Identifying ribosome heterogeneity using ribosome profiling

Recent studies have revealed multiple mechanisms that can lead to heterogeneity in ribosomal composition. This heterogeneity can lead to preferential translation of specific panels of mRNAs, and is defined in large part by the ribosomal protein (RP) content, amongst other things. However, it is currently unknown to what extent ribosomal composition is heterogeneous across tissues, which is compounded by a lack of tools available to study it. Here we present dripARF, a method for detecting differential RP incorporation into the ribosome using Ribosome Profiling (Ribo-seq) data. We combine the ‘waste’ rRNA fragment data generated in Ribo-seq with the known 3D structure of the human ribosome to predict differences in the composition of ribosomes in the material being studied. We have validated this approach using publicly available data, and have revealed a potential role for eS25/RPS25 in development. Our results indicate that ribosome heterogeneity can be detected in Ribo-seq data, providing a new method to study this phenomenon. Furthermore, with dripARF, previously published Ribo-seq data provides a wealth of new information, allowing the identification of RPs of interest in many disease and normal contexts. dripARF is available as part of the ARF R package and can be accessed through https://github.com/fallerlab/ARF.     

Chaperoning ribosome assembly

Reconstitution of bacterial ribosomes in vitro from RNA and protein constituents requires a heating step to rearrange conformation of an intermediate. In this issue of Molecular Cell, Maki et al. demonstrate that the DnaK chaperone system circumvents the requirement for heating.     

Crystal structure of the ribosome recycling factor bound to the ribosome

In bacteria, disassembly of the ribosome at the end of translation is facilitated by an essential protein factor termed ribosome recycling factor (RRF), which works in concert with elongation factor G. Here we describe the crystal structure of the Thermus thermophilus RRF bound to a 70S ribosomal complex containing a stop codon in the A site, a transfer RNA anticodon stem-loop in the P site and tRNA(fMet) in the E site. The work demonstrates that structures of translation factors bound to 70S ribosomes can be determined at reasonably high resolution. Contrary to earlier reports, we did not observe any RRF-induced changes in bridges connecting the two subunits. This suggests that such changes are not a direct requirement for or consequence of RRF binding but possibly arise from the subsequent stabilization of a hybrid state of the ribosome.     

The primitive ribosome model

A new model is proposed on the origin of crystallizable ribosomes and the kinetics of ribosome crystallization. The model assumes that ribosome crystallizability is a property the ribosomes have only in a well-defined period of their life cycle and implies a close relationship between cellular differentiation and structural rearrangements at the ribosome level. The model is used to interpret a variety of cases in which ribosome crystallization occurs, such as chick embryo tissues during development, tissue cultures treated with different antibiotics, chick adult tissues infected by viruses, lizard oocytes, degenerating cells and dedifferentiated cellular systems.     

Programmed drug-dependent ribosome stalling

The ribosome has the intrinsic capacity to monitor the sequence and structure of the nascent peptide. This fundamental property of the ribosome is often exploited in regulation of gene expression, in particular, for activation of expression of genes conferring resistance to ribosome-targeting antibiotics. Induction of expression of these genes is controlled by the programmed stalling of the ribosome at a regulatory open reading frame located upstream of the resistance cistron. Formation of the stalled translation complex depends on the presence of an antibiotic in the ribosome exit tunnel and the sequence of the nascent peptide. In this review, we summarize our current understanding of the molecular mechanisms of drug- and nascent peptide-dependent ribosome stalling.     

SRP meets the ribosome

Cotranslational targeting directly couples synthesis of proteins to their translocation across or insertion into membranes. The signal recognition particle (SRP) and its membrane-bound receptor facilitate the targeting of the translation machinery, the ribosome, via recognition of a signal sequence in the nascent peptide chain. By combining structures of free and ribosome-bound SRP we derive a structural model describing the dynamic nature of SRP when it meets the ribosome.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

Powering through ribosome assembly

Ribosome assembly is required for cell growth in all organisms. Classic in vitro work in bacteria has led to a detailed understanding of the biophysical, thermodynamic, and structural basis for the ordered and correct assembly of ribosomal proteins on ribosomal RNA. Furthermore, it has enabled reconstitution of active subunits from ribosomal RNA and proteins in vitro. Nevertheless, recent work has shown that eukaryotic ribosome assembly requires a large macromolecular machinery in vivo. Many of these assembly factors such as ATPases, GTPases, and kinases hydrolyze nucleotide triphosphates. Because these enzymes are likely regulatory proteins, much work to date has focused on understanding their role in the assembly process. Here, we review these factors, as well as other sources of energy, and their roles in the ribosome assembly process. In addition, we propose roles of energy-releasing enzymes in the assembly process, to explain why energy is used for a process that occurs largely spontaneously in bacteria. Finally, we use literature data to suggest testable models for how these enzymes could be used as targets for regulation of ribosome assembly.     

Decoding a ribosome uncertainty

The ribosome is among the most ancient macromolecular complexes. Throughout evolution, the function of the ribosome has remained essential and conserved: the decoding of an mRNA template with tRNA-linked amino acids, to synthesize a protein. In a recent study, Holm et al. capture evolutionary distinctions in the structure and kinetics of ‘mRNA decoding’ by the human ribosome.     

Drugs targeting the ribosome

Several classes of clinically important antibiotics target the bacterial ribosome, where they interfere with microbial protein synthesis. Structural studies of the interaction of antibiotics with the ribosome have revealed that these small molecules recognize predominantly the rRNA components. Over the past two years, three-dimensional structures of ribosome-antibiotic complexes have been determined, providing a detailed picture of the binding sites and mechanism of action of antibacterials, including 'blockbuster' drugs such as the macrolides. Structure-based approaches have come to fruition that comprise the design and crystal structure analysis of novel semi-synthetic antibiotics that target the ribosome decoding site.     

Drop-off during ribosome hopping

Ribosomes bypass a 50 nucleotide non-coding segment of mRNA between the two open reading frames of bacteriophage T4 gene 60 in order to synthesize a topoisomerase subunit. While nearly all ribosomes appear to initiate bypassing, only 50 % resume translation in the second open reading frame. Failure to bypass is shown here to be independent of the stop codon at the end of the first open reading frame and to be amplified by mutant variants of tRNA(Gly)(2) known to diminish bypassing efficiency. Unproductive bypassing may result from premature dissociation of peptidyl-tRNAs from ribosomes (drop-off) or resumption of translation at inappropriate sites. Assessment of the influence of factors known to induce drop-off reveals that ribosome recycling factor accounts for a small fraction of unproductive bypassing products, but none of the other known factors appear to play a significant role. Resumption of translation at inappropriate sites appears to be minimal, which suggests that spontaneous release of the peptidyl-tRNA may account for the remaining unproductive bypassing products and may be inherent to the gene 60 bypassing mechanism.