The landscape of translational stall sites in bacteria revealed by monosome and disome profiling

Ribosome pauses are associated with various cotranslational events and determine the fate of mRNAs and proteins. Thus, the identification of precise pause sites across the transcriptome is desirable; however, the landscape of ribosome pauses in bacteria remains ambiguous. Here, we harness monosome and disome (or collided ribosome) profiling strategies to survey ribosome pause sites in Escherichia coli. Compared to eukaryotes, ribosome collisions in bacteria showed remarkable differences: a low frequency of disomes at stop codons, collisions occurring immediately after 70S assembly on start codons, and shorter queues of ribosomes trailing upstream. The pause sites corresponded with the biochemical validation by integrated nascent chain profiling (iNP) to detect polypeptidyl-tRNA, an elongation intermediate. Moreover, the subset of those sites showed puromycin resistance, presenting slow peptidyl transfer. Among the identified sites, the ribosome pause at Asn586 of ycbZ was validated by biochemical reporter assay, tRNA sequencing (tRNA-Seq), and cryo-electron microscopy (cryo-EM) experiments. Our results provide a useful resource for ribosome stalling sites in bacteria

trans-Translation inhibitors bind to a novel site on the ribosome and clear Neisseria gonorrhoeae in vivo

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways.

N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

Protein-Free ribosomal RNA scaffolds can assemble poly-lysine oligos from charged tRNA fragments

Ribosomal protein synthesis is a central process of the modern biological world. Because the ribosome contains proteins itself, it is very important to understand its precursor and evolution. Small ribozymes have demonstrated the principle of “RNA world” hypothesis, but protein free peptide ligase remains elusive. In this report, we have identified two fragments in the peptidyl transfer center that can synthesize a 9-mer poly-lysine in a solution contains Mg2+. This result is deduced from isotope-shifting in high resolution MS. To our best knowledge, this is the longest peptide oligo that can be synthesized by a pure ribozyme. Via single molecule FRET experiments, we have demonstrated the ligase mechanism was probably by substrate proximity via dimerization. We prospect that these RNA fragments can be useful to synthesize template free natural and non-natural peptides, to be model system for peptidyl transfer reaction mechanism and can shed light to the evolution of ribosome.Table of Content Graph

Evolution of ribosomal protein network architectures

To perform an accurate protein synthesis, ribosomes accomplish complex tasks involving the long-range communication between its functional centres such as the peptidyl transfer centre, the tRNA bindings sites and the peptide exit tunnel. How information is transmitted between these sites remains one of the major challenges in current ribosome research. Many experimental studies have revealed that some r-proteins play essential roles in remote communication and the possible involvement of r-protein networks in these processes have been recently proposed. Our phylogenetic, structural and mathematical study reveals that of the three kingdom’s r-protein networks converged towards non-random graphs where r-proteins collectively coevolved to optimize interconnection between functional centres. The massive acquisition of conserved aromatic residues at the interfaces and along the extensions of the newly connected eukaryotic r-proteins also highlights that a strong selective pressure acts on their sequences probably for the formation of new allosteric pathways in the network.

Partial spontaneous intersubunit rotations in actively translating ribosomes

SummaryThe ribosome is a molecular machine that adopts at least two global states during translation. Two main steps of translation, peptidyl transfer and translocation, are accompanied by counterclockwise and clockwise rotations of the two ribosomal subunits. However, when and why the ribosome alternates between these states remains unclear, with two well supported but conflicting hypotheses. Ribosomes may undergo a single cycle of forward and backward rotations per codon read. Alternatively, in addition to rotations caused by peptidyl transfer and translocation, ribosomes may undergo multiple full spontaneous rotations, with these rotations playing a critical role in elongation and specifically in translocation mechanism. We applied high-speed single-molecule TIRF microscopy to follow translation in real-time. Actively translating ribosomes undergo partial spontaneous rotations between three different rotational states. Spontaneous rotations are restricted prior to A-site tRNA decoding. Peptidyl transfer unlocks spontaneous rotations. Consequently, translocation proceeds via a novel rotational state induced by EF-G. Our results bridge both models and provide a coherent view of ribosome dynamics during translation.

Elongational stalling activates mitoribosome-associated quality control

The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo–electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.

Molecular Functions of Conserved Developmentally-Regulated GTP-Binding Protein Drg1 in Translation

SUMMARYDevelopmentally-regulated GTP-binding (Drg) proteins are important for embryonic development, cell growth, proliferation, and differentiation. Despite their highly conserved nature, the functions of Drg proteins in translation are unknown. Here, we demonstrate the yeast Drg ortholog, Rbg1, alleviates ribosome pausing at Arginine/Lysine-rich regions in mRNAs, and mainly targets genes related to ribonucleoprotein complex biogenesis and non-coding RNA processing pathways. Furthermore, we reveal the global architecture of the ribosome and the molecular interactions involved when Rbg1 and its binding partner, Tma46, associate with the ribosome using biochemistry and single particle reconstruction using cryoEM. Our data show that Rbg1/Tma46 associate with the larger subunit of ribosome via the N-terminal zinc finger domain in Tma46, and that the protein complex helps to enrich translating ribosomes in the post-peptidyl transfer state, after peptide-bond formation, but before elongation factor binding and tRNA translocation. Based on our results and the conserved nature of Drg proteins, broader functions of the Drg proteins in the protein synthesis and quality control pathways of eukaryotic cells are proposed.