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.     

Amino acid starvation and colicin D treatment induce A-site mRNA cleavage in Escherichia coli

Escherichia coli possesses a unique RNase activity that cleaves stop codons in the ribosomal aminoacyl-tRNA binding site (A-site) during inefficient translation termination. This A-site mRNA cleavage allows recycling of arrested ribosomes by facilitating recruitment of the tmRNA•SmpB ribosome rescue system. To test whether A-site nuclease activity also cleaves sense codons, we induced ribosome pausing at each of the six arginine codons using three strategies; rare codon usage, arginine starvation, and inactivation of arginine tRNAs with colicin D. In each instance, ribosome pausing induced mRNA cleavage within the target arginine codons, and resulted in tmRNA-mediated SsrA-peptide tagging of the nascent polypeptide. A-site mRNA cleavage did not require the stringent factor ppGpp, or bacterial toxins such as RelE, which mediates a similar nuclease activity. However, the efficiency of A-site cleavage was modulated by the identity of the two codons immediately upstream (5′ side) of the A-site codon. Starvation for histidine and tryptophan also induced A-site cleavage at histidine and tryptophan codons, respectively. Thus, A-site mRNA cleavage is a general response to ribosome pausing, capable of cleaving a variety of sense and stop codons. The induction of A-site cleavage during amino acid starvation suggests this nuclease activity may help to regulate protein synthesis during nutritional stress.     

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.     

Optimization of Translation Profiles Enhances Protein Expression and Solubility

mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5’-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.     

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.     

Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites

The initiation of translation is a fundamental and highly regulated process in gene expression. Translation initiation in prokaryotic systems usually requires interaction between the ribosome and an mRNA sequence upstream of the initiation codon, the so-called ribosome-binding site (Shine-Dalgarno sequence). However, a large number of genes do not possess Shine-Dalgarno sequences, and it is unknown how start codon recognition occurs in these mRNAs. We have performed genome-wide searches in various groups of prokaryotes in order to identify sequence elements and/or RNA secondary structural motifs that could mediate translation initiation in mRNAs lacking Shine-Dalgarno sequences. We find that mRNAs without a Shine-Dalgarno sequence are generally less structured in their translation initiation region and show a minimum of mRNA folding at the start codon. Using reporter gene constructs in bacteria, we also provide experimental support for local RNA unfoldedness determining start codon recognition in Shine-Dalgarno--independent translation. Consistent with this, we show that AUG start codons reside in single-stranded regions, whereas internal AUG codons are usually in structured regions of the mRNA. Taken together, our bioinformatics analyses and experimental data suggest that local absence of RNA secondary structure is necessary and sufficient to initiate Shine-Dalgarno--independent translation. Thus, our results provide a plausible mechanism for how the correct translation initiation site is recognized in the absence of a ribosome-binding site.     

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.     

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.     

Effect of codon adaptation on codon-level and gene-level translation efficiency in vivo

Background

There is a significant difference between synonymous codon usage in many organisms, and it is known that codons used more frequently generally showed efficient decoding rate. At the gene level, however, there are conflicting reports on the existence of a correlation between codon adaptation and translation efficiency, even in the same organism.

Results

To resolve this issue, we cultured Escherichia coli under conditions designed to maintain constant levels of mRNA and protein and subjected the cells to ribosome profiling (RP) and mRNA-seq analyses. We showed that the RP results correlated more closely with protein levels generated under similar culture conditions than with the mRNA abundance from the mRNA-seq. Our result indicated that RP/mRNA ratio could be used as a measure of translation efficiency at gene level. On the other hand, the RP data showed that codon-specific ribosome density at the decoding site negatively correlated with codon usage, consistent with the hypothesis that preferred codons display lower ribosome densities due to their faster decoding rate. However, highly codon-adapted genes showed higher ribosome densities at the gene level, indicating that the efficiency of translation initiation, rather than higher elongation efficiency of preferred codons, exerted a greater effect on ribosome density and thus translation efficiency.

Conclusions

These findings indicate that evolutionary pressure on highly expressed genes influenced both codon bias and translation initiation efficiency and therefore explains contradictory findings that codon usage bias correlates with translation efficiency of native genes, but not with the artificially created gene pool, which was not subjected to evolution pressure.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1115) contains supplementary material, which is available to authorized users.     

The Effects of Codon Context on In Vivo Translation Speed

We developed a bacterial genetic system based on translation of the his operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons in vivo. Low frequency effects of so-called “silent” codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 5′- and 3′- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected in vivo translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated in vivo. This system allowed us to determine the effects of different factors on in vivo translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels.     

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.     

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。     

Folding at the rhythm of the rare codon beat

The persistent difficulties in the production of protein at high levels in heterologous systems, as well as the inability to understand pathologies associated with protein aggregation, highlight our limited knowledge on the mechanisms of protein folding in vivo. Attempts to improve yield and quality of recombinant proteins are diverse, frequently involving optimization of the cell growth temperature, the use of synonymous codons and/or the co-expression of tRNAs, chaperones and folding catalysts among others. Although protein secondary structure can be determined largely by the amino acid sequence, protein folding within the cell is affected by a range of factors beyond amino acid sequence. The folding pathway of a nascent polypeptide can be affected by transient interactions with other proteins and ligands, the ribosome, translocation through a pore membrane, redox conditions, among others. The translation rate as well as the translation machinery itself can dramatically affect protein folding, and thus the structure and function of the protein product. This review addresses current efforts to better understand how the use of synonymous codons in the mRNA and the availability of tRNAs can modulate translation kinetics, affecting the folding, the structure and the biological activity of proteins.     

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.     

Translation attenuation regulation of chloramphenicol resistance in bacteria — a review

The chloramphenicol (Cm)-inducible cat and cmlA genes are regulated by translation attenuation, a regulatory device that modulates mRNA translation. In this form of gene regulation, translation of the Cm^R coding sequence is prevented by mRNA secondary structure that sequesters its ribosome-binding site (RBS). A translated leader of nine codons precedes the secondary structure, and induction results when a ribosome becomes stalled at a specific site in the leader. Here we demonstrate that the site of ribosome stalling in the leader is selected by a cis effect of the nascent leader peptide on its translating ribosome.     

A computational framework for the design of optimal protein synthesis

Despite the establishment of design principles to optimize codon choice for heterologous expression vector design, the relationship between codon sequence and final protein yield remains poorly understood. In this work, we present a computational framework for the identification of a set of mutant codon sequences for optimized heterologous protein production, which uses a codon-sequence mechanistic model of protein synthesis. Through a sensitivity analysis on the optimal steady state configuration of protein synthesis we are able to identify the set of codons, that are the most rate limiting with respect to steady state protein synthesis rate, and we replace them with synonymous codons recognized by charged tRNAs more efficient for translation, so that the resulting codon-elongation rate is higher. Repeating this procedure, we iteratively optimize the codon sequence for higher protein synthesis rate taking into account multiple constraints of various types. We determine a small set of optimized synonymous codon sequences that are very close to each other in sequence space, but they have an impact on properties such as ribosomal utilization or secondary structure. This limited number of sequences can then be offered for further experimental study. Overall, the proposed method is very valuable in understanding the effects of the different properties of mRNA sequences on the final protein yield in heterologous protein production and it can find applications in synthetic biology and biotechnology.     

The functional half-life of an mRNA depends on the ribosome spacing in an early coding region

Bacterial mRNAs are translated by closely spaced ribosomes and degraded from the 5′-end, with half-lives of around 2 min at 37 °C in most cases. Ribosome-free or “naked” mRNA is known to be readily degraded, but the initial event that inactivates the mRNA functionally has not been fully described. Here, we characterize a determinant of the functional stability of an mRNA, which is located in the early coding region. Using literature values for the mRNA half-lives of variant lacZ mRNAs in Escherichia coli, we modeled how the ribosome spacing is affected by the translation rate of the individual codons. When comparing the ribosome spacing at various segments of the mRNA to its functional half-life, we found a clear correlation between the functional mRNA half-life and the ribosome spacing in the mRNA region approximately between codon 20 and codon 45. From this finding, we predicted that inserts of slowly translated codons before codon 20 or after codon 45 should shorten or prolong, respectively, the functional mRNA half-life by altering the ribosome density in the important region. These predictions were tested on eight new lacZ variants, and their experimentally determined mRNA half-lives all supported the model. We thus suggest that translation-rate-mediated differences in the spacing between ribosomes in this early coding region is a parameter that determines the mRNAs functional half-life. We present a model that is in accordance with many earlier observations and that allows a prediction of the functional half-life of a given mRNA sequence.     

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.