Indentification of conformational changes of RF1

The ribosome is one of the largest cellular machines known to date and its task is to translate the genetic information from messenger RNAs (mRNAs) into proteins with the help of amino acylated transfer RNAs (tRNAs). This process can be dissected into four main steps: initiation, elongation, termination and recycling. The catalytic heart of the ribosome, the peptidyl transferase center, is in the large ribosomal subunit (50S subunit) and composed only of ribosomal RNA. The small ribosomal subunit (30S subunit) is home of the decoding center. While the tRNA is passing through the ribosome, three major binding sites have been identified: the A-, P- and E-sites. The entry of a stop codon into the A site initiates the termination phase of protein synthesis. The stop codon in the A site is recognized by class I release factors (RF1 and RF2 in E. coli) that bind to the ribosome and catalyze the release of the newly synthesized protein attached to the peptidyl-tRNA. Because premature translation termination at sense codons is costly to the cell, it is essential that RFs recognize the stop codons with high fidelity. Translation termination is a dynamic process that remains incompletely understood from static structures of the ribosomeRF complexes. The goal of this proposal is to determine the mechanism used by release factors to achieve high fidelity stop codon recognition. The proposed hypothesis is that conformational changes in RF1/RF2 play a critical role in discriminating between stop and sense codons. Recent findings strongly support the idea that conformational changes in the ribosome regulate specific steps in protein synthesis. Thus, the principal mechanism used to achieve high termination fidelity may be by linking stop codon recognition to conformational changes in RF1/RF2 that stabilize the interaction with the ribosome and trigger peptide release. It is not known in what conformation RF1 and RF2 initially bind to the ribosome. By proposing new fluorescence resonance energy transfer (FRET) experiments, the changes in RF1/RF2 structure following binding to the ribosome will be determined. After stop codon recognition and peptide release, release factor 3 (RF3) is required to promote the dissociation of RF1 and RF2 from the ribosome. We here present a new fluorescence-based assay that will enable us to monitor this event and should visualize the state RF1/RF2 adopts when released from the ribosome. These studies have medical significance because ribosomes are a well-known target of antibiotics. Furthermore, the here proposed studies would present novel insights in translation termination that cannot be provided by crystal structures and computational studies. Ribosomal translation is a dynamic process and kinetic data is required to fully understand it.

The aim of my project was to identify the conditions, properties, and chronology of a conformational change in RF1. High-resolution crystal structures showed that RF1 is in an open conformation when bound to the ribosome but could adopt a closed conformation when not bound to the ribosome. It is not clear whether only the open form of RF1 binds to the ribosome or the closed form of RF1 binds to the ribosome and then undergoes a conformational change into the open state. I used transition metal ion FRET experiments (tmFRET) to precisely monitor the conformation of RF1 in the absence and in the presence of the ribosome. This is a cutting-edge technology that has never been used in such a setting. The results indicate that RF1 undergoes a large conformational change from a closed to an open form upon binding to the ribosome. We propose that high termination fidelity is achieved by linking stop codon recognition by RF1 to a change in conformation from closed to open state, which increases the binding affinity of RF1 to the ribosome and induce peptide release. The results of this project are not only contributing to the knowledge of this research field but has also potential to impact future antibiotic development.