Ribosome-mediated translational pause and protein domain organization.

Because regions on the messenger ribonucleic acid differ in the rate at which they are translated by the ribosome and because proteins can fold cotranslationally on the ribosome, a question arises as to whether the kinetics of translation influence the folding events in the growing nascent polypeptide chain. Translationally slow regions were identified on mRNAs for a set of 37 multidomain proteins from Escherichia coli with known three-dimensional structures. The frequencies of individual codons in mRNAs of highly expressed genes from E. coli were taken as a measure of codon translation speed. Analysis of codon usage in slow regions showed a consistency with the experimentally determined translation rates of codons; abundant codons that are translated with faster speeds compared with their synonymous codons were found to be avoided; rare codons that are translated at an unexpectedly higher rate were also found to be avoided in slow regions. The statistical significance of the occurrence of such slow regions on mRNA spans corresponding to the oligopeptide domain termini and linking regions on the encoded proteins was assessed. The amino acid type and the solvent accessibility of the residues coded by such slow regions were also examined. The results indicated that protein domain boundaries that mark higher-order structural organization are largely coded by translationally slow regions on the RNA and are composed of such amino acids that are stickier to the ribosome channel through which the synthesized polypeptide chain emerges into the cytoplasm. The translationally slow nucleotide regions on mRNA possess the potential to form hairpin secondary structures and such structures could further slow the movement of ribosome. The results point to an intriguing correlation between protein synthesis machinery and in vivo protein folding. Examination of available mutagenic data indicated that the effects of some of the reported mutations were consistent with our hypothesis.     

同义密码子使用模式对多肽链共翻译折叠的影响研究进展

同义密码子使用模式作为核苷酸与氨基酸的纽带,其多样性介导了核糖体扫描速率,同时扩充了基因的遗传信息存储量。随着新型技术的应用,发现特异性密码子和密码子结合力可调节核糖体扫描速率并影响蛋白质构象。同义密码子使用模式通过多种方式在不同环节影响着核糖体扫描速率,同时还影响着自身mRNA的稳定性。本文简述了密码子使用模式如何在核糖体扫描翻译mRNA的过程中实现对多肽链翻译延伸的调控,为今后生物工程学领域如何优化蛋白高效表达提供可参考的思路与理念。     

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。     

Protein secondary structural types are differentially coded on messenger RNA.

Tricodon regions on messenger RNAs corresponding to a set of proteins from Escherichia coli were scrutinized for their translation speed. The fractional frequency values of the individual codons as they occur in mRNAs of highly expressed genes from Escherichia coli were taken as an indicative measure of the translation speed. The tricodons were classified by the sum of the frequency values of the constituent codons. Examination of the conformation of the encoded amino acid residues in the corresponding protein tertiary structures revealed a correlation between codon usage in mRNA and topological features of the encoded proteins. Alpha helices on proteins tend to be preferentially coded by translationally fast mRNA regions while the slow segments often code for beta strands and coil regions. Fast regions correspondingly avoid coding for beta strands and coil regions while the slow regions similarly move away from encoding alpha helices. Structural and mechanistic aspects of the ribosome peptide channel support the relevance of sequence fragment translation and subsequent conformation. A discussion is presented relating the observation to the reported kinetic data on the formation and stabilization of protein secondary structural types during protein folding. The observed absence of such strong positive selection for codons in non-highly expressed genes is compatible with existing theories that mutation pressure may well dominate codon selection in non-highly expressed genes.     

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.     

Control of translation efficiency in yeast by codon-anticodon interactions

The choice of synonymous codons used to encode a polypeptide contributes to substantial differences in translation efficiency between genes. However, both the magnitude and the mechanisms of codon-mediated effects are unknown, as neither the effects of individual codons nor the parameters that modulate codon-mediated regulation are understood, particularly in eukaryotes. To explore this problem in Saccharomyces cerevisiae, we performed the first systematic analysis of codon effects on expression. We find that the arginine codon CGA is strongly inhibitory, resulting in progressively and sharply reduced expression with increased CGA codon dosage. CGA-mediated inhibition of expression is primarily due to wobble decoding of CGA, since it is nearly completely suppressed by coexpression of an exact match anticodon-mutated tRNA(Arg(UCG)), and is associated with generation of a smaller RNA fragment, likely due to endonucleolytic cleavage at a stalled ribosome. Moreover, CGA codon pairs are more effective inhibitors of expression than individual CGA codons. These results directly implicate decoding by the ribosome and interactions at neighboring sites within the ribosome as mediators of codon-specific translation efficiency.     

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.     

Synonymous codon usage influences the local protein structure observed

Translation of mRNA into protein is a unidirectional information flow process. Analysing the input (mRNA) and output (protein) of translation, we find that local protein structure information is encoded in the mRNA nucleotide sequence. The Coding Sequence and Structure (CSandS) database developed in this work provides a detailed mapping between over 4000 solved protein structures and their mRNA. CSandS facilitates a comprehensive analysis of codon usage over many organisms. In assigning translation speed, we find that relative codon usage is less informative than tRNA concentration. For all speed measures, no evidence was found that domain boundaries are enriched with slow codons. In fact, genes seemingly avoid slow codons around structurally defined domain boundaries. Translation speed, however, does decrease at the transition into secondary structure. Codons are identified that have structural preferences significantly different from the amino acid they encode. However, each organism has its own set of ‘significant codons’. Our results support the premise that codons encode more information than merely amino acids and give insight into the role of translation in protein folding.     

Sequence similarity is more relevant than species specificity in probabilistic backtranslation

Background  

Backtranslation is the process of decoding a sequence of amino acids into the corresponding codons. All synthetic gene design systems include a backtranslation module. The degeneracy of the genetic code makes backtranslation potentially ambiguous since most amino acids are encoded by multiple codons. The common approach to overcome this difficulty is based on imitation of codon usage within the target species.     

Codon usage and gene expression.

The hypothesis that codon usage regulates gene expression at the level of translation is tested. Codon usage of Escherichia coli and phage lambda is compared by correspondence analysis, and the basis of this hypothesis is examined by connecting codon and tRNA distributions to polypeptide elongation kinetics. Both approaches indicate that if codon usage was random tRNA limitation would only affect the rarest tRNA species. General discrimination against their cognate codons indicates that polypeptide elongation rates are maintained constant. Thus, differences in expression of E. coli genes are not a consequence of their variable codon usage. The preference of codons recognized by the most abundant tRNAs in E. coli genes encoding abundant proteins is explained by a constraint on the cost of proof-reading.     

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.     

Rationalization and prediction of selective decoding of pseudouridine-modified nonsense and sense codons

A stop or nonsense codon is an in-frame triplet within a messenger RNA that signals the termination of translation. One common feature shared among all three nonsense codons (UAA, UAG, and UGA) is a uridine present at the first codon position. It has been recently shown that the conversion of this uridine into pseudouridine (Ψ) suppresses translation termination, both in vitro and in vivo. Furthermore, decoding of the pseudouridylated nonsense codons is accompanied by the incorporation of two specific amino acids in a nonsense codon-dependent fashion. Ψ differs from uridine by a single N1H group at the C5 position; how Ψ suppresses termination and, more importantly, enables selective decoding is poorly understood. Here, we provide molecular rationales for how pseudouridylated stop codons are selectively decoded. Our analysis applies crystal structures of ribosomes in varying states of translation to consider weakened interaction of Ψ with release factor; thermodynamic and geometric considerations of the codon-anticodon base pairs to rank and to eliminate mRNA-tRNA pairs; the mechanism of fidelity check of the codon-anticodon pairing by the ribosome to evaluate noncanonical codon-anticodon base pairs and the role of water. We also consider certain tRNA modifications that interfere with the Ψ-coordinated water in the major groove of the codon-anticodon mini-helix. Our analysis of nonsense codons enables prediction of potential decoding properties for Ψ-modified sense codons, such as decoding ΨUU potentially as Cys and Tyr. Our results provide molecular rationale for the remarkable dynamics of ribosome decoding and insights on possible reprogramming of the genetic code using mRNA modifications.     

Effect of Correlated tRNA Abundances on Translation Errors and Evolution of Codon Usage Bias

Despite the fact that tRNA abundances are thought to play a major role in determining translation error rates, their distribution across the genetic code and the resulting implications have received little attention. In general, studies of codon usage bias (CUB) assume that codons with higher tRNA abundance have lower missense error rates. Using a model of protein translation based on tRNA competition and intra-ribosomal kinetics, we show that this assumption can be violated when tRNA abundances are positively correlated across the genetic code. Examining the distribution of tRNA abundances across 73 bacterial genomes from 20 different genera, we find a consistent positive correlation between tRNA abundances across the genetic code. This work challenges one of the fundamental assumptions made in over 30 years of research on CUB that codons with higher tRNA abundances have lower missense error rates and that missense errors are the primary selective force responsible for CUB.     

New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae

Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5′ nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNA^Gln were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.     

Codon usage bias in prokaryotic pyrimidine-ending codons is associated with the degeneracy of the encoded amino acids

Synonymous codons are unevenly distributed among genes, a phenomenon termed codon usage bias. Understanding the patterns of codon bias and the forces shaping them is a major step towards elucidating the adaptive advantage codon choice can confer at the level of individual genes and organisms. Here, we perform a large-scale analysis to assess codon usage bias pattern of pyrimidine-ending codons in highly expressed genes in prokaryotes. We find a bias pattern linked to the degeneracy of the encoded amino acid. Specifically, we show that codon-pairs that encode two- and three-fold degenerate amino acids are biased towards the C-ending codon while codons encoding four-fold degenerate amino acids are biased towards the U-ending codon. This codon usage pattern is widespread in prokaryotes, and its strength is correlated with translational selection both within and between organisms. We show that this bias is associated with an improved correspondence with the tRNA pool, avoidance of mis-incorporation errors during translation and moderate stability of codon-anticodon interaction, all consistent with more efficient translation.     

Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system

The translation machinery recognizes codons that enter the ribosomal A site with remarkable accuracy to ensure that polypeptide synthesis proceeds with a minimum of errors. When a termination codon enters the A site of a eukaryotic ribosome, it is recognized by the release factor eRF1. It has been suggested that the recognition of translation termination signals in these organisms is not limited to a simple trinucleotide codon, but is instead recognized by an extended tetranucleotide termination signal comprised of the stop codon and the first nucleotide that follows. Interestingly, pharmacological agents such as aminoglycoside antibiotics can reduce the efficiency of translation termination by a mechanism that alters this ribosomal proofreading process. This leads to the misincorporation of an amino acid through the pairing of a near-cognate aminoacyl tRNA with the stop codon. To determine whether the sequence context surrounding a stop codon can influence aminoglycoside-mediated suppression of translation termination signals, we developed a series of readthrough constructs that contained different tetranucleotide termination signals, as well as differences in the three bases upstream and downstream of the stop codon. Our results demonstrate that the sequences surrounding a stop codon can play an important role in determining its susceptibility to suppression by aminoglycosides. Furthermore, these distal sequences were found to influence the level of suppression in remarkably distinct ways. These results suggest that the mRNA context influences the suppression of stop codons in response to subtle differences in the conformation of the ribosomal decoding site that result from aminoglycoside binding.     

The role of tRNA as a molecular spring in decoding, accommodation, and peptidyl transfer

Translation is the process by which the genetic information contained in mRNA is used to link amino acids in a predetermined sequential order into a polypeptide chain, which then folds into a protein. Transfer RNAs (tRNAs) are the adapter molecules designed to provide the "lookup" from codons to amino acids. Cryo-EM has provided evidence that the ribosome, as a molecular machine, undergoes many structural changes during translation. Recent findings show that the tRNA structure itself undergoes large conformational changes as well, and that the decoding process must be seen as a complex dynamic interplay between tRNA and the ribosome.