SPECIFIC AIMS:
Accuracy of translational decoding is influenced by the tRNA, mRNA and ribosomal proteins. I am going to investigate elements of each of these factors to help understand their impact on translation.

Specific Aim I:
Ribosomal proteins S3, S4 and S5 create an entrance channel in the ribosome that the mRNA must pass through to reach the decoding center. I hypothesize that charged residues from each of these proteins contribute to accuracy by contacting the phosphate backbone of the mRNA. This contribution would result in slower tRNA association times and therefore increase ribosomal discrimination and accuracy. I will test the effect of each residue by monitoring frameshifting and readthrough in E. coli.

Specific Aim II:
The P site codons for both the Ty1 +1 frameshift site and the Ty3 +1 frameshift site are decoded by tRNAs that result in non-canonical wobble pairing and disruption of the structure of the P site. This disruption potentially causes the mRNA in the A site to displace a base and move into the +1 frame. I hypothesize that non-canonical pairing at the first and second bases in the P site could also cause this disruption in the A site. I will test the effect by forcing non-canonical pairing and monitoring frameshift levels in S. cerevisiae.

Specific Aim III:
I will attempt to clearly define whether or not S. cerevisiae uses slippage during frameshifting. For each site at which frameshifting can be explained by slippage, it could also be explained by out of frame binding. There is little evidence that slippage occurs in yeast. The only data come from the CUX AGG C family of codons, which show a correlation between ability of the tRNA to slip, and the ability to cause +1 frameshifting. I hypothesize that this correlation could also be explained by out of frame binding. I will use the CUX AGG C family of codons to test the hypothesis.