Accuracy of protein synthesis and its tuning by mRNA modifications

  • Date:
  • Location: A1:111a, BMC, Husargatan 3, Uppsala
  • Doctoral student: Indrisiunaite, Gabriele
  • About the dissertation
  • Organiser: Molekylärbiologi
  • Contact person: Indrisiunaite, Gabriele
  • Disputation

This thesis addresses two questions regarding the accuracy of protein synthesis. How do bacterial and eukaryotic release factors ensure accurate termination? How do mRNA modifications affect the accuracy of bacterial protein synthesis?

The ribosome is a large macromolecular complex that synthesizes all proteins in the cell in all kingdoms of life. Proteins perform many vital functions, ranging from catalysis of biochemical reactions to muscle movement. It is essential for cells and organisms that proteins are synthesized rapidly and accurately.

This thesis addresses two questions regarding the accuracy of protein synthesis. How do bacterial and eukaryotic release factors ensure accurate termination? How do mRNA modifications affect the accuracy of bacterial protein synthesis?

Bacterial release factors 1 (RF1) and 2 (RF2) are proteins that recognize the stop codons of mRNA and catalyze the release of a synthesized protein chain from the ribosome. It has been proposed that RFs ensure accurate termination by binding to the ribosome in an inactive, compact conformation and acquire a catalytically active, extended conformation only after recognizing a correct stop codon. However, the native compact conformation was too short-lived to be captured by conventional structural methods. We have developed a fast-kinetics approach for determining when the RFs are in a compact conformation on the ribosome and then used time-resolved cryogenic electron microscopy to capture the compact conformations of native RF1 and RF2 bound to a stop codon. We have also measured the effect of eukaryotic release factor 3 (eRF3) on the rate and accuracy of peptide release by eukaryotic release factor 1 (eRF1) in a yeast (Saccharomyces cerevisiaein vitro translation system.

Modifications of mRNA nucleotides are post-transcriptional regulators of gene expression, but little is known about their role in protein synthesis. We have studied the effect on accuracy of protein synthesis by two of these modifications: 2’-O-methylation and N6-methylation of adenosine. 2’-O-methylation greatly reduced the maximal rate (kcat) and efficiency (kcat/Km) of cognate (correct) codon reading by decreasing the initial GTPase activity in elongation factor Tu and enhancing proofreading losses of cognate aminoacyl-tRNAs. Remarkably, N6-methylation reduced the efficiency of codon reading by cognate aminoacyl-tRNAs and release factors, leaving the efficiency of the corresponding non-cognate reactions much less affected.