Namit Ranjan

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.

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.

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 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.

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.

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 tRNA^Lys, tRNA^Glu, and tRNA^Gln 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 tRNA^Lys affects mRNA decoding and tRNA–mRNA translocation. Using real-time kinetic analysis we show that mcm⁵-modified tRNA^Lys lacking the s² group has a lower affinity of binding to the cognate codon and is more efficiently rejected than the fully modified tRNA^Lys. The lack of the s² modification also slows down the rearrangements in the ribosome–EF-Tu–GDP–Pi–Lys-tRNA^Lys 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.

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 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.