Translation Elongation Rates: Insights and Implications
In order to maintain a healthy proteome, the ribosome must not only generate sequences of peptides in sufficient quantities to serve their biological purpose but must also ensure that these polymers correctly fold into their native structures to yield fully functional proteins. Somewhat counter-intuitively, these mutually beneficial processes of protein synthesis and folding are favoured by opposing kinetic properties. To strike the optimum balance between these processes ribosomes must move across transcripts at varying rates, speeding up over highly conserved structural domains where accurate translation is important and slowing down at structural boundaries to facilitate proper folding of downstream domain regions. Perturbations in the translation elongation kinetics of protein synthesis is, in and of itself, a powerful pathophysiological process that drives changes in cellular phenotype and cause disease, by either a), interfering with normal expression levels of native proteins, or b), leading to an overabundance of aberrant misfolded proteins which aggregate together giving rise to significant cytotoxicity. Differences in the supply of cognate tRNAs and demand for their use (termed codon usage, which is dependent upon differences in the frequency of codons in the transcriptome) is a critical determinant of the translation rate of individual transcripts.
The composition of the tRNA pool is dynamic in nature with alterations accompanying diverse disease states, while synonymous mutations (which changes the codon without changing the amino acid that is encoded) can drive cancer development by exchanging a frequently occurring codon for a rare one. Ribo-Seq allows for the investigation of global translation kinetics using a pulse-chase strategy, with Ingolia et al., (2011) being the first to implement this approach. A summary of the key findings and implications of this article along with two others that exemplify the use of ribosome profiling in measuring translation elongation rates are discussed below.
Translation elongation rate varies among organs and decreases with age
Nucleic Acids Research, 2021; 49(2), pp.e9-e9.
Gerashchenko, M.V., Peterfi, Z., Yim, S.H. and Gladyshev, V.N.
While there has been a large interest in the study of protein synthesis, especially in areas of disease treatment, the accurate monitoring of protein synthesis in animals is complex due to variability between organs. Here, the authors developed a method to directly assess translation in vivo capable of measuring cell and organ specific translation elongation rates by using two separate translation inhibitors in a time dependent manner. First the inhibitor Harringtonine is injected (as this blocks translation initiation without affecting elongation) into the bloodstream, followed by an injection of the second inhibitor Cycloheximide after a short period of time (cycloheximide is used to block elongation). By detecting the time dependent run off of ribosomes after these injections using a simple linear model of distance ~time the authors could calculate the rate of translation elongation.
- The authors measured the elongation rates in liver, kidney, skeletal muscle, heart, pancreas, testis and lung in mice and found that the elongation rates differ more than 50% among organs, with liver being the highest and skeletal muscle being the lowest, consistent with the metabolic rates of these organs.
- The elongation rate of liver ribosomes in mice was reduced to 20% (approx. 1.3 amino acids per second from 5.6 amino acids per second) between young adulthood (approx. 3 months old) and mid-life (approx. 18 months old). The authors state that given the average lifespan of a mouse is 30 months, a larger decline is expected nearer to the end of its lifespan.
The authors have developed a method that is capable of assessing translation elongation rates in vivo in rodents (and potentially other animals). This presents new opportunities for studies on the effects of ribosomal protein mutations, dietary interventions and the implication of other factors on translation. The authors proved that there was a decline in the elongation rate of certain organs as they age, falling in line with the consensus that the regulation of biological processes become less robust as an animal ages.
Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes
Cell, 2011; 147(4), pp.789-802.
Ingolia, N.T., Lareau, L.F. and Weissman, J.S.,
In this paper, the authors use a series of techniques based on a simplified ribosome profiling protocol to map protein synthesis at a genome level and to help determine the rate of translation elongation. To do this they employed the translational inhibitor drug harringtonine which stalls ribosomes at the start codon. The use of harringtonine in conjunction with treatment of the elongation inhibitors emetine or cycloheximide allowed for a pulse-chase strategy where run-off elongation could be investigated.
- The use of ribosome-profiling reveals the complexity of the mammalian proteome as the authors observed widespread translation upstream of many protein-coding genes and alternate start sites that allowed for the production of extended or truncated isoforms. The authors also report the translation of polycistronic ribosome-associated coding RNAs that encode for small proteins (known as sprcRNAs), a type of RNA whose protein-coding potential was not known prior to this.
- The data established that many sites of translation initiation occur at non canonical start codons, especially in the case of upstream initiation. The authors revealed that initiation at CUG and GUG codons were widespread throughout the data suggesting biological significance.
- Translation proceeds at 5.6 codons per second and stalls at Pro-Pro-Glu motifs as revealed in figure 3. Metagene analysis revealed a progressive depletion of ribosomes after harringtonine treatment.
This study established that many sites of translation initiation occur at non-AUG codons such as initiation at CUG and GUG. This non-canonical initiation appears to impact many aspects of translation, as the extensive upstream initiation observed is likely to regulate main ORF protein synthesis. How, and if, CUG and GUG codon initiation differ mechanistically from AUG initiation remains an open question.
Causal signals between codon bias, mRNA structure, and the efficiency of translation and elongation
Molecular Systems Biology, 2014; 10(12), p.770
Pop, C., Rouskin, S., Ingolia, N.T., Han, L., Phizicky, E.M., Weissman, J.S. and Koller, D.
A main feature of ribosome profiling is its ability to detect protein synthesis at codon-level resolution. this is achieved by arresting ribosomes as it resides over a part of mRNA and removing flanking mRNA by RNAse digestion, leaving ribosome-protected fragments (RPFs) that can be made into NGS libraries. Thus, analysing data generated by ribosome profiling allows for the accurate measurement of the protein synthesis rate and the rate of translation for each codon. In this paper, the authors focused on identifying causal signals that can impede or help the efficiency of translation and the rate at which it occurs. They test this by performing ribosome profiling experiments in both wild-type yeast and in mutants that have altered tRNA levels to check if the rate of elongation or the translational efficiency is altered by tRNA.
- The authors directly manipulated the amount of available tRNA by creating three mutant strains (listed below) and found that it does not significantly affect the elongation rate of cognate codons or the overall TE.
- The first mutant “AGG-OE”, overexpressed the tRNA that recognises AGG.
- The second mutant “AGG-QC” in which the body sequence of the tRNA that recognises AGG was swapped with a different body sequence, to check if the tRNA body itself was the cause of the tRNA-dependent rate effect.
- The third mutant “ACA-K” in which they deleted 3 out of 4 copies of the tRNA that recognises ACA from the genome.
- The authors found that other sequence signals like mRNA structures and the Kozak sequence correlate to translation efficiency and as such may be causal determinants for initiation regulation.
- The wobble base in the CGA codon was suggested to cause significant pausing.
- Clusters of slowly translated codons could stall ribosomes more than the sum of the individual decoding times for each codon.
- The authors suggest that there is a compendium of biological features that interact with each other, which dictate elongation rate.
- An example would be that the effects from the nascent peptide leaving a ribosome could stall elongation at interactions with prolines.
The robust framework developed from this paper allows for the visualization of new information regarding the regulation for translation in cells and characterize the features that are associated with efficient elongation and translation.
