Functional Studies of Transfer RNA Interactions in the Ribosome
During translation, tRNAs must move rapidly to their adjacent sites on the ribosome, while maintaining precise pairing with mRNA. In this work, the interactions of tRNA with the ribosome were examined in order to gain insights into the mechanisms by which the ribosome maintains the correct reading frame, and those interactions which contribute to rapid translocation.
Mutant tRNAs with expanded anticodon loops have been isolated, which are capable of decoding a four-nucleotide sequence of mRNA. Using an in vitro assay with purified components, the position of mRNA was mapped with respect to the ribosome to determine the molecular basis by which these expanded-anticodon loop tRNAs recognize and position a quadruplet frameshift sequence. The results of these experiments suggest a new model for frameshift suppression by tRNAs with expanded anticodon loops, and provide insights into how the ribosome normally maintains the reading frame.
Movement of tRNAs through the ribosome is thought to take place in a stepwise manner, with hybrid intermediates. This work demonstrates that movement of tRNA into one of these hybrid-tRNA binding sites, the P/E site, destabilizes the mRNA. In order to determine how these hybrid states relate to kinetically defined events of translocation, substitutions in tRNA or rRNA were examined which inhibited movement into either the P/E site or the A/P site. These data suggest that movement of tRNA into the P/E and A/P sites are separable events and allow tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:ribosome trna gtpase ef g hybrid states translocation frameshift suppression
Date of Publication:01/01/2008