Low Complexity Regions behave as tRNA sponges to help co-translational folding of plasmodial proteins

In most organisms, the information necessary to specify the native 3D-structures of proteins is encoded in the corresponding mRNA sequences. Translational accuracy and efficiency are coupled and sequences that are slowly translated play an essential role in the concomitant folding of protein domains. Here, we suggest that the well-known mechanisms for the regulation of translational efficiency, which involves mRNA structure and/or asymmetric tRNA abundance, do not apply to all organisms. We propose that Plasmodium, the parasite responsible for malaria, uses an alternative strategy to slow down ribosomal speed and avoid multidomain protein misfolding during translation. In our model, the abundant Low Complexity Regions present in Plasmodium proteins replace the codon preferences, which influence the assembly of protein secondary structures.     

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.     

Reinvestigating the codon and amino acid usage of S. cerevisiae genome: a new insight from protein secondary structure analysis

Biased usage of synonymous codons has been elucidated under the perspective of cellular tRNA abundance for quite a long time now. Taking advantage of publicly available gene expression data for Saccharomyces cerevisiae, a systematic analysis of the codon and amino acid usages in two different coding regions corresponding to the regular (helix and strand) as well as the irregular (coil) protein secondary structures, have been performed. Our analyses suggest that apart from tRNA abundance, mRNA folding stability is another major evolutionary force in shaping the codon and amino acid usage differences between the highly and lowly expressed genes in S. cerevisiae genome and surprisingly it depends on the coding regions corresponding to the secondary structures of the encoded proteins. This is obviously a new paradigm in understanding the codon usage in S. cerevisiae. Differential amino acid usage between highly and lowly expressed genes in the regions coding for the irregular protein secondary structure in S. cerevisiae is expounded by the stability of the mRNA folded structure. Irrespective of the protein secondary structural type, the highly expressed genes always tend to encode cheaper amino acids in order to reduce the overall biosynthetic cost of production of the corresponding protein. This study supports the hypothesis that the tRNA abundance is a consequence of and not a reason for the biased usage of amino acid between highly and lowly expressed genes.     

Conservation of the relative tRNA composition in healthy and cancerous tissues

Elongation in protein translation is strongly dependent on the availability of mature transfer RNAs (tRNAs). The relative concentrations of the tRNA isoacceptors determine the translation efficiency in unicellular organisms. However, the degree of correspondence of codons and the relevant tRNA isoacceptors serves as an estimator for translation efficiency in all organisms. In this study, we focus on the translational capacity of the human proteome. We show that the correspondence between the codon usage and tRNAs can be improved by combining experimental measurements with the genomic copy number of isoacceptor groups. We show that there are technologies of tRNA measurements that are useful for our analysis. However, fragments of tRNAs do not agree with translational capacity. It was shown that there is a significant increase in the absolute levels of tRNA genes in cancerous cells in comparison to healthy cells. However, we find that the relative composition of tRNA isoacceptors in healthy, cancerous, or transformed cells remains almost identical. This result may indicate that maintaining the relative tRNA composition in cancerous cells is advantageous via its stabilizing of the effectiveness of translation.     

同义密码子使用模式对蛋白产物表达及构象形成的影响*

鉴于遗传密码子的简并性能够将基因遗传信息的容量提升,同义密码子使用偏嗜性得以在生物体的基因组中广泛存在。虽然同义密码子之间碱基的变化并不能导致氨基酸种类的改变,在研究mRNA半衰期、编码多肽翻译效率及肽链空间构象正确折叠的准确性和翻译等这一系列过程中发现,同义密码子使用的偏嗜性在某种程度上通过精微调控翻译机制体现其遗传学功能。同义密码子指导tRNA在翻译过程中识别核糖体的速率变化是由氨基酸的特定顺序决定,并且在新生多肽链合成时,蛋白质共翻译转运机制同时调节其空间构象的正确折叠从而保证蛋白的正常生物学功能。某些同义密码子使用偏嗜性与特定蛋白结构的形成具有显著相关性,密码子使用偏嗜性一旦改变将可能导致新生多肽空间构象出现错误折叠。结合近些年来国内外在此领域的研究成果,阐述同义密码子使用偏嗜性如何发挥精微调控翻译的生物学功能与作用。     

Efficient translation initiation dictates codon usage at gene start

The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5–10 codons of protein‐coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate.     

On finding poorly translated codons based on their usage frequency

Long stretches of “rare” codons are known to severely inhibit the efficiency of translation. Understanding the distribution of such rare codons is of critical importance in improving the efficiency of heterologous gene expression systems. Accurate estimates of codon usage take the abundance of each protein into consideration. In this paper, we analyze the correlation between approximate measures of codon usage and the availability of tRNA at various growth rates in E coli. We show that the computationally derived estimates of tRNA isoacceptor concentration enable the finding of poorly translated codons.     

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the message^{for review see 1}. However, it''s now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates¹. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others². Codon bias is organism as well as tissue specific^2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs⁴. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes^5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein folding^{for review see 9}. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding^1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems^6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.     

Modelling the Efficiency of Codon–tRNA Interactions Based on Codon Usage Bias

The tRNA adaptation index (tAI) is a widely used measure of the efficiency by which a coding sequence is recognized by the intra-cellular tRNA pool. This index includes among others weights that represent wobble interactions between codons and tRNA molecules. Currently, these weights are based only on the gene expression in Saccharomyces cerevisiae. However, the efficiencies of the different codon–tRNA interactions are expected to vary among different organisms. In this study, we suggest a new approach for adjusting the tAI weights to any target model organism without the need for gene expression measurements. Our method is based on optimizing the correlation between the tAI and a measure of codon usage bias. Here, we show that in non-fungal the new tAI weights predict protein abundance significantly better than the traditional tAI weights. The unique tRNA–codon adaptation weights computed for 100 different organisms exhibit a significant correlation with evolutionary distance. The reported results demonstrate the usefulness of the new measure in future genomic studies.     

The effects of codon usage on the formation of secondary structures of nucleocapsid protein of peste des petits ruminants virus

The nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) with a conserved amino acid usage pattern plays an important role in viral replication. The primary objective of this study was to estimate roles of synonymous codon usages of PPRV N gene and tRNA abundances of host in the formation of secondary structure of N protein. The potential effects of synonymous codon usages of N gene and tRNA abundances of host on shaping different folding units (α-helix, β-strand and the coil) in N protein were estimated, based on the information about the modeling secondary structure of PPRV N protein. The synonymous codon usage bias was found in different folding units in PPRV N protein. To better understand the role of translation speed caused by variant tRNA abundances in shaping the specific folding unit in N protein, we modeled the changing trends of tRNA abundance at the transition boundaries from one folding unit to another folding unit (β-strand → coil, coil → β-strand, α-helix → coil, coil → α-helix). The obvious fluctuations of tRNA abundance were identified at the two transition boundaries (β-strand → coil and coil → β-strand) in PPRV N protein. Our findings suggested that viral synonymous codon usage bias and cellular tRNA abundance variation might have potential effects on the formation of secondary structure of PPRV N protein.     

Causal signals between codon bias,mRNA structure,and the efficiency of translation and elongation

Ribosome profiling data report on the distribution of translating ribosomes, at steady‐state, with codon‐level resolution. We present a robust method to extract codon translation rates and protein synthesis rates from these data, and identify causal features associated with elongation and translation efficiency in physiological conditions in yeast. We show that neither elongation rate nor translational efficiency is improved by experimental manipulation of the abundance or body sequence of the rare AGG tRNA. Deletion of three of the four copies of the heavily used ACA tRNA shows a modest efficiency decrease that could be explained by other rate‐reducing signals at gene start. This suggests that correlation between codon bias and efficiency arises as selection for codons to utilize translation machinery efficiently in highly translated genes. We also show a correlation between efficiency and RNA structure calculated both computationally and from recent structure probing data, as well as the Kozak initiation motif, which may comprise a mechanism to regulate initiation.     

Strong association between mRNA folding strength and protein abundance in S. cerevisiae

One of the open questions in regulatory genomics is how the efficiency of gene translation is encoded in the coding sequence. Here we analyse recently generated measurements of folding energy in Saccharomyces cerevisiae, showing that genes with high protein abundance tend to have strong mRNA folding (mF; R=0.68). mF strength also strongly correlates with ribosomal density and mRNA levels, suggesting that this relation at least partially pertains to the efficiency of translation elongation, presumably by preventing aggregation of mRNA molecules.     

Slowing Bacterial Translation Speed Enhances Eukaryotic Protein Folding Efficiency

The mechanisms for de novo protein folding differ significantly between bacteria and eukaryotes, as evidenced by the often observed poor yields of native eukaryotic proteins upon recombinant production in bacterial systems. Polypeptide synthesis rates are faster in bacteria than in eukaryotes, but the effects of general variations in translation rates on protein folding efficiency have remained largely unexplored. By employing Escherichia coli cells with mutant ribosomes whose translation speed can be modulated, we show here that reducing polypeptide elongation rates leads to enhanced folding of diverse proteins of eukaryotic origin. These results suggest that in eukaryotes, protein folding necessitates slow translation rates. In contrast, folding in bacteria appears to be uncoupled from protein synthesis, explaining our findings that a generalized reduction in translation speed does not adversely impact the folding of the endogenous bacterial proteome. Utilization of this strategy has allowed the production of a native eukaryotic multidomain protein that has been previously unattainable in bacterial systems and may constitute a general alternative to the production of aggregation-prone recombinant proteins.     

Over expression of a tRNA(Leu) isoacceptor changes charging pattern of leucine tRNAs and reveals new codon reading

During mRNA translation, synonymous codons for one amino acid are often read by different isoaccepting tRNAs. The theory of selective tRNA charging predicts greatly varying percentages of aminoacylation among isoacceptors in cells starved for their common amino acid. It also predicts major changes in tRNA charging patterns upon concentration changes of single isoacceptors, which suggests a novel type of translational control of gene expression. We therefore tested the theory by measuring with Northern blots the charging of Leu-tRNAs in Escherichia coli under Leu limitation in response to over expression of tRNA(GAG)(Leu). As predicted, the charged level of tRNA(GAG)(Leu) increased and the charged levels of four other Leu isoacceptors decreased or remained unchanged, but the charged level of tRNA(UAG)(Leu) increased unexpectedly. To remove this apparent inconsistency between theory and experiment we postulated a previously unknown common codon for tRNA(GAG)(Leu) and tRNA(UAG)(Leu). Subsequently, we demonstrated that the tRNA(GAG)(Leu) codon CUU is, in fact, read also by tRNA(UAG)(Leu), due to a uridine-5-oxyacetic acid modification.     

Deciphering the rules by which dynamics of mRNA secondary structure affect translation efficiency in Saccharomyces cerevisiae

Messenger RNA (mRNA) secondary structure decreases the elongation rate, as ribosomes must unwind every structure they encounter during translation. Therefore, the strength of mRNA secondary structure is assumed to be reduced in highly translated mRNAs. However, previous studies in vitro reported a positive correlation between mRNA folding strength and protein abundance. The counterintuitive finding suggests that mRNA secondary structure affects translation efficiency in an undetermined manner. Here, we analyzed the folding behavior of mRNA during translation and its effect on translation efficiency. We simulated translation process based on a novel computational model, taking into account the interactions among ribosomes, codon usage and mRNA secondary structures. We showed that mRNA secondary structure shortens ribosomal distance through the dynamics of folding strength. Notably, when adjacent ribosomes are close, mRNA secondary structures between them disappear, and codon usage determines the elongation rate. More importantly, our results showed that the combined effect of mRNA secondary structure and codon usage in highly translated mRNAs causes a short ribosomal distance in structural regions, which in turn eliminates the structures during translation, leading to a high elongation rate. Together, these findings reveal how the dynamics of mRNA secondary structure coupling with codon usage affect translation efficiency.