Structural studies of arrested ribosome nascent chain complexes prepared using a flexizyme-based approach – RiboFLEX

The major roadblock in determining the structures of RNCs by X-ray crystallography is obtaining large quantities of homogeneous RNCs for crystallization. We will overcome current limitations in sample preparation by attaching activated peptides prepared synthetically to tRNAs overexpressed in a bacterial host with the help of small RNA enzymes known as flexizymes (Goto et al. (2011) Nat Protocols 6, 779). By pairing a suitable leaving group on the peptide with a compatible flexizyme, most short peptides 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 produce RNCs, thus sidestepping some of the problems associated with peptide synthesis in cis by the ribosome. Using this approach, we expect to obtain structures of stalled RNCs that diffract to a resolution of 2.5–3.0 Å.
Our main objective will be accomplished in three stages:
1. Synthesize activated peptides and peptide mimics for charging onto tRNA with flexizymes
We will produce various peptides of interest with different leaving groups added to their C-terminus, so that virtually any peptidyl-tRNA conjugate can subsequently be prepared using the flexizyme technology.
2. Charge activated peptides and peptide mimics onto tRNA using the flexizyme technology
Non-hydrolyzable peptidyl-tRNAs featuring an amide bond instead of the usual ester bond will be prepared. This modification is needed to ensure that the peptidyl-tRNAs can withstand the crystallization process.
3. Prepare stalled RNCs and determine their structures by X-ray crystallography
We will elucidate the atomic structures of the bacterial ribosome in complex with various short arrest peptides and use the resulting models as a starting point for structure–function experiments.

The success of each of these stages will be ensured by the combined expertise in structural biology, peptide/peptidomimetic chemistry and biochemistry of the riboFLEX consortium.

To date, we have prepared most of the flexizymes, tRNAs and activated peptides needed for the project. Six of the 10 peptide–leaving group combinations tested could be successfully attached to the 3’-OH group of tRNAiMet with efficiencies ranging from 20% to 100%. However, work is underway to attach these peptides to the 3’-NH2 group of 3’NH2-tRNAiMet as flexizymes appear to have difficulties catalyzing the formation of amide bonds compared to ester bonds.

In parallel, we have begun to assess whether the various arrest peptides that we have chosen to focus on are capable of inducing translational arrest on the Thermus thermophilus 70S ribosomes that we use for crystallization. Once this is confirmed, structural characterization of various RNCs will reveal how short arrest peptides cause translational arrest in bacteria.

Interestingly, our crystallographic analyses of peptide-mediated translational inhibition took us into an unexpected direction, as we demonstrated that certain proline-rich antimicrobial peptides (PrAMPs) produced by the host immune response of insects and mammals block translation by binding to the exit tunnel of the bacterial ribosome. The overlapping binding sites for arrest peptides, PrAMPs and antibiotics that target the large ribosomal subunit suggest that an increased understanding of these three types of ribosomal ligands could help us design improved translation inhibitors.

The findings made during the first 18 months of the riboFLEX project have resulted in two major research publications in top-tier international peer-reviewed journals. In addition, we have presented our work at various international meetings and at research institutes in France and abroad. It is, however, too soon to establish what the impact and direct applications of the project will be.

1. Seefeldt, A.C., Nguyen, F., Antunes, S., Pérébaskine, N., Graf, M., Arenz, S., Inampudi, K.K., Douat, C., Guichard, G., Wilson, D.N., Innis, C.A. (2015). The proline-rich antimicrobial peptide Onc112 inhibits translation by blocking and destabilizing the initiation complex. Nat Struct Mol Biol 22, 470-475.

2. Seefeldt, A.C., Graf, M., Nguyen, F., Pérébaskine, N., Arenz, S., Mardirossian, M., Scocchi, M., Wilson, D.N., Innis, C.A. (2016). Structure of the mammalian antimicrobial peptide Bac7(1-16) bound within the exit tunnel of a bacterial ribosome. Nucleic Acids Res. 44, 2429-2438.

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.




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