Global and local depletion of ternary complex limits translational elongation

The translation of genetic information according to the sequence of the mRNA template occurs with high accuracy and fidelity. Critical events in each single step of translation are selection of transfer RNA (tRNA), codon reading and tRNA-regeneration for a new cycle. We developed a model that accurately describes the dynamics of single elongation steps, thus providing a systematic insight into the sensitivity of the mRNA translation rate to dynamic environmental conditions. Alterations in the concentration of the aminoacylated tRNA can transiently stall the ribosomes during translation which results, as suggested by the model, in two outcomes: either stress-induced change in the tRNA availability triggers the premature termination of the translation and ribosomal dissociation, or extensive demand for one tRNA species results in a competition between frameshift to an aberrant open-reading frame and ribosomal drop-off. Using the bacterial Escherichia coli system, we experimentally draw parallels between these two possible mechanisms.     

Protein Synthesis in E. coli: Dependence of Codon-Specific Elongation on tRNA Concentration and Codon Usage

To synthesize a protein, a ribosome moves along a messenger RNA (mRNA), reads it codon by codon, and takes up the corresponding ternary complexes which consist of aminoacylated transfer RNAs (aa-tRNAs), elongation factor Tu (EF-Tu), and GTP. During this process of translation elongation, the ribosome proceeds with a codon-specific rate. Here, we present a general theoretical framework to calculate codon-specific elongation rates and error frequencies based on tRNA concentrations and codon usages. Our theory takes three important aspects of in-vivo translation elongation into account. First, non-cognate, near-cognate and cognate ternary complexes compete for the binding sites on the ribosomes. Second, the corresponding binding rates are determined by the concentrations of free ternary complexes, which must be distinguished from the total tRNA concentrations as measured in vivo. Third, for each tRNA species, the difference between total tRNA and ternary complex concentration depends on the codon usages of the corresponding cognate and near-cognate codons. Furthermore, we apply our theory to two alternative pathways for tRNA release from the ribosomal E site and show how the mechanism of tRNA release influences the concentrations of free ternary complexes and thus the codon-specific elongation rates. Using a recently introduced method to determine kinetic rates of in-vivo translation from in-vitro data, we compute elongation rates for all codons in Escherichia coli. We show that for some tRNA species only a few tRNA molecules are part of ternary complexes and, thus, available for the translating ribosomes. In addition, we find that codon-specific elongation rates strongly depend on the overall codon usage in the cell, which could be altered experimentally by overexpression of individual genes.     

Selective constraints in yeast genes with differential expressivity: codon pair usage and mRNA stability perspectives

Protein translation has been elucidated to be dictated by evolutionary constraints, namely, variations in tRNA availabilities and/or variations in codon-anticodon binding that is manifested in biased codon usage. Taking advantage of publicly available mRNA expression and protein abundance data for Saccharomyces cerevisiae, we have performed a comprehensive analysis of the diverse factors guiding translation leading to desired protein levels irrespective of the corresponding high or low mRNA levels. It has been elucidated in this study that different combinations of most abundant/non abundant tRNA isoacceptors are selected for in S. cerevisiae that helps in achieving the optimum speed and accuracy in the protein translation process. This is also accompanied by the strategic location of codon pairs in coherence to mRNA secondary structure folding stability for the above mentioned combinations of tRNA isoacceptors. We thus find that codon pair contextual effects; in addition to tRNA abundance and mRNA folding stability during translation elongation process play plausible roles in maintaining translation accuracy and speed that can achieve desired protein levels.     

The rates of macromolecular chain elongation modulate the initiation frequencies for transcription and translation inEscherichia coli

Here we show that most macromolecular biosynthesis reactions in growing bacteria are sub-saturated with substrate. The experiments should in part test predictions from a previously proposed model (Jensen & Pedersen 1990) which proposed a central role for the rates of the RNA and peptide chain elongation reactions in determining the concentration of initiation competent RNA polymerases and ribosomes and thereby the initiation frequencies for these reactions. We have shown that synthesis of ribosomal RNA and the concentration of ppGpp did not exhibit the normal inverse correlation under balanced growth conditions in batch cultures when the RNA chain elongation rate was limited by substrate supply. The RNA chain elongation rate for the polymerase transcribinglacZ mRNA was directly measured and found to be reduced by two-fold under conditions of high ppGpp levels. In the case of translation, we have shown that the peptide elongation rate varied at different types of codons and even among codons read by the same tRNA species. The faster translated codons probably have the highest cognate tRNA concentration and the highest affinity to the tRNA. Thus, the ribosome may operate close to saturation at some codons and be unsaturated at synonymous codons. Therefore, not only translation of the codons for the seven amino acids, whose biosynthesis is regulated by attenuation, but also a substantial fraction of the other translation reactions may be unsaturated. Recently, we have obtained results which indicate that also many ribosome binding sites are unsaturated with their substrate, i.e. with ribosomes. This observation affects the interpretation of many results obtained by use of reporter genes, because the expression from such genes is strongly influenced by the general physiology of the cell.     

Translation rate modification by preferential codon usage: intragenic position effects

We present a model for calculating the protein production rate as a function of the translation rate. The model takes into account that the elongation rate along an mRNA molecule is non-uniform as a result of different tRNA availabilities for different codons. Initiation of ribosomes on an mRNA is normally the rate-limiting step in the translation process, and blocking of the initiation site can be avoided if the codons closest to this site allow fast translation by the ribosome. Hence, different selective forces may act on the choice of synonymous codons in the initiation region than elsewhere on a given mRNA. We show that the elongation rate along the whole mRNA influences the production rate of abundant proteins, whereas only the elongation rate in the initiation region is of importance for the production rate of rare proteins. We also present an analysis of the codon distribution along known mRNAs coding for abundant and rare proteins.     

The role of tRNA and ribosome competition in coupling the expression of different mRNAs in Saccharomyces cerevisiae

Protein synthesis translates information from messenger RNAs into functional proteomes. Because of the finite nature of the resources required by the translational machinery, both the overall protein synthesis activity of a cell and activity on individual mRNAs are controlled by the allocation of limiting resources. Upon introduction of heterologous sequences into an organism-for example for the purposes of bioprocessing or synthetic biology-limiting resources may also become overstretched, thus negatively affecting both endogenous and heterologous gene expression. In this study, we present a mean-field model of translation in Saccharomyces cerevisiae for the investigation of two particular translational resources, namely ribosomes and aminoacylated tRNAs. We firstly use comparisons of experiments with heterologous sequences and simulations of the same conditions to calibrate our model, and then analyse the behaviour of the translational system in yeast upon introduction of different types of heterologous sequences. Our main findings are that: competition for ribosomes, rather than tRNAs, limits global translation in this organism; that tRNA aminoacylation levels exert, at most, weak control over translational activity; and that decoding speeds and codon adaptation exert strong control over local (mRNA specific) translation rates.     

Effects of codon distributions and tRNA competition on protein translation

Translation is a central cellular process and the complexity of its mechanism necessitates mathematical frameworks to better understand system properties and make quantitative predictions. We have developed a gene sequence-specific mechanistic model for translation which accounts for all the elementary steps of translation elongation. Included in our model is the nonspecific binding of tRNAs to the ribosomal A site, and we find that the competitive, nonspecific binding of the tRNAs is the rate-limiting step in the elongation cycle for every codon. By introducing our model in terms of the Michaelis-Menten kinetic framework, we determine that these results are due to the tRNAs that do not recognize the ribosomal A site codon acting as competitive inhibitors to the tRNAs that do recognize the ribosomal A site codon. We present the results of a sensitivity analysis to determine the contribution of elongation cycle kinetic parameters of each codon on the overall translation rate, and observe that the translation rates of mRNAs are controlled by segments of rate-limiting codons that are sequence-specific. Along these lines, we find that the relative position of codons along the mRNA determines the optimal protein synthesis rate.     

Activation of human mitochondrial lysyl-tRNA synthetase upon maturation of its premitochondrial precursor

The cytoplasmic and mitochondrial species of human lysyl-tRNA synthetase are encoded by a single gene by means of alternative splicing of the KARS1 gene. The cytosolic enzyme possesses a eukaryote-specific N-terminal polypeptide extension that confers on the native enzyme potent tRNA binding properties required for the vectorial transfer of tRNA from the synthetase to elongation factor EF1A within the eukaryotic translation machinery. The mitochondrial enzyme matures from its precursor upon being targeted to that organelle. To understand how the cytosolic and mitochondrial enzymes are adapted to participate in two distinct translation machineries, of eukaryotic or bacterial origin, we characterized the mitochondrial LysRS species. Here we report that cleavage of the precursor of mitochondrial LysRS leads to a mature enzyme with reduced tRNA binding properties compared to those of the cytoplasmic counterpart. This adaptation mechanism may prevent inhibition of translation through sequestration of lysyl-tRNA on the synthetase in a compartment where the bacterial-like elongation factor EF-Tu could not assist in its dissociation from the synthetase. We also observed that the RxxxKRxxK tRNA-binding motif of mitochondrial LysRS is not functional in the precursor form of that enzyme and becomes operational after cleavage of the mitochondrial targeting sequence. The finding that maturation of the precursor is needed to reveal the potent tRNA binding properties of this enzyme has strong implications for the spatiotemporal regulation of its activities and is consistent with previous studies suggesting that the only LysRS species able to promote packaging of tRNA(Lys) into HIV-1 viral particles is the mature form of the mitochondrial enzyme.     

Release of the inhibition of messenger RNA translation in extracts of interferon-treated Ehrlich ascites tumor cells by added transfer RNA

In an extract of Ehrlich ascites tumor (EAT) cells which had been “preincubated” for 45 min to lower endogenous protein synthesis (S30_C) the translation of exogenous encephalomyocarditis (EMC) viral mRNA proceeds at a constant rate for over 90 min. In a similarly treated extract of interferon-treated EAT cells (S30_INT) the translation proceeds at a lower rate than in the S30_C for about 30 min and then stops. The impairment of the translation in the S30_INT is mediated by one or more inhibitors. After the cessation of translation the viral mRNA in the S30_INT is in large polysomes. The size of these changes little (if any) during a further 15 min incubation. The addition of mouse tRNA (but not ribosomal RNA or $\text{E. coli}$ tRNA) to the S30_INT after the cessation of viral mRNA translation results in the restart of translation at a rate close to that in the S30_C. This effect of tRNA is diminished by pactamycin, which inhibits peptide chain initiation but not elongation. These results indicate that addition of tRNA allows the elongation of incomplete peptide chains and the initiation of new chains. The need for added tRNA may be due to the fact that in S30_INT the amino acid acceptance of some of the endogenous tRNA species (but not of added tRNAs) is impaired. This impairment is pronounced for leucine and very slight, if any, for five other amino acids tested (i.e. isoleucine, methionine, phenylalanine, threonine, and valine).     

Trade-offs between tRNA abundance and mRNA secondary structure support smoothing of translation elongation rate

Translation of protein from mRNA is a complex multi-step process that occurs at a non-uniform rate. Variability in ribosome speed along an mRNA enables refinement of the proteome and plays a critical role in protein biogenesis. Detailed single protein studies have found both tRNA abundance and mRNA secondary structure as key modulators of translation elongation rate, but recent genome-wide ribosome profiling experiments have not observed significant influence of either on translation efficiency. Here we provide evidence that this results from an inherent trade-off between these factors. We find codons pairing to high-abundance tRNAs are preferentially used in regions of high secondary structure content, while codons read by significantly less abundant tRNAs are located in lowly structured regions. By considering long stretches of high and low mRNA secondary structure in Saccharomyces cerevisiae and Escherichia coli and comparing them to randomized-gene models and experimental expression data, we were able to distinguish clear selective pressures and increased protein expression for specific codon choices. The trade-off between secondary structure and tRNA-concentration based codon choice allows for compensation of their independent effects on translation, helping to smooth overall translational speed and reducing the chance of potentially detrimental points of excessively slow or fast ribosome movement.     

Ribosomes are stalled during in vitro translation of alfalfa mosaic virus RNA 1

In the presence of plant tRNAs the full-length translation product of alfalfa mosaic virus RNA 1 is produced in rabbit reticulocytes only at low mRNA concentration. At higher mRNA concentration translation is restricted to the 5' half of RNA 1. At high mRNA concentration the full-length product can be formed when additional plant tRNA and glutamine are supplied to the translation mixture. In contrast, in the presence of yeast or calf liver tRNA the translation pattern of alfalfa mosaic virus RNA 1 always results in the synthesis of the full-length product. Pulse-chase experiments in the presence of plant tRNAs show that the ribosomes pause at several positions in the 5' half of RNA 1. The pausing time is different at the different 'halting places'. Protein synthesis is resumed upon addition of glutamine, even when the addition is delayed for more than 3 h after the start of protein synthesis. Only one tRNA species, purified from wheat germ or tobacco, could promote full-length translation of RNA 1. This tRNA can be charged with glutamine. Analysis of the position of glutamine codons on RNA 1 shows a correlation between the positions of the CAA codons and the halting places of the ribosomes. The CAA codon (for any other codon) on its own cannot be responsible for the pausing of the ribosomes, since a variety of RNAs, known to contain all sense codons, are translated efficiently in rabbit reticulocyte lysates in the presence of plant tRNAs. Apparently other elements can restrict decoding of normal codons during protein chain elongation.     

Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors

Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions but also re-associated yeast ribosomal subunits. On the other hand, sordarin or fusidic acid, when applied together with eEF2/GTP, specifically inhibited ribosome splitting without blocking of tRNA/mRNA release. From these inhibitor studies, we propose that, in accordance with eEF3’s known function in elongation, the release of tRNA via exit site occurs first, then mRNA is released, followed by the splitting of ribosomes during the disassembly of post-termination complexes catalyzed by eEF3 and ATP.