Distinct responses to rare codons in select Drosophila tissues

Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. Here, we use codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however, we find the testis and brain are uniquely capable of expressing rare codon-enriched reporters. We define a new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index, to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. We further demonstrate a role for rare codons in restricting protein expression of an evolutionarily young gene, RpL10Aa, to the Drosophila testis. Rare codon-mediated restriction of this testis-specific protein is critical for female fertility. Our work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology.

Modification of Transfer RNA Levels Affects Cyclin Aggregation and the Correct Duplication of Yeast Cells

Codon usage bias (the preferential use of certain synonymous codons (optimal) over others is found at the organism level (intergenomic) within specific genomes (intragenomic) and even in certain genes. Whether it is the result of genetic drift due to GC/AT content and/or natural selection is a topic of intense debate. Preferential codons are mostly found in genes encoding highly-expressed proteins, while lowly-expressed proteins usually contain a high proportion of rare (lowly-represented) codons. While optimal codons are decoded by highly expressed tRNAs, rare codons are usually decoded by lowly-represented tRNAs. Whether rare codons play a role in controlling the expression of lowly- or temporarily-expressed proteins is an open question. In this work we approached this question using two strategies, either by replacing rare glycine codons with optimal counterparts in the gene that encodes the cell cycle protein Cdc13, or by overexpression the tRNAGly that decodes rare codons from the fission yeast, Schizosaccharomyces pombe. While the replacement of synonymous codons severely affected cell growth, increasing tRNA levels affected the aggregation status of Cdc13 and cell division. These lead us to think that rare codons in lowly-expressed cyclin proteins are crucial for cell division, and that the overexpression of tRNA that decodes rare codons affects the expression of proteins containing these rare codons. These codons may be the result of the natural selection of codons in genes that encode lowly-expressed proteins.

Expression of transgenes enriched in rare codons is enhanced by the MAPK pathway

The ability to translate three nucleotide sequences, or codons, into amino acids to form proteins is conserved across all organisms. All but two amino acids have multiple codons, and the frequency that such synonymous codons occur in genomes ranges from rare to common. Transcripts enriched in rare codons are typically associated with poor translation, but in certain settings can be robustly expressed, suggestive of codon-dependent regulation. Given this, we screened a gain-of-function library for human genes that increase the expression of a GFPrare reporter encoded by rare codons. This screen identified multiple components of the mitogen activated protein kinase (MAPK) pathway enhancing GFPrare expression. This effect was reversed with inhibitors of this pathway and confirmed to be both codon-dependent and occur with ectopic transcripts naturally coded with rare codons. Finally, this effect was associated, at least in part, with enhanced translation. We thus identify a potential regulatory module that takes advantage of the redundancy in the genetic code to modulate protein expression.

EXPRESSION OF NUCLEOCAPSID VIRAL PROTEINS IN THE BACTERIAL SYSTEM OF Escherichia coli: THE INFLUENCE OF THE CODON COMPOSITION AND THE UNIFORMITY OF ITS DISTRIBUTION WITHIN GENE

A heterologous host has got a unique expression ability of each gene. Differences between the synonymous sequences play an important role in regulation of protein expression in organisms from Escherichia coli to human, and many details of this process remain unclear. The work was aimed to study the composition of codons, its distribution over the sequence and the effect of rare codons on the expression of viral nucleocapsid proteins and their fragments in the heterologous system of E.coli. The plasmid vector pJC 40 and the BL 21 (DE 3) E. coli strain were used for protein expression. The codon composition analysis was performed using the online resource (www.biologicscorp.com). 10 recombinant polypeptides were obtained encoding the complete nucleotide sequence of nucleocapsid proteins (West Nile and hepatitis C viruses) and the fragments including antigenic determinants (Lassa virus, Marburg, Ebola, Crimean-Congo hemorrhagic fever (CCHF), Puumaravala, Hantaan, and lymphocytic choriomeningitis (LHM)). Hybrid plasmid DNAs provide efficient production of these proteins in the prokaryotic system with the recombinant protein yield varying by a factor of 8: from 5 to 40 mg per 1 liter of bacterial culture. No correlation was found between the level of protein expression and the frequency of occurrence of rare codons in the cloned sequence: the maximum frequency of occurrence of rare codons per cloned sequence was observed for the West Nile virus (14.6%), the minimum was for the CCHF virus (6.6%), whereas the expression level for these proteins was 30 and 5 mg/L culture, respectively. The codon adaptation index (CAI) values, calculated on the basis of the codon composition in E. coli, for the cloned viral sequences were in the range from 0.50 to 0.58, which corresponded to the average expressed proteins. The analysis of the distribution profiles of CAI in the cloned sequences indicated the absence of clusters of rare codons that could create difficulties in translation. A statistically significant difference between the frequencies of the distribution of amino acids in the cloned sequences and their content in E. coli was observed for the nucleocapsid proteins of the Marburg, Ebola, West Nile, and hepatitis C viruses.

In silico analysis of relative rareness, codon usage, and enzyme-substrate docking of Lampyroidea maculata luciferase

: Bioluminescence is the production and emission of light by the luciferase enzymes in a living organism. The luciferases were identified in different domains of life, but the Lampyridae luciferases are considered for biotechnological and clinical applications. Recently, the new Iranian luciferase gene from the Lampyroidea maculata has been cloned and characterized. In this study, in silico analysis of this enzyme as the codon usage bias parameters (CAI, CBI, ENC, and rare codons) were conducted. Furthermore, the 3D structure of this enzyme was modeled in the I-TASSER web server and the status of these rare codons in this model was studied using SPDBV and PyMOL software. In the following, the substrate-binding site was studied using the AutoDock Vina. By molecular modeling, some rare codons were identified that may have a critical role in the structure and function of this enzyme. The GC3% of the CDs was 17/304 and GC3 Skewness was 0.115. The molecular docking analysis recognizes some residues that yield closely related to the DLSA binding site. By these analyses, a new understanding of the sequence and structure of this enzyme was created, and our findings can be used in some fields of clinical and industrial biotechnology. This bioinformatics analysis plays an important role in the design of the new recombinant enzyme.

Network analysis of synonymous codon usage

Motivation Most amino acids are encoded by multiple synonymous codons, some of which are used more rarely than others. Analyses of positions of such rare codons in protein sequences revealed that rare codons can impact co-translational protein folding and that positions of some rare codons are evolutionarily conserved. Analyses of their positions in protein 3-dimensional structures, which are richer in biochemical information than sequences alone, might further explain the role of rare codons in protein folding. Results We model protein structures as networks and use network centrality to measure the structural position of an amino acid. We first validate that amino acids buried within the structural core are network-central, and those on the surface are not. Then, we study potential differences between network centralities and thus structural positions of amino acids encoded by conserved rare, non-conserved rare and commonly used codons. We find that in 84% of proteins, the three codon categories occupy significantly different structural positions. We examine protein groups showing different codon centrality trends, i.e. different relationships between structural positions of the three codon categories. We see several cases of all proteins from our data with some structural or functional property being in the same group. Also, we see a case of all proteins in some group having the same property. Our work shows that codon usage is linked to the final protein structure and thus possibly to co-translational protein folding. Availability and implementation https://nd.edu/∼cone/CodonUsage/. Supplementary information Supplementary data are available at Bioinformatics online.

Effect of protein structure on evolution of cotranslational folding

Cotranslational folding is expected to occur when the folding speed of the nascent chain is faster than the translation speed of the ribosome, but it is difficult to predict which proteins cotranslationally fold. Here, we simulate evolution of model proteins to investigate how native structure influences evolution of cotranslational folding. We developed a model that connects protein folding during and after translation to cellular fitness. Model proteins evolved improved folding speed and stability, with proteins adopting one of two strategies for folding quickly. Low contact order proteins evolve to fold cotranslationally. Such proteins adopt native conformations early on during the translation process, with each subsequently translated residue establishing additional native contacts. On the other hand, high contact order proteins tend not to be stable in their native conformations until the full chain is nearly extruded. We also simulated evolution of slowly translating codons, finding that slowing translation at certain positions enhances cotranslational folding. Finally, we investigated real protein structures using a previously published dataset that identified evolutionarily conserved rare codons in E. coli genes and associated such codons with cotranslational folding intermediates. We found that protein substructures preceding conserved rare codons tend to have lower contact orders, in line with our finding that lower contact order proteins are more likely to fold cotranslationally. Our work shows how evolutionary selection pressure can cause proteins with local contact topologies to evolve cotranslational folding.Statement of significanceSubstantial evidence exists for proteins folding as they are translated by the ribosome. Here we developed a biologically intuitive evolutionary model to show that avoiding premature protein degradation can be a sufficient evolutionary force to drive evolution of cotranslational folding. Furthermore, we find that whether a protein’s native fold consists of more local or more nonlocal contacts affects whether cotranslational folding evolves. Proteins with local contact topologies are more likely to evolve cotranslational folding through nonsynonymous mutations that strengthen native contacts as well as through synonymous mutations that provide sufficient time for cotranslational folding intermediates to form.

In Silico Evaluation of the ATP7B Protein: Insights from the Role of Rare Codon Clusters and Mutations that Affect Protein Structure and Function

Background: Wilson’s disease is a rare autosomal recessive genetic disorder of copper metabolism, which is characterized by hepatic and neurological disease. ATP7B encodes a transmembrane protein ATPase (ATP7B), which functions as a copper-dependent P-type ATPase. The mutations in the gene ATP7B (on chromosome 13) lead to Wilson’s disease and is highly expressed in the liver, kidney, and placenta. Consequently, this enzyme was considered a special topic in clinical and biotechnological research. For in silico analysis, the 3D molecular modeling of this enzyme was conducted in the I-TASSER web server. Methods: For a better evaluation, the important characteristics of this enzyme such as the rare codons of the ATP7B gene were evaluated by online software, including a rare codon calculator (RCC), ATGme, LaTcOm, and Sherlocc program. Additionally, the multiple sequence alignment of this enzyme was studied. Finally, for evaluation of the effects of rare codons, the 3D structure of ATP7B was modeled in the Swiss Model and I-TASSER web server. Results: The results showed that the ATP7B gene has 35 single rare codons for Arg. Additionally, RCC detected two rare codons for Leu, 13 single rare codons for Ile and 28 rare codons for the Pro. ATP7B gene analysis in minmax and sliding_window algorithm resulted in the identification of 16 and 17 rare codon clusters, respectively, indicating the different features of these algorithms in the detection of RCCs. Analyzing the 3D model of ATP7B protein showed that Arg816 residue constitutes hydrogen bonds with Glu810 and Glu816. Mutation of this residue to Ser816 cause these hydrogen bonds not to be formed and may interfere in the proper folding of ATP7B protein. Furthermore, the side chain of Arg1228 does not form any bond with other residues. By mutation of Arg1228 to Thr1228, a new hydrogen bond is formed with the side chain of Arg1228. The addition and deletion of hydrogen bonds alter the proper folding of ATP7B protein and interfere with the proper function of the ATP7B position. On the other hand, His1069 forms the hydrogen bonds with the His880 and this hydrogen bond adhere two regions of the protein together, which is critical in the final structural folding of ATP7B protein. Conclusion: Previous studies show that synonymous and silent mutations have been linked to numerous diseases. Given the importance of synonymous and silent mutations in diseases, the aim of this study was to investigate the rare codons (synonymous codons) in the structure of ATP7B enzyme. By these analyses, a new understanding was developed and our findings can further be used in some fields of the clinical and industrial biotechnology.

Cotranslational folding allows misfolding-prone proteins to circumvent deep kinetic traps

Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins’ sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins’ sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily.

In silicoEvaluation of Substrate Binding Site and Rare Codons in the Structure of CYP152A1

Background:The Cytochromes P450 (CYPs) have an essential role in the oxidation of endogenous and exogenous molecules. The CYPs are identified in all domains of life, but the CYP152A1 from Bacillus subtilis is specially considered for clinical and industrial applications. The molecular cloning of a new type of CYP from Bacillus subtilis was reported, previously. Here, we describe the hidden layer of biological information of the CYP152A1 enzyme, which can help researchers for better understanding of enzyme application. In this study, four rare codons of enzyme, including Arg63, Arg187, Arg276, and Arg338 were identified and evaluated using the bioinformatics web servers.Methods:Through in silico modeling of CYP152A1 via the I-TASSER server, the above-mentioned rare codons were studied in the structure of enzyme that may have an important role in the proper folding of CYP152A1. In the following, the substrate binding site of CYP152A1 was studied by AutoDock Vina, and the heme and palmitic acid were considered as the substrates.Results:The results of docking study elucidated the Arg242 in the active site is closely related to the substrate binding site of CYP152A1, which help us to further clarify the mechanism of the enzyme reaction.Conclusion:Studies of these hidden information’s can enhance our understanding of CYP152A1 folding and protein expression challenges. Moreover, identification of rare codons can help in the rational design of new and effective drugs.