Yeast gene KTI13 (alias DPH8) operates in the initiation step of diphthamide synthesis on elongation factor 2.

In yeast, Elongator-dependent tRNA modifications are regulated by the Kti11•Kti13 dimer and hijacked for cell killing by zymocin, a tRNase ribotoxin. Kti11 (alias Dph3) also controls modification of elongation factor 2 (EF2) with diphthamide, the target for lethal ADP-ribosylation by diphtheria toxin (DT). Diphthamide formation on EF2 involves four biosynthetic steps encoded by the DPH1-DPH7 network and an ill-defined KTI13 function. On further examining the latter gene in yeast, we found that kti13Δ null-mutants maintain unmodified EF2 able to escape ADP-ribosylation by DT and to survive EF2 inhibition by sordarin, a diphthamide-dependent antifungal. Consistently, mass spectrometry shows kti13Δ cells are blocked in proper formation of amino-carboxyl-propyl-EF2, the first diphthamide pathway intermediate. Thus, apart from their common function in tRNA modification, both Kti11/Dph3 and Kti13 share roles in the initiation step of EF2 modification. We suggest an alias KTI13/DPH8 nomenclature indicating dual-functionality analogous to KTI11/DPH3.

Modulation of translational decoding by m6A modification of mRNA.

N6-methyladenosine (m6A) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. m6A in the mRNA coding regions inhibits translation elongation. Here, we show how m6A modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of m6A fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. m6A does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs.

Translation Phases in Eukaryotes.

Protein synthesis in eukaryotes is carried out by 80S ribosomes with the help of many specific translation factors. Translation comprises four major steps: initiation, elongation, termination, and ribosome recycling. In this review, we provide a comprehensive list of translation factors required for protein synthesis in yeast and higher eukaryotes and summarize the mechanisms of each individual phase of eukaryotic translation.

In Vitro Assembly of a Fully Reconstituted Yeast Translation System for Studies of Initiation and Elongation Phases of Protein Synthesis.

Protein synthesis is an essential and highly regulated cellular process. To facilitate the understanding of eukaryotic translation, we have assembled an in vitro translation system from yeast using purified components to recapitulate the initiation and elongation phases of protein synthesis. Here, we describe methods to express and purify the components of the translation system and the assays for their functional characterization.

Ribosome-bound Get4/5 facilitates the capture of tail-anchored proteins by Sgt2 in yeast.

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.

Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation.

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA-tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of native eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states, as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.

The epitranscriptome in translation regulation: mRNA and tRNA modifications as the two sides of the same coin?

Translation of mRNA is a highly regulated process that is tightly coordinated with cotranslational protein maturation. Recently, mRNA modifications and tRNA modifications - the so called epitranscriptome - have added a new layer of regulation that is still poorly understood. Both types of modifications can affect codon-anticodon interactions, thereby affecting mRNA translation and protein synthesis in similar ways. Here, we describe an updated view on how the different types of modifications can be mapped, how they affect translation, how they trigger phenotypes and discuss how the combined action of mRNA and tRNA modifications coordinate translation in health and disease.

Thio-Modification of tRNA at the Wobble Position as Regulator of the Kinetics of Decoding and Translocation on the Ribosome.

Uridine 34 (U34) at the wobble position of the tRNA anticodon is post-transcriptionally modified, usually to mcm5s2, mcm5, or mnm5. The lack of the mcm5 or s2 modification at U34 of tRNALys, tRNAGlu, and tRNAGln causes ribosome pausing at the respective codons in yeast. The pauses occur during the elongation step, but the mechanism that triggers ribosome pausing is not known. Here, we show how the s2 modification in yeast tRNALys affects mRNA decoding and tRNA-mRNA translocation. Using real-time kinetic analysis we show that mcm5-modified tRNALys lacking the s2 group has a lower affinity of binding to the cognate codon and is more efficiently rejected than the fully modified tRNALys. The lack of the s2 modification also slows down the rearrangements in the ribosome-EF-Tu-GDP-Pi-Lys-tRNALys complex following GTP hydrolysis by EF-Tu. Finally, tRNA-mRNA translocation is slower with the s2-deficient tRNALys. These observations explain the observed ribosome pausing at AAA codons during translation and demonstrate how the s2 modification helps to ensure the optimal translation rates that maintain proteome homeostasis of the cell.

NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation.

Mitochondrial gene expression uses a non-universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt-)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m5C34 mt-tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNAMet function. Together, our data reveal how modifications in mt-tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine.

tRNA wobble modifications and protein homeostasis.

tRNA is a central component of the protein synthesis machinery in the cell. In living cells, tRNAs undergo numerous post-transcriptional modifications. In particular, modifications at the anticodon loop play an important role in ensuring efficient protein synthesis, maintaining protein homeostasis, and helping cell adaptation and survival. Hypo-modification of the wobble position of the tRNA anticodon loop is of particular relevance for translation regulation and is implicated in various human diseases. In this review we summarize recent evidence of how methyl and thiol modifications in eukaryotic tRNA at position 34 affect cellular fitness and modulate regulatory circuits at normal conditions and under stress.

tRNA tKUUU, tQUUG, and tEUUC wobble position modifications fine-tune protein translation by promoting ribosome A-site binding.

tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic (tK(UUU)), (tQ(UUG)), and (tE(UUC)). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1- and ELP-dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tK(UUU) to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content.

Solution structure and activation mechanism of ubiquitin-like small archaeal modifier proteins.

In archaea, two ubiquitin-like small archaeal modifier protein (SAMPs) were recently shown to be conjugated to proteins in vivo. SAMPs display homology to bacterial MoaD sulfur transfer proteins and eukaryotic ubiquitin-like proteins, and they share with them the conserved C-terminal glycine-glycine motif. Here, we report the solution structure of SAMP1 from Methanosarcina acetivorans and the activation of SAMPs by an archaeal protein with homology to eukaryotic E1 enzymes. Our results show that SAMP1 possesses a ß-grasp fold and that its hydrophobic and electrostatic surface features are similar to those of MoaD. M. acetivorans SAMP1 exhibits an extensive flexible surface loop between helix-2 and the third strand of the ß-sheet, which contributes to an elongated surface groove that is not observed in bacterial ubiquitin homologues and many other SAMPs. We provide in vitro biochemical evidence that SAMPs are activated in an ATP-dependent manner by an E1-like enzyme that we have termed E1-like SAMP activator (ELSA). We show that activation occurs by formation of a mixed anhydride (adenylate) at the SAMP C-terminus and is detectable by SDS-PAGE and electrospray ionization mass spectrometry.

Studying chaperone-proteases using a real-time approach based on FRET.

Chaperone-proteases are responsible for the processive breakdown of proteins in eukaryotic, archaeal and bacterial cells. They are composed of a cylinder-shaped protease lined on the interior with proteolytic sites and of ATPase rings that bind to the apical sides of the protease to control substrate entry. We present a real-time FRET-based method for probing the reaction cycle of chaperone-proteases, which consists of substrate unfolding, translocation into the protease and degradation. Using this system we show that the two alternative bacterial ClpAP and ClpXP complexes share the same mechanism: after initial tag recognition, fast unfolding of substrate occurs coinciding with threading through the chaperone. Subsequent slow substrate translocation into the protease chamber leads to formation of a transient compact substrate intermediate presumably close to the chaperone-protease interface. Our data for ClpX and ClpA support the mechanical unfolding mode of action proposed for these chaperones. The general applicability of the designed FRET system is demonstrated here using in addition an archaeal PAN-proteasome complex as model for the more complex eukaryotic proteasome.