A new look at the fluorescent protein-based approach for identifying optimal coding sequence for recombinant protein expression in E. coli.

Due to the degeneracy of the genetic code, most amino acids are encoded by several codons. The choice among synonymous codons at the N-terminus of genes has a profound effect on protein expression in Escherichia coli. This is often explained by the different contributions of synonymous codons to mRNA secondary structure formation. Strong secondary structures at the 5'-end of mRNA interfere with ribosome binding and affect the process of translation initiation. In silico optimization of the gene 5'-end can significantly increase the level of protein expression; however, this method is not always effective due to the uncertainty of the exact mechanism by which synonymous substitutions affect expression; thus, it may produce nonoptimal variants as well as miss some of the best producers. In this paper, an alternative approach is proposed based on screening a partially randomized library of expression constructs comprising hundreds of selected synonymous variants. The effect of such substitutions was evaluated using the gene of interest fused to the reporter gene of the fluorescent protein with subsequent screening for the most promising candidates according to the reporter's signal intensity. The power of the approach is demonstrated by a significant increase in the prokaryotic expression of three proteins: canine cystatin C, human BCL2-associated athanogene 3 and human cardiac troponin I. This simple approach was suggested which may provide an efficient, easy, and inexpensive optimization method for poorly expressed proteins in bacteria.

Translation velocity determines the efficacy of engineered suppressor tRNAs on pathogenic nonsense mutations.

Nonsense mutations - the underlying cause of approximately 11% of all genetic diseases - prematurely terminate protein synthesis by mutating a sense codon to a premature stop or termination codon (PTC). An emerging therapeutic strategy to suppress nonsense defects is to engineer sense-codon decoding tRNAs to readthrough and restore translation at PTCs. However, the readthrough efficiency of the engineered suppressor tRNAs (sup-tRNAs) largely varies in a tissue- and sequence context-dependent manner and has not yet yielded optimal clinical efficacy for many nonsense mutations. Here, we systematically analyze the suppression efficacy at various pathogenic nonsense mutations. We discover that the translation velocity of the sequence upstream of PTCs modulates the sup-tRNA readthrough efficacy. The PTCs most refractory to suppression are embedded in a sequence context translated with an abrupt reversal of the translation speed leading to ribosomal collisions. Moreover, modeling translation velocity using Ribo-seq data can accurately predict the suppression efficacy at PTCs. These results reveal previously unknown molecular signatures contributing to genotype-phenotype relationships and treatment-response heterogeneity, and provide the framework for the development of personalized tRNA-based gene therapies.

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria.

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

Three complete chloroplast genomes from two north American Rhus species and phylogenomics of Anacardiaceae.

BACKGROUND: The suamc genus Rhus (sensu stricto) includes two subgenera, Lobadium (ca. 25 spp.) and Rhus (ca. 10 spp.). Their members, R. glabra and R. typhina (Rosanae: Sapindales: Anacardiaceae), are two economic important species. Chloroplast genome information is of great significance for the study of plant phylogeny and taxonomy. RESULTS: The three complete chloroplast genomes from two Rhus glabra and one R. typhina accessions were obtained with a total of each about 159k bp in length including a large single-copy region (LSC, about 88k bp), a small single-copy regions (SSC, about 19k bp) and a pair of inverted repeats regions (IRa/IRb, about 26k bp), to form a canonical quadripartite structure. Each genome contained 88 protein-coding genes, 37 transfer RNA genes, eight ribosomal RNA genes and two pseudogenes. The overall GC content of the three genomes all were same (37.8%), and RSCU values showed that they all had the same codon prefers, i.e., to use codon ended with A/U (93%) except termination codon. Three variable hotspots, i.e., ycf4-cemA, ndhF-rpl32-trnL and ccsA-ndhD, and a total of 152-156 simple sequence repeats (SSR) were identified. The nonsynonymous (Ka)/synonymous (Ks) ratio was calculated, and cemA and ycf2 genes are important indicators of gene evolution. The phylogenetic analyses of the family Anacardiaceae showed that the eight genera were grouped into three clusters, and supported the monophyly of the subfamilies and all the genera. The accessions of five Rhus species formed four clusters, while, one individual of R. typhina grouped with the R. glabra accessions instead of clustering into the two other individuals of R. typhina in the subgenus Rhus, which showed a paraphyletic relationship. CONCLUSIONS: Comparing the complete chloroplast genomes of the Rhus species, it was found that most SSRs were A/T rich and located in the intergenic spacer, and the nucleotide divergence exhibited higher levels in the non-coding region than in the coding region. The Ka/Ks ratio of cemA gene was > 1 for species collected in America, while it was < 1 for other species in China, which dedicated that the Rhus species from North America and East Asia have different evolutionary pressure. The phylogenetic analysis of the complete chloroplast genome clarified the Rhus placement and relationship. The results obtained in this study are expected to provide valuable genetic resources to perform species identification, molecular breeding, and intraspecific diversity of the Rhus species.

Decomposing bulk signals to reveal hidden information in processive enzyme reactions: A case study in mRNA translation.

Processive enzymes like polymerases or ribosomes are often studied in bulk experiments by monitoring time-dependent signals, such as fluorescence time traces. However, due to biomolecular process stochasticity, ensemble signals may lack the distinct features of single-molecule signals. Here, we demonstrate that, under certain conditions, bulk signals from processive reactions can be decomposed to unveil hidden information about individual reaction steps. Using mRNA translation as a case study, we show that decomposing a noisy ensemble signal generated by the translation of mRNAs with more than a few codons is an ill-posed problem, addressable through Tikhonov regularization. We apply our method to the fluorescence signatures of in-vitro translated LepB mRNA and determine codon-position dependent translation rates and corresponding state-specific fluorescence intensities. We find a significant change in fluorescence intensity after the fourth and the fifth peptide bond formation, and show that both codon position and encoded amino acid have an effect on the elongation rate. This demonstrates that our approach enhances the information content extracted from bulk experiments, thereby expanding the range of these time- and cost-efficient methods.

Riboformer: a deep learning framework for predicting context-dependent translation dynamics.

Translation elongation is essential for maintaining cellular proteostasis, and alterations in the translational landscape are associated with a range of diseases. Ribosome profiling allows detailed measurements of translation at the genome scale. However, it remains unclear how to disentangle biological variations from technical artifacts in these data and identify sequence determinants of translation dysregulation. Here we present Riboformer, a deep learning-based framework for modeling context-dependent changes in translation dynamics. Riboformer leverages the transformer architecture to accurately predict ribosome densities at codon resolution. When trained on an unbiased dataset, Riboformer corrects experimental artifacts in previously unseen datasets, which reveals subtle differences in synonymous codon translation and uncovers a bottleneck in translation elongation. Further, we show that Riboformer can be combined with in silico mutagenesis to identify sequence motifs that contribute to ribosome stalling across various biological contexts, including aging and viral infection. Our tool offers a context-aware and interpretable approach for standardizing ribosome profiling datasets and elucidating the regulatory basis of translation kinetics.

Blastocrithidia nonstop mitochondrial genome and its expression are remarkably insulated from nuclear codon reassignment.

The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.

Codon usage bias and phylogenetic analysis of chloroplast genome in 36 gracilariaceae species.

Gracilariaceae is a group of marine large red algae and main source of agar with important economic and ecological value. The codon usage patterns of chloroplast genomes in 36 species from Graciliaceae show that GC range from 0.284 to 0.335, the average GC3 range from 0.135 to 0.243 and the value of ENC range from 35.098 to 42.327, which indicates these genomes are rich in AT and prefer to use codons ending with AT in these species. Nc plot, PR2 plot, neutrality plot analyses and correlation analysis indicate that these biases may be caused by multiple factors, such as natural selection and mutation pressure, but prolonged natural selection is the main driving force influencing codon usage preference. The cluster analysis and phylogenetic analysis show that the differentiation relationship of them is different and indicate that codons with weak or unbiased preferences may also play an irreplaceable role in these species' evolution. In addition, we identified 26 common high-frequency codons and 8-18 optimal codons all ending in A/U in these 36 species. Our results will not only contribute to carrying out transgenic work in Gracilariaceae species to maximize the protein yield in the future, but also lay a theoretical foundation for further exploring systematic classification of them.

GC Content Across Insect Genomes: Phylogenetic Patterns, Causes and Consequences.

The proportions of A:T and G:C nucleotide pairs are often unequal and can vary greatly between animal species and along chromosomes. The causes and consequences of this variation are incompletely understood. The recent release of high-quality genome sequences from the Darwin Tree of Life and other large-scale genome projects provides an opportunity for GC heterogeneity to be compared across a large number of insect species. Here we analyse GC content along chromosomes, and within protein-coding genes and codons, of 150 insect species from four holometabolous orders: Coleoptera, Diptera, Hymenoptera, and Lepidoptera. We find that protein-coding sequences have higher GC content than the genome average, and that Lepidoptera generally have higher GC content than the other three insect orders examined. GC content is higher in small chromosomes in most Lepidoptera species, but this pattern is less consistent in other orders. GC content also increases towards subtelomeric regions within protein-coding genes in Diptera, Coleoptera and Lepidoptera. Two species of Diptera, Bombylius major and B. discolor, have very atypical genomes with ubiquitous increase in AT content, especially at third codon positions. Despite dramatic AT-biased codon usage, we find no evidence that this has driven divergent protein evolution. We argue that the GC landscape of Lepidoptera, Diptera and Coleoptera genomes is influenced by GC-biased gene conversion, strongest in Lepidoptera, with some outlier taxa affected drastically by counteracting processes.

Significance of genetic code module structure in gene expression and GC content enhancement in RNA sequences.

The existent algebraic models of the genetic code contribute to the understanding of the physio-chemical characteristics of the amino acids. However, the process of translating a gene into a phenotype is highly complex. Moreover, the intricacy of gene expression gets further multiplied due to the biases in the codon usage. This paper explores an algebraic structure called module on the set of codons as well as on that of RNA sequences. We study the potential implications of these structures on gene expression and the GC content of an RNA sequence. The base order {C,U,G,A} appears to possess greater biological significance than many of the orders previously studied. We have developed a novel algorithm to generate RNA sequences with high GC content, aiming to enhance the thermostability of biomolecules. The insights gained from this investigation may have applications in biomolecular modeling and docking, protein engineering, drug development, and related fields.

Alter codon bias of the P. pastoris genome to overcome a bottleneck in codon optimization strategy development and improve protein expression.

Apart from its role in translation, codon bias is also an important mechanism to regulate mRNA levels. The traditional frequency-based codon optimization strategy is rather efficient in organisms such as N. crassa, but much less in yeast P. pastoris which is a popular host for heterologous protein expression. This is because that unlike N. crassa, the preferred codons of P. pastoris are actually AU-rich and hence codon optimization for extremely low GC content comes with issues of pre-mature transcriptional termination or low RNA stability in spite of translational advantages. To overcome this bottleneck, we focused on three reporter genes in P. pastoris first and confirmed the great advantage of GC-prone codon optimization on mRNA levels. Then we altered the codon bias profile of P. pastoris by introducing additional rare tRNA gene copies. Prior to that we constructed IPTG-regulated tRNA species to enable chassis cells to switch between different codon bias status. As demonstrated again with reporter genes, protein yield of luc and 0788 was successfully increased by 4-5 folds in chassis cells. In summary, here we provide an alternative codon optimization strategy for genes with unsatisfactory performance under traditional codon frequency-based optimization.

Codon Pattern and Context Analysis in Genes Triggering Alzheimer's Disease and Latent Tau Protein Aggregation Post-Anesthesia Exhibited Unique Molecular Patterns Associated with Functional Aspects.

Background: Previous reports have demonstrated post-operative dementia and Alzheimer's disease (AD), and increased amyloid-ß levels and tau hyperphosphorylation have been observed in animal models post-anesthesia. Objective: After surgical interventions, loss in memory has been observed that has been found linked with genes modulated after anesthesia. Present study aimed to study molecular pattern present in genes modulated post anesthesia and involved in characters progressing towards AD. Methods: In the present study, 17 transcript variants belonging to eight genes, which have been found to modulate post-anesthesia and contribute to AD progression, were envisaged for their compositional features, molecular patterns, and codon and codon context-associated studies. Results: The sequences' composition was G/C rich, influencing dinucleotide preference, codon preference, codon usage, and codon context. The G/C nucleotides being highly occurring nucleotides, CpGdinucleotides were also preferred; however, CpG was highly disfavored at p3-1 at the codon junction. The nucleotide composition of Cytosine exhibited a unique feature, and unlike other nucleotides, it did not correlate with codon bias. Contrarily, it correlated with the sequence lengths. The sequences were leucine-rich, and multiple leucine repeats were present, exhibiting the functional role of neuroprotection from neuroinflammation post-anesthesia. Conclusions: The analysis pave the way to elucidate unique molecular patterns in genes modulated during anesthetic treatment and might help ameliorate the ill effects of anesthetics in the future.

Evolving precision: rRNA expansion segment 7S modulates translation velocity and accuracy in eukaryal ribosomes.

Ribosome-enhanced translational miscoding of the genetic code causes protein dysfunction and loss of cellular fitness. During evolution, open reading frame length increased, necessitating mechanisms for enhanced translation fidelity. Indeed, eukaryal ribosomes are more accurate than bacterial counterparts, despite their virtually identical, conserved active centers. During the evolution of eukaryotic organisms ribosome expansions at the rRNA and protein level occurred, which potentially increases the options for translation regulation and cotranslational events. Here we tested the hypothesis that ribosomal RNA expansions can modulate the core function of the ribosome, faithful protein synthesis. We demonstrate that a short expansion segment present in all eukaryotes' small subunit, ES7S, is crucial for accurate protein synthesis as its presence adjusts codon-specific velocities and guarantees high levels of cognate tRNA selection. Deletion of ES7S in yeast enhances mistranslation and causes protein destabilization and aggregation, dramatically reducing cellular fitness. Removal of ES7S did not alter ribosome architecture but altered the structural dynamics of inter-subunit bridges thus affecting A-tRNA selection. Exchanging the yeast ES7S sequence with the human ES7S increases accuracy whereas shortening causes the opposite effect. Our study demonstrates that ES7S provided eukaryal ribosomes with higher accuracy without perturbing the structurally conserved decoding center.

Variably protease-sensitive prionopathy with methionine homozygosity at codon 129 in the prion protein gene.

Variably protease-sensitive prionopathy (VPSPr) is a recently characterised rare subtype of sporadic prion disease, mainly affecting individuals with valine homozygosity at codon 129 in the prion protein gene, with only seven methionine homozygote cases reported to date. This case presents clinical, neuropathological and biochemical features of the eighth VPSPr case worldwide with methionine homozygosity at codon 129 and compares the features with the formerly presented cases.The patient, a woman in her 70s, presented with cognitive decline, impaired balance and frequent falls. Medical history and clinical presentation were suggestive of a rapidly progressive dementia disorder. MRI showed bilateral thalamic hyperintensity. Cerebrospinal fluid real-time quaking-induced conversion was negative, and the electroencephalogram was unremarkable. The diagnosis was established through post-mortem pathological examinations. VPSPr should be suspected in rapidly progressive dementia lacking typical features or paraclinical results of protein misfolding diseases.

C2-methyladenosine in tRNA promotes protein translation by facilitating the decoding of tandem m2A-tRNA-dependent codons.

RNA modification C2-methyladenosine (m2A) exists in both rRNA and tRNA of Escherichia coli (E. coli), installed by the methyltransferase RlmN using a radical-S-adenosylmethionine (SAM) mechanism. However, the precise function of m2A in tRNA and its ubiquity in plants have remained unclear. Here we discover the presence of m2A in chloroplast rRNA and tRNA, as well as cytosolic tRNA, in multiple plant species. We identify six m2A-modified chloroplast tRNAs and two m2A-modified cytosolic tRNAs across different plants. Furthermore, we characterize three Arabidopsis m2A methyltransferases-RLMNL1, RLMNL2, and RLMNL3-which methylate chloroplast rRNA, chloroplast tRNA, and cytosolic tRNA, respectively. Our findings demonstrate that m2A37 promotes a relaxed conformation of tRNA, enhancing translation efficiency in chloroplast and cytosol by facilitating decoding of tandem m2A-tRNA-dependent codons. This study provides insights into the molecular function and biological significance of m2A, uncovering a layer of translation regulation in plants.

Characterization of the chloroplast genome of Gleditsia species and comparative analysis.

The genus Gleditsia has significant medicinal and economic value, but information about the chloroplast genomic characteristics of Gleditsia species has been limited. Using the Illumina sequencing, we assembled and annotated the whole chloroplast genomes of seven Gleditsia species (Gleditsia sinensis, Gleditsia japonica var. delavayi (G. delavayi), G. fera, G. japonica, G. microphylla, Fructus Gleditsiae Abnormalis (Zhu Yá Zào), G. microphylla mutant). The assembled genomes revealed that Gleditsia species have a typical circular tetrad structure, with genome sizes ranging from 162,746 to 170,907 bp. Comparative genomic analysis showed that most (65.8-75.8%) of the abundant simple sequence repeats in Gleditsia and Gymnocladus species were located in the large single copy region. The Gleditsia chloroplast genome prefer T/A-ending codons and avoid C/G-ending codons, positive selection was acting on the rpoA, rpl20, atpB, ndhA and ycf4 genes, most of the chloroplast genes of Gleditsia species underwent purifying selection. Expansion and contraction of the inverted repeat (IR)/single copy (SC) region showed similar patterns within the Gleditsia genus. Polymorphism analysis revealed that coding regions were more conserved than non-coding regions, and the IR region was more conserved than the SC region. Mutational hotspots were mostly found in intergenic regions such as "rps16-trnQ", "trnT-trnL", "ndhG-ndhI", and "rpl32-trnL" in Gleditsia. Phylogenetic analysis showed that G. fera is most closely related to G. sinensis,G. japonica and G. delavayi are relatively closely related. Zhu Yá Zào can be considered a bud mutation of the G. sinensis. The albino phenotype of G. microphylla mutant is not caused by variations in the chloroplast genome, and that the occurrence of the albino phenotype may be due to mutations in chloroplast-related genes involved in splicing or localization functions. This study will help us enhance our exploration of the genetic evolution and geographical origins of the Gleditsia genus.

Relative synonymous codon usage and codon pair analysis of depression associated genes.

Depression negatively impacts mood, behavior, and mental and physical health. It is the third leading cause of suicides worldwide and leads to decreased quality of life. We examined 18 genes available at the genetic testing registry (GTR) from the National Center for Biotechnological Information to investigate molecular patterns present in depression-associated genes. Different genotypes and differential expression of the genes are responsible for ensuing depression. The present study, investigated codon pattern analysis, which might play imperative roles in modulating gene expression of depression-associated genes. Of the 18 genes, seven and two genes tended to up- and down-regulate, respectively, and, for the remaining genes, different genotypes, an outcome of SNPs were responsible alone or in combination with differential expression for different conditions associated with depression. Codon context analysis revealed the abundance of identical GTG-GTG and CTG-CTG pairs, and the rarity of methionine-initiated codon pairs. Information based on codon usage, preferred codons, rare, and codon context might be used in constructing a deliverable synthetic construct to correct the gene expression level of the human body, which is altered in the depressive state. Other molecular signatures also revealed the role of evolutionary forces in shaping codon usage.

Expanding the synthetic biology toolbox of Cupriavidus necator for establishing fatty acid production.

The Gram-negative betaproteobacterium Cupriavidus necator is a chemolithotroph that can convert carbon dioxide into biomass. Cupriavidus necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack of a well-characterized toolbox for gene expression and genome engineering. Development and optimization of biosynthetic pathways in metabolically engineered microorganisms necessitates control of gene expression via functional genetic elements such as promoters, ribosome binding sites (RBSs), and codon optimization. In this work, a set of inducible and constitutive promoters were validated and characterized in C. necator, and a library of RBSs was designed and tested to show a 50-fold range of expression for green fluorescent protein (gfp). The effect of codon optimization on gene expression in C. necator was studied by expressing gfp and mCherry genes with varied codon-adaptation indices and was validated by expressing codon-optimized variants of a C12-specific fatty acid thioesterase to produce dodecanoic acid. We discuss further hurdles that will need to be overcome for C. necator to be widely used for biosynthetic processes.

Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human.

Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.

Antibody production relies on the tRNA inosine wobble modification to meet biased codon demand.

Antibodies are produced at high rates to provide immunoprotection, which puts pressure on the B cell translational machinery. Here, we identified a pattern of codon usage conserved across antibody genes. One feature thereof is the hyperutilization of codons that lack genome-encoded Watson-Crick transfer RNAs (tRNAs), instead relying on the posttranscriptional tRNA modification inosine (I34), which expands the decoding capacity of specific tRNAs through wobbling. Antibody-secreting cells had increased I34 levels and were more reliant on I34 for protein production than naïve B cells. Furthermore, antibody I34-dependent codon usage may influence B cell passage through regulatory checkpoints. Our work elucidates the interface between the tRNA pool and protein production in the immune system and has implications for the design and selection of antibodies for vaccines and therapeutics.