RiboFLEX

Ribosomes are large ribonucleoprotein complexes responsible for decoding and translating genetic information into protein in all living organisms. As part of the process of translation, nascent polypeptides must first transit through a long exit tunnel spanning the large subunit of the ribosome in order to reach the intracellular milieu. Initially viewed as a passive conduit for proteins, this cavity has emerged in recent years as a functional microenvironment in which signals encoded by specific nascent peptides are relayed back to the ribosomal active site to modulate its activity. In some instances, nascent chain feedback brings protein synthesis to a complete halt, causing a ribosome nascent chain complex (RNC) to become stalled on the mRNA. This process, which is referred to as nascent chain-mediated translational arrest, invariably depends upon the sequence of the peptide being synthesized, but may additionally require a small molecule such as a drug or an amino acid to act as a co-inducer. It has been reported in both prokaryotes and eukaryotes, where stalled RNCs regulate the expression of downstream open reading frames by facilitating or impeding their accessibility by the translational machinery. This proposal deals with two of the better-characterized examples of nascent chain-mediated arrest: (i) drug-dependent arrest by the Erm family of peptides in response to inducing concentrations of macrolide antibiotics, and (ii) ribosome stalling on stretches of mRNA encoding multiple consecutive prolines and their subsequent rescue by the translation factor EF-P. Our current knowledge of processes involving the nascent polypeptide comes from two main sources: biochemical studies and medium-resolution cryo-electron microscopy (cryo-EM) models of RNCs. These have shown that arrest peptides within the exit tunnel are structured, form specific interactions with the ribosome and ultimately affect the geometry of the ribosomal active site to either block peptide bond formation or peptidyl-tRNA hydrolysis by release factors. However, a number of key questions remain concerning the manner in which these various events give rise to the arrest process. Moreover, a full mechanistic picture of this fundamental aspect of ribosome biology is not only essential for understanding an underappreciated facet of translational control, but could also lead to the design of improved antibiotics that target the ribosome by mimicking the physicochemical properties of inhibitory nascent peptides and their co-inducers. Atomic resolution structures of stalled RNCs featuring various peptidyl-tRNAs are therefore needed to gain a deeper insight into the process of nascent chain-mediated arrest. The goal of this project is to obtain high-resolution structures of RNCs by X-ray crystallography and to use the information derived from these structures to guide structure-function studies that address how the ribosome senses and responds to different nascent chains. Up until now, RNCs bearing a nascent peptide of defined sequence have typically been prepared using an in vitro translation system derived from a cell extract or reconstituted from purified components of the translational machinery. While this method allows large quantities of RNCs to be prepared, it also presents a number of drawbacks for high resolution X-ray crystallography, including some degree of sample heterogeneity. In order to overcome these limitations, we propose to attach activated peptides prepared synthetically to tRNAs overexpressed in bacteria using small RNA enzymes known as flexizymes. By pairing a suitable leaving group on the peptide substrate with a compatible flexizyme, virtually any amino acid or short peptide can be attached onto a tRNA with an intact 3’ end. Peptidyl-tRNAs obtained in this manner will be added to the ribosome in trans to obtain RNCs for structural studies, thereby sidestepping some of the problems associated with peptide synthesis in cis by the ribosome. .