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。     

The imprint of codons on protein structure

The "central dogma" of biology outlines the unidirectional flow of interpretable data from genetic sequence to protein sequence. This has led to the idea that a protein's structure is dependent only on its amino acid sequence and not its genetic sequence. Recently, however, a more than transient link between the coding genetic sequence and the protein structure has become apparent. The two interact at the ribosome via the process of co-translational protein folding. Evidence for co-translational folding is growing rapidly, but the influence of codons on the protein structure attained is still highly contentious. It is theorised that the speed of codon translation modulates the time available for protein folding and hence the protein structure. Here, past and present research regarding synonymous codons and codon translation speed are reviewed within the context of protein structure attainment.     

Manipulating the genetic code for membrane protein production: What have we learnt so far?

With synthetic gene services, molecular cloning is as easy as ordering a pizza. However choosing the right RNA code for efficient protein production is less straightforward, more akin to deciding on the pizza toppings. The possibility to choose synonymous codons in the gene sequence has ignited a discussion that dates back 50years: Does synonymous codon use matter? Recent studies indicate that replacement of particular codons for synonymous codons can improve expression in homologous or heterologous hosts, however it is not always successful. Furthermore it is increasingly apparent that membrane protein biogenesis can be codon-sensitive. Single synonymous codon substitutions can influence mRNA stability, mRNA structure, translational initiation, translational elongation and even protein folding. Synonymous codon substitutions therefore need to be carefully evaluated when membrane proteins are engineered for higher production levels and further studies are needed to fully understand how to select the codons that are optimal for higher production. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

The secondary structure of mRNAs from Escherichia coli: its possible role in increasing the accuracy of translation.

A secondary structure model was proposed for mRNAs during translation (in a polysome) where the secondary structure is described by a set of small unbranched hairpins. Computer simulation experiments reveal that the number of hairpins is much greater (P less than 10(-6) in highly expressed mRNAs from E. coli as compared with the random sequences coding for the same amino acid sequence, i.e. certain synonymous codons are used in definite mRNA positions to increase the number of hairpins. No constraints on the amino acid sequence, which would affect the secondary structure of mRNAs, were found. The codons UGU, UGC (Cys), GCC (Ala), ACA, ACG (Thr), CCU, CCC (Pro), etc. translated by minor tRNAs were found to occur significantly more frequently in the position 5' to the hairpins than the other codons translated by major tRNAs (P less than 5.10(-6). This correlation leads to the hypothesis that the process of hairpin unfolding can increase the time of translocation from the A to P ribosome site of the codon 5' to the hairpin, thus decreasing the probability of translational error (the latter would likely occur more frequently in the codons translated by minor tRNAs).     

Protein folding and tRNA biology

Polypeptides can fold into tertiary structures while they are synthesized by the ribosome. In addition to the amino acid sequence, protein folding is determined by several factors within the cell. Among others, the folding pathway of a nascent polypeptide can be affected by transient interactions with other proteins, ligands, or the ribosome, as well as by the translocation through membrane pores. Particularly, the translation machinery and the population of tRNA under different physiological or adaptive responses can dramatically affect protein folding. This review summarizes the scientific evidence describing the role of translation kinetics and tRNA populations on protein folding and addresses current efforts to better understand tRNA biology. It is organized into three main parts, which are focused on: (i) protein folding in the cellular context; (ii) tRNA biology and the complexity of the tRNA population; and (iii) available methods and technical challenges in the characterization of tRNA pools. In this manner, this work illustrates the ways by which functional properties of proteins may be modulated by cellular tRNA populations.     

Adjustment of the tRNA population to the codon usage in chloroplasts.

In chloroplasts there is a correlation between the amounts of tRNAs specific for a given amino acid and the codons specifying this amino acid. Furthermore, for the amino acids coded for by more than one codon, the population of isoaccepting tRNAs is adjusted to the frequency of synonymous codons used in chloroplast protein genes. A comparison by two-dimensional gel electrophoresis of the tRNA populations extracted from chloroplasts and from chloroplast polysomes shows that all chloroplast tRNAs are involved in protein biosynthesis.     

The influence of protein coding sequences on protein folding rates of all-β proteins

It is currently believed that the protein folding rate is related to the protein structures and its amino acid sequence. However, few studies have been done on the problem that whether the protein folding rate is influenced by its corresponding mRNA sequence. In this paper, we analyzed the possible relationship between the protein folding rates and the corresponding mRNA sequences. The content of guanine and cytosine (GC content) of palindromes in protein coding sequence was introduced as a new parameter and added in the Gromiha's model of predicting protein folding rates to inspect its effect in protein folding process. The multiple linear regression analysis and jack-knife test show that the new parameter is significant. The linear correlation coefficient between the experimental and the predicted values of the protein folding rates increased significantly from 0.96 to 0.99, and the population variance decreased from 0.50 to 0.24 compared with Gromiha's results. The results show that the GC content of palindromes in the corresponding protein coding sequence really influences the protein folding rate. Further analysis indicates that this kind of effect mostly comes from the synonymous codon usage and from the information of palindrome structure itself, but not from the translation information from codons to amino acids.     

Levels of tRNAs in bacterial cells as affected by amino acid usage in proteins.

Transfer RNAs of Mycoplasma capricolum were separated by two-dimensional polyacrylamide gel electrophoresis, and the relative abundance of each of the 28 known tRNA species was measured. There existed a correlation between the relative amount of isoacceptor tRNAs and the frequency in choosing synonymous codons that could be translated by the isoacceptors. Furthermore, it was observed that the total amount of tRNAs for a particular amino acid was paralleled by the composition of the amino acid in ribosomal proteins. A similar relationship was obtained from reexamination of the previous data on Escherichia coli tRNAs, suggesting that the amount of tRNAs for an amino acid is affected by the usage of the amino acid in proteins.     

Studies on synonymous codon and amino acid usage biases in the broad-host range bacteriophage KVP40

In this study, the relative synonymous codon and amino acid usage biases of the broad-host range phage, KVP40, were investigated in an attempt to understand the structure and function of its proteins/protein-coding genes, as well as the role of its tRNAs. Synonymous codons in KVP40 were determined to be ATrich at the third codon positions, and their variations are dictated principally by both mutational bias and translational selection. Further analysis revealed that the RSCU of KVP40 is distinct from that of its Vibrio hosts, V. cholerae and V. parahaemolyticus. Interestingly, the expression of the putative highly expressed genes of KVP40 appear to be preferentially influenced by the abundant host tRNA species, whereas the tRNAs expressed by KVP40 may be required for the efficient synthesis of all its proteins in a diverse array of hosts. The data generated in this study also revealed that KVP40 proteins are rich in low molecular weight amino acid residues, and that these variations are influenced primarily by hydropathy, mean molecular weight, aromaticity, and cysteine content.     

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.     

Rare codons cluster

Most amino acids are encoded by more than one codon. These synonymous codons are not used with equal frequency: in every organism, some codons are used more commonly, while others are more rare. Though the encoded protein sequence is identical, selective pressures favor more common codons for enhanced translation speed and fidelity. However, rare codons persist, presumably due to neutral drift. Here, we determine whether other, unknown factors, beyond neutral drift, affect the selection and/or distribution of rare codons. We have developed a novel algorithm that evaluates the relative rareness of a nucleotide sequence used to produce a given protein sequence. We show that rare codons, rather than being randomly scattered across genes, often occur in large clusters. These clusters occur in numerous eukaryotic and prokaryotic genomes, and are not confined to unusual or rarely expressed genes: many highly expressed genes, including genes for ribosomal proteins, contain rare codon clusters. A rare codon cluster can impede ribosome translation of the rare codon sequence. These results indicate additional selective pressures govern the use of synonymous codons, and specifically that local pauses in translation can be beneficial for protein biogenesis.     

Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes

By considering the nucleotide sequence of several highly expressed coding regions in bacteriophage MS2 and mRNAs from Escherichia coli, it is possible to deduce some rules which govern the selection of the most appropriate synonymous codons NNU or NNC read by tRNAs having GNN, QNN or INN as anticodon. The rules fit with the general hypothesis that an efficient in-phase translation is facilitated by proper choice of degenerate codewords promoting a codon-anticodon interaction with intermediate strength (optimal energy) over those with very strong or very weak interaction energy. Moreover, codons corresponding to minor tRNAs are clearly avoided in these efficiently expressed genes. These correlations are clearcut in the normal reading frame but not in the corresponding frameshift sequences +1 and +2. We hypothesize that both the optimization of codon-anticodon interaction energy and the adaptation of the population to codon frequency or vice versa in highly expressed mRNAs of E. coli are part of a strategy that optimizes the efficiency of translation. Conversely, codon usage in weakly expressed genes such as repressor genes follows exactly the opposite rules. It may be concluded that, in addition to the need for coding an amino acid sequence, the energetic consideration for codon-anticodon pairing, as well as the adaptation of codons to the tRNA population, may have been important evolutionary constraints on the selection of the optimal nucleotide sequence.     

Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system

The relative quantities of 26 known transfer RNAs of Escherichia coli have been measured previously (Ikemura, 1981). Based on this relative abundance, the usage of cognate codons in E. coli genes as well as in transposon and coliphage genes was examined. A strong positive correlation between tRNA content and the occurrence of respective codons was found for most E. coli genes that had been sequenced, although the correlation was less significant for transposon and phage genes. The dependence of the usage of isoaccepting tRNA, in E. coli genes encoding abundant proteins, on tRNA content was especially noticeable and was greater than that expected from the proportional relationship between the two variables, i.e. these genes selectively use codons corresponding to major tRNAs but almost completely avoid using codons of minor tRNAs. Therefore, codon choice in E. coli genes was considered to be largely constrained by tRNA availability and possibly by translational efficiency. Based on the content of isoaccepting tRNA and the nature of codon-anticodon interaction, it was then possible to predict for most amino acids the order of preference among synonymous codons. The synonymous codon predicted in this way to be the most preferred codon was thought to be optimized for the E. coli translational system and designated as the “Optimal codon”. E. coli genes encoding abundant protein species use the optimal codons selectively, and other E. coli genes, such as amino acid synthesizing genes, use optimal and “non-optimal” codons to a roughly equal degree. The finding that the frequency of usage of optimal codons is closely correlated with the production levels of individual genes was discussed from an evolutionary viewpoint.     

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.     

Factors influencing synonymous codon and amino acid usage biases in Mimivirus

Synonymous codon and amino acid usage biases have been investigated in 903 Mimivirus protein-coding genes in order to understand the architecture and evolution of Mimivirus genome. As expected for an AT-rich genome, third codon positions of the synonymous codons of Mimivirus carry mostly A or T bases. It was found that codon usage bias in Mimivirus genes is dictated both by mutational pressure and translational selection. Evidences show that four factors such as mean molecular weight (MMW), hydropathy, aromaticity and cysteine content are mostly responsible for the variation of amino acid usage in Mimivirus proteins. Based on our observation, we suggest that genes involved in translation, DNA repair, protein folding, etc., have been laterally transferred to Mimivirus a long ago from living organism and with time these genes acquire the codon usage pattern of other Mimivirus genes under selection pressure.     

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.     

Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm ("codon harmonization") for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the "recoded" genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli.     

Human tRNAs with inosine 34 are essential to efficiently translate eukarya-specific low-complexity proteins

The modification of adenosine to inosine at the wobble position (I34) of tRNA anticodons is an abundant and essential feature of eukaryotic tRNAs. The expansion of inosine-containing tRNAs in eukaryotes followed the transformation of the homodimeric bacterial enzyme TadA, which generates I34 in tRNA^Arg and tRNA^Leu, into the heterodimeric eukaryotic enzyme ADAT, which modifies up to eight different tRNAs. The emergence of ADAT and its larger set of substrates, strongly influenced the tRNA composition and codon usage of eukaryotic genomes. However, the selective advantages that drove the expansion of I34-tRNAs remain unknown. Here we investigate the functional relevance of I34-tRNAs in human cells and show that a full complement of these tRNAs is necessary for the translation of low-complexity protein domains enriched in amino acids cognate for I34-tRNAs. The coding sequences for these domains require codons translated by I34-tRNAs, in detriment of synonymous codons that use other tRNAs. I34-tRNA-dependent low-complexity proteins are enriched in functional categories related to cell adhesion, and depletion in I34-tRNAs leads to cellular phenotypes consistent with these roles. We show that the distribution of these low-complexity proteins mirrors the distribution of I34-tRNAs in the phylogenetic tree.     

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its “codon-preferred” DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ∼2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10⁻⁸ m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.