these proteins have been proposed to promote disruption of the interface, facilitating domain closure even in the absence of the cognate tRNA leading to error- prone phenotype. I used a yeast two hybrid system to look at the effects of the ram mutations on the interactions between rpS4 and rpS5. My results confirm the predicted interactions between rpS4 and rpS5. But the fact that some of the error- prone mutations do not affect the protein- protein interaction at the interface contradicts the proposed model. I have also studied the efficiency of stop codon recognition by variant- code ciliate chimeric release factors. Stop codon recognition at a shifty stop frameshift site influences frameshifting efficiency. Using a yeast genetic system, I showed that the chimeric yeast release factors with domain 1 replaced by that of Euplotes and Tetrahymena, impaired recognition of all three stop codons. My results support our hypothesis that poor recognition of UAA and UAG stop codons at the shifty stop frameshift site AAA-UAA/ UAG- A promotes frequent frameshifting in Euplotes spp