The genetics of ribosomal reading frame maintenance

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DESCRIPTION (provided by applicant): Translation of mRNA sequences by the ribosome is normally a faithful process, yet mistakes occur. Sequences in mRNAs have evolved that modulate the accuracy of translation to increase the frequency of translational frameshifting errors. Such sites are termed programmed translational frameshift sites. The yeast Ty elements, a class of retrotransposons, express the enzymes needed for retrotransposition using a programmed+1 frameshifting mechanism. We find evidence that frameshifting occurs because the mRNA disrupts the function of the ribosomal accuracy center by inserting peptidyl-tRNAs into the ribosomal P site that Make a non-canonical interaction with the mRNA. The unusual codon*anticodon complex formed disrupts the ability of the ribosome to accurately recognize in-frame cognate tRNAs in the A site. Also, a sequence derived from the Ty3 retrotransposon appears to directly interact with a structure in the rRNA necessary for accurate decoding in the A site. This sequence, termed the Ty3 stimulator, appears to base pair with part of the loop of Helix 18. A nucleotide in that base paired region, G530, directly contacts both mRNA and tRNA nucleotides in the A site codon*anticodon complex to assure that they are correctly paired. By sequestering that nucleotide in an rnRNA*rRNA complex, the Ty3 stimulator could reduce overall discrimination in the A site. Recently, detailed molecular structures of the 30S, 50S and 70S ribosomes were solved. These structures provide an unprecedented level of detail about the structure of the ribosome, and can be used to predict the mechanisms used during translation, including the error correction mechanisms. The structures clearly show how cognate, in-frame tRNAs are recognized, but gives little direct information about how the ribosome avoids errors leading to changes in translational frame. Arguably, maintenance of frame is the most critical role for the ribosome, since frame errors almost always result in production of inactive protein products while missense errors rarely do. To address how the ribosome maintains reading frame we have been using the tools provided by the programmed translational frameshift sites. We will continue that work attempting to determine how error-correcting elements of the ribosome function in this process. Recently, we found that mRNA sequences that stimulate frame errors also induce errors during translational initiation. This suggests that the accuracy of these processes has shared aspects. We will use the twin tools afforded by frameshifting and translation initiation to dissect the ribosomal functions insuring accuracy.