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1.
Gene ; 747: 144673, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32304783

RESUMO

Krabbe disease is one of the rarest autosomal recessive disorders in human, caused by mutation in the GALC (ß-galactosylceramidase) gene, resulting in several mental and physical health issues. Due to its rarity and phenotypic heterogeneity, diagnosis rate of this disease is very low. This study generated information on the recessive allele frequency dynamics of GALC gene across 15 global populations, with the highest frequency detected in Druze (Israel) population and the lowest frequency in Turkey and the United States. The recessive allele would take more time period (about 24,975 years) to be completely removed from the population having the lowest frequency and vice versa. The codon usage patterns of four isoforms of GALC gene revealed that a few synonymous codons were used more frequently than others in the isoforms. The codon AGA (arginine) was found to be overrepresented in GALC gene, except for galactocerebrosidase isoform a precursor. Further, GALC gene showed low codon usage bias (CUB) as evident from high ENC values (55.7-58.2), with A/T ending codons more preferred to G/C ending codons. CUB analysis elucidated the dual role of mutational pressure (major role) and natural selection (minor role) in GALC gene evolution.


Assuntos
Uso do Códon/genética , Galactosilceramidase/genética , Frequência do Gene/genética , Leucodistrofia de Células Globoides/enzimologia , Leucodistrofia de Células Globoides/genética , Aminoácidos/genética , Composição de Bases/genética , Códon/genética , Evolução Molecular , Galactosilceramidase/metabolismo , Humanos , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
2.
PLoS One ; 15(3): e0218302, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32191710

RESUMO

This study demonstrates that novel polymer production can be achieved by introducing pTAM, a broad-host-range plasmid expressing codon-optimized genes encoding Clostridium propionicum propionate CoA transferase (PctCp, Pct532) and a modified Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19, PhaC1400), into phaC mutant strains of the native polymer producers Sinorhizobium meliloti and Pseudomonas putida. Both phenotypic analysis and gas chromatography analysis indicated the synthesis and accumulation of biopolymers in S. meliloti and P. putida strains. Expression in S. meliloti resulted in the production of PLA homopolymer up to 3.2% dried cell weight (DCW). The quaterpolymer P (3HB-co-LA-co-3HHx-co-3HO) was produced by expression in P. putida. The P. putida phaC mutant strain produced this type of polymer the most efficiently with polymer content of 42% DCW when cultured in defined media with the addition of sodium octanoate. This is the first report, to our knowledge, of the production of a range of different biopolymers using the same plasmid-based system in different backgrounds. In addition, it is the first time that the novel polymer (P(3HB-co-LA-co-3HHx-co-3HO)), has been reported being produced in bacteria.


Assuntos
Engenharia Genética , Ácido Láctico/metabolismo , Polímeros/metabolismo , Pseudomonas putida/metabolismo , Sinorhizobium meliloti/metabolismo , Caprilatos/farmacologia , Códon/genética , Fluorescência , Genes Bacterianos , Glucuronidase/metabolismo , Isopropiltiogalactosídeo/farmacologia , Fenótipo , Plasmídeos/metabolismo , Poliésteres/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas putida/efeitos dos fármacos , Pseudomonas putida/genética , Sinorhizobium meliloti/efeitos dos fármacos , Sinorhizobium meliloti/genética
3.
J Microbiol Immunol Infect ; 53(3): 419-424, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32178970

RESUMO

Translation of a genetic codon without a cognate tRNA gene is affected by both the cognate tRNA availability and the interaction with non-cognate isoacceptor tRNAs. Moreover, two consecutive slow codons (slow di-codons) lead to a much slower translation rate. Calculating the composition of host specific slow codons and slow di-codons in the viral protein coding sequences can predict the order of viral protein synthesis rates between different virus strains. Comparison of human-specific slow codon and slow di-codon compositions in the genomes of 590 coronaviruses infect humans revealed that the protein synthetic rates of 2019 novel coronavirus (2019-nCoV) and severe acute respiratory syndrome-related coronavirus (SARS-CoV) may be much faster than other coronaviruses infect humans. Analysis of host-specific slow codon and di-codon compositions provides links between viral genomic sequences and capability of virus replication in host cells that may be useful for surveillance of the transmission potential of novel viruses.


Assuntos
Betacoronavirus/genética , Códon/genética , Biossíntese de Proteínas/genética , Vírus da SARS/genética , Genoma Viral/genética , Humanos , Filogenia , RNA de Transferência/genética , Replicação Viral/fisiologia
4.
PLoS One ; 15(2): e0225352, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32102090

RESUMO

INTRODUCTION: At low mutation-detection thresholds, next generation sequencing (NGS) for HIV-1 genotypic resistance testing is susceptible to artifactual detection of mutations arising from PCR error and APOBEC-mediated G-to-A hypermutation. METHODS: We analyzed published HIV-1 pol Illumina NGS data to characterize the distribution of mutations at eight NGS mutation detection thresholds: 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, and 0.1%. At each threshold, we determined proportions of amino acid mutations that were unusual (defined as having a prevalence <0.01% in HIV-1 group M sequences) or signature APOBEC mutations. RESULTS: Eight studies, containing 855 samples, in the NCBI Sequence Read Archive were analyzed. As detection thresholds were lowered, there was a progressive increase in the proportion of positions with usual and unusual mutations and in the proportion of all mutations that were unusual. The median proportion of positions with an unusual mutation increased gradually from 0% at the 20% threshold to 0.3% at the 1% threshold and then exponentially to 1.3% (0.5% threshold), 6.9% (0.2% threshold), and 23.2% (0.1% threshold). In two of three studies with available plasma HIV-1 RNA levels, the proportion of positions with unusual mutations was negatively associated with virus levels. Although the complete set of signature APOBEC mutations was much smaller than that of unusual mutations, the former outnumbered the latter in one-sixth of samples at the 0.5%, 1%, and 2% thresholds. CONCLUSIONS: The marked increase in the proportion of positions with unusual mutations at thresholds below 1% and in samples with lower virus loads suggests that, at low thresholds, many unusual mutations are artifactual, reflecting PCR error or G-to-A hypermutation. Profiling the numbers of unusual and signature APOBEC pol mutations at different NGS mutation detection thresholds may be useful to avoid selecting a threshold that is too low and poses an unacceptable risk of identifying artifactual mutations.


Assuntos
Desaminases APOBEC/genética , Infecções por HIV/genética , HIV-1/genética , Mutação , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética , Aminoácidos/genética , Códon/genética , Farmacorresistência Viral/genética , Genótipo , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Viral/sangue , RNA Viral/genética , Carga Viral/genética
5.
BMC Infect Dis ; 20(1): 87, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32000702

RESUMO

BACKGROUND: Xpert® MTB/RIF assay is currently used in Ethiopia for the rapid diagnosis of Mycobacterium tuberculosis (MTB) and mutations that confer Rifampicin resistance. Rifampicin resistance is determined based on any mutation in the 81 bp of rpoB gene using five overlapping probes represented as Probe A (codons 507-511), Probe B (codons 512-518), Probe C (codons 518-523), Probe D (codons 523-529) and Probe E (codons 529-533). In this review, we assessed the frequency of missed probe types for Rifampicin Resistance results. METHODS: Data were reviewed from specimens received and tested using Xpert® MTB/RIF assay at Ethiopian National Tuberculosis Reference Laboratory, in Addis Ababa from 15 July 2016 to 31 December 2018 retrospectively. All archived data were reviewed carefully to describe missed probe types and the quantity of DNA in the sample. RESULTS: A total of 100 specimens were reported as MTB Detected Rifampicin Resistance Detected by Xpert® MTB/RIF assay. More than half (55%) of these results were reported from male patients. The median age was 28.0 years (5 months to 88 years). Majorities (62%) of the cases were detected from sputum. Among the total of 38 extrapulmonary samples, lymph node aspirates were accounted for 50% (19/38). The most common mutations (81.0%) were found in the Probe E region followed by Probe D (10.0%), and Probe B (3.0%). Mutations in Probe A and Probe C regions were not observed. However, six (6.0%) Rifampicin resistance cases were found without any missed probe type. The delta Ct max is ≥4.3. No specimen yielded Rifampicin resistance associated with more than one probe failure or mutation combinations. CONCLUSION: Mutations associated with Probe E (codons 529-533) region were identified as the commonest rpoB gene mutations. The Rifampicin resistance results found without any identified missing probe needs further study. The lower DNA amount was observed in extrapulmonary specimens compared with sputum.


Assuntos
Farmacorresistência Bacteriana/genética , Testes Genéticos/métodos , Mutação , Mycobacterium tuberculosis/genética , Rifampina/uso terapêutico , Tuberculose/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Códon/genética , DNA/análise , Etiópia , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Rifampina/efeitos adversos , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose/microbiologia , Adulto Jovem
6.
Proc Natl Acad Sci U S A ; 117(7): 3528-3534, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015130

RESUMO

In the cell, proteins are synthesized from N to C terminus and begin to fold during translation. Cotranslational folding mechanisms are therefore linked to elongation rate, which varies as a function of synonymous codon usage. However, synonymous codon substitutions can affect many distinct cellular processes, which has complicated attempts to deconvolve the extent to which synonymous codon usage can promote or frustrate proper protein folding in vivo. Although previous studies have shown that some synonymous changes can lead to different final structures, other substitutions will likely be more subtle, perturbing predominantly the protein folding pathway without radically altering the final structure. Here we show that synonymous codon substitutions encoding a single essential enzyme lead to dramatically slower cell growth. These mutations do not prevent active enzyme formation; instead, they predominantly alter the protein folding mechanism, leading to enhanced degradation in vivo. These results support a model in which synonymous codon substitutions can impair cell fitness by significantly perturbing cotranslational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.


Assuntos
Cloranfenicol O-Acetiltransferase/química , Cloranfenicol O-Acetiltransferase/genética , Códon/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Mutação Silenciosa , Cloranfenicol O-Acetiltransferase/metabolismo , Uso do Códon , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Biossíntese de Proteínas , Dobramento de Proteína
7.
Nucleic Acids Res ; 48(5): 2777-2789, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32009164

RESUMO

The synthetic capability of the Escherichia coli ribosome has attracted efforts to repurpose it for novel functions, such as the synthesis of polymers containing non-natural building blocks. However, efforts to repurpose ribosomes are limited by the lack of complete peptidyl transferase center (PTC) active site mutational analyses to inform design. To address this limitation, we leverage an in vitro ribosome synthesis platform to build and test every possible single nucleotide mutation within the PTC-ring, A-loop and P-loop, 180 total point mutations. These mutant ribosomes were characterized by assessing bulk protein synthesis kinetics, readthrough, assembly, and structure mapping. Despite the highly-conserved nature of the PTC, we found that >85% of the PTC nucleotides possess mutational flexibility. Our work represents a comprehensive single-point mutant characterization and mapping of the 70S ribosome's active site. We anticipate that it will facilitate structure-function relationships within the ribosome and make possible new synthetic biology applications.


Assuntos
Domínio Catalítico , Escherichia coli/metabolismo , Mutação/genética , Ribossomos/química , Ribossomos/genética , Códon/genética , Modelos Moleculares , Peptidil Transferases/metabolismo , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Ribossômico/metabolismo
8.
Cell Mol Life Sci ; 77(1): 149-160, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31175370

RESUMO

Protein-coding nucleic acids exhibit composition and codon biases between sequences coding for intrinsically disordered regions (IDRs) and those coding for structured regions. IDRs are regions of proteins that are folding self-insufficient and which function without the prerequisite of folded structure. Several authors have investigated composition bias or codon selection in regions encoding for IDRs, primarily in Eukaryota, and concluded that elevated GC content is the result of the biased amino acid composition of IDRs. We substantively extend previous work by examining GC content in regions encoding IDRs, from 44 species in Eukaryota, Archaea, and Bacteria, spanning a wide range of GC content. We confirm that regions coding for IDRs show a significantly elevated GC content, even across all domains of life. Although this is largely attributable to the amino acid composition bias of IDRs, we show that this bias is independent of the overall GC content and, most importantly, we are the first to observe that GC content bias in IDRs is significantly different than expected from IDR amino acid composition alone. We empirically find compensatory codon selection that reduces the observed GC content bias in IDRs. This selection is dependent on the overall GC content of the organism. The codon selection bias manifests as use of infrequent, AT-rich codons in encoding IDRs. Further, we find these relationships to be independent of the intrinsic disorder prediction method used, and independent of estimated translation efficiency. These observations are consistent with the previous work, and we speculate on whether the observed biases are causal or symptomatic of other driving forces.


Assuntos
Códon/química , Proteínas Intrinsicamente Desordenadas/química , Animais , Composição de Bases , Códon/genética , Uso do Códon , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Biossíntese de Proteínas , Conformação Proteica
9.
Nucleic Acids Res ; 48(2): e7, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31777932

RESUMO

Recently, newly developed ribosome profiling methods based on high-throughput sequencing of ribosome-protected mRNA footprints allow to study genome-wide translational changes in detail. However, computational analysis of the sequencing data still represents a bottleneck for many laboratories. Further, specific pipelines for quality control and statistical analysis of ribosome profiling data, providing high levels of both accuracy and confidence, are currently lacking. In this study, we describe automated bioinformatic and statistical diagnoses to perform robust quality control of ribosome profiling data (RiboQC), to efficiently visualize ribosome positions and to estimate ribosome speed (RiboMine) in an unbiased way. We present an R pipeline to setup and undertake the analyses that offers the user an HTML page to scan own data regarding the following aspects: periodicity, ligation and digestion of footprints; reproducibility and batch effects of replicates; drug-related artifacts; unbiased codon enrichment including variability between mRNAs, for A, P and E sites; mining of some causal or confounding factors. We expect our pipeline to allow an optimal use of the wealth of information provided by ribosome profiling experiments.


Assuntos
Biologia Computacional , Ribossomos/genética , Software , Códon/genética , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética
10.
Sci Adv ; 5(12): eaax3124, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31840062

RESUMO

Different amino acid pairs have drastically different relative exchangeabilities (REs), and accounting for this variation is an important and common practice in inferring phylogenies, testing selection, and predicting mutational effects, among other analyses. In all such endeavors, REs have been generally considered invariant among species; this assumption, however, has not been scrutinized. Using maximum likelihood to analyze 180 genome sequences, we estimated REs from 90 clades representing all three domains of life, and found numerous instances of substantial between-clade differences in REs. REs show more differences between orthologous proteins of different clades than unrelated proteins of the same clade, suggesting that REs are genome-wide, clade-specific features, probably a result of proteome-wide evolutionary changes in the physicochemical environments of amino acid residues. The discovery of among-clade RE variations cautions against assuming constant REs in various analyses and demonstrates a higher-than-expected complexity in mechanisms of proteome evolution.


Assuntos
Aminoácidos/metabolismo , Filogenia , Animais , Viés , Códon/genética , Simulação por Computador , Evolução Molecular , Genoma , Humanos , Funções Verossimilhança , Proteoma/metabolismo
11.
PLoS One ; 14(12): e0225633, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31800603

RESUMO

The 3'-end of the coding sequence in several species is known to show specific codon usage bias. Several factors have been suggested to underlie this phenomenon, including selection against translation efficiency, selection for translation accuracy, and selection against RNA folding. All are supported by some evidence, but there is no general agreement as to which factors are the main determinants. Nor is it known how universal this phenomenon is, and whether the same factors explain it in different species. To answer these questions, we developed a measure that quantifies the codon usage bias at the gene end, and used it to compute this bias for 91 species that span the three domains of life. In addition, we characterized the codons in each species by features that allow discrimination between the different factors. Combining all these data, we were able to show that there is a universal trend to favor AT-rich codons toward the gene end. Moreover, we suggest that this trend is explained by avoidance from forming RNA secondary structures around the stop codon, which may interfere with normal translation termination.


Assuntos
Composição de Bases/genética , Uso do Códon/genética , Nucleotídeos/genética , Viés , Códon/genética , Regulação da Expressão Gênica , Humanos , Dobramento de RNA , RNA Mensageiro/química , RNA Mensageiro/genética
12.
PLoS One ; 14(12): e0226865, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31860647

RESUMO

Siraitia grosvenorii fruit, known as Luo-Han-Guo, has been used as a traditional Chinese medicine for many years, and mogrosides are its primary active ingredients. Unfortunately, Siraitia siamensis, its wild relative, might be misused due to its indistinguishable appearance, not only threatening the reliability of the medication but also partly exacerbating wild resource scarcity. Therefore, high-resolution genetic markers must be developed to discriminate between these species. Here, the complete chloroplast genomes of S. grosvenorii and S. siamensis were assembled and analyzed for the first time; they were 158,757 and 159,190 bp in length, respectively, and possessed conserved quadripartite circular structures. Both contained 134 annotated genes, including 8 rRNA, 37 tRNA and 89 protein-coding genes. Twenty divergences (Pi > 0.03) were found in the intergenic regions. Nine protein-coding genes, accD, atpA, atpE, atpF, clpP, ndhF, psbH, rbcL, and rpoC2, underwent selection within Cucurbitaceae. Phylogenetic relationship analysis indicated that these two species originated from the same ancestor. Finally, four pairs of molecular markers were developed to distinguish the two species. The results of this study will be beneficial for taxonomic research, identification and conservation of Siraitia Merrill wild resources in the future.


Assuntos
Cloroplastos/genética , Cucurbitaceae/genética , Genes de Plantas , Marcadores Genéticos/genética , Genoma de Cloroplastos , Códon/genética , Cucurbitaceae/classificação , Frutas/genética , Medicina Tradicional Chinesa , Anotação de Sequência Molecular , Filogenia , Repetições de Trinucleotídeos/genética , Sequenciamento Completo do Genoma
13.
BMC Bioinformatics ; 20(Suppl 24): 678, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861979

RESUMO

BACKGROUND: Ribosome profiling brings insight to the process of translation. A basic step in profile construction at transcript level is to map Ribo-seq data to transcripts, and then assign a huge number of multiple-mapped reads to similar isoforms. Existing methods either discard the multiple mapped-reads, or allocate them randomly, or assign them proportionally according to transcript abundance estimated from RNA-seq data. RESULTS: Here we present DeepShape, an RNA-seq free computational method to estimate ribosome abundance of isoforms, and simultaneously compute their ribosome profiles using a deep learning model. Our simulation results demonstrate that DeepShape can provide more accurate estimations on both ribosome abundance and profiles when compared to state-of-the-art methods. We applied DeepShape to a set of Ribo-seq data from PC3 human prostate cancer cells with and without PP242 treatment. In the four cell invasion/metastasis genes that are translationally regulated by PP242 treatment, different isoforms show very different characteristics of translational efficiency and regulation patterns. Transcript level ribosome distributions were analyzed by "Codon Residence Index (CRI)" proposed in this study to investigate the relative speed that a ribosome moves on a codon compared to its synonymous codons. We observe consistent CRI patterns in PC3 cells. We found that the translation of several codons could be regulated by PP242 treatment. CONCLUSION: In summary, we demonstrate that DeepShape can serve as a powerful tool for Ribo-seq data analysis.


Assuntos
Ribossomos/metabolismo , Análise de Sequência de RNA/métodos , Linhagem Celular Tumoral , Códon/genética , Códon/metabolismo , Humanos , Isoformas de Proteínas/genética , Software
14.
Nat Commun ; 10(1): 5774, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852903

RESUMO

Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.


Assuntos
Códon/genética , Elongação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Aminoácidos/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Biblioteca Gênica , Genes Reporter/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nucleotídeos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas RGS/genética , Proteínas RGS/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula
15.
PLoS One ; 14(12): e0219231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31841523

RESUMO

The deluge of sequence information in the recent times provide us with an excellent opportunity to compare organisms on a large genomic scale. In this study we have tried to decipher the variation in the gene organization and structuring of a vital bacterial gene called ftsZ which codes for an integral component of the bacterial cell division, the FtsZ protein. FtsZ is homologous to tubulin protein and has been found to be ubiquitous in eubacteria. FtsZ is showing increasing promise as a target for antibacterial drug discovery. Our study of ftsZ protein from 143 different bacterial species spanning a wider range of morphological and physiological type demonstrates that the ftsZ gene of about ninety three percent of the organisms show relatively biased codon usage profile and significant GC deviation from their genomic GC content. Comparative codon usage analysis of ftsZ and a core housekeeping gene rpoB demonstrated that codon usage pattern of ftsZ CDS is shaped by natural selection to a large extent and mimics that of a housekeeping gene. We have also detected a tendency among the different organisms to utilize a core set of codons in structuring the ftsZ coding sequence. We observed that the compositional frequency of the amino acid serine in the FtsZ protein appears to be a indicator of the bacterial lifestyle. Our meticulous analysis of the ftsZ gene linked with the corresponding FtsZ protein show that there is a bias towards the use of specific synonymous codons particularly in the helix and strand regions of the multi-domain FtsZ protein. Overall our findings suggest that in an indispensable and vital protein such as FtsZ, there is an inherent tendency to maintain form for optimized performance in spite of the extrinsic variability in coding features.


Assuntos
Proteínas de Bactérias/genética , Uso do Códon/genética , Códon/genética , Proteínas do Citoesqueleto/genética , Sequência de Aminoácidos , Bactérias/genética , Proteínas de Bactérias/metabolismo , Composição de Bases , Simulação por Computador , Proteínas do Citoesqueleto/metabolismo , Genes Bacterianos/genética , Genômica/métodos , Fases de Leitura Aberta , Seleção Genética , Tubulina (Proteína)/genética
16.
Mol Biol (Mosk) ; 53(6): 883-898, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31876270

RESUMO

In the cell, protein folding begins during protein synthesis/translation and thus is a co-translational process. Co-translational protein folding is tightly linked to translation elongation, which is not a uniform process. While there are many reasons for translation non-uniformity, it is generally believed that non-uniform synonymous codon usage is one of the key factors modulating translation elongation rates. Frequent/optimal codons as a rule are translated more rapidly than infrequently used ones and vice versa. Over 30 years ago, it was hypothesized that changes in synonymous codon usage affecting translation elongation rates could impinge on co-translation protein folding and that many synonymous codons are strategically placed within mRNA to ensure a particular translation kinetics facilitating productive step-by-step co-translational folding of proteins. It was suggested that this particular translation kinetics (and, specifically, translation pause sites) may define the window of opportunity for the protein parts to fold locally, particularly at the critical points where folding is far from equilibrium. It was thus hypothesized that synonymous codons may provide a secondary code for protein folding in the cell. Although, mostly accepted now, this hypothesis appeared to be difficult to prove and many convincing results were obtained only relatively recently. Here, I review the progress in the field and explain, why this simple idea appeared to be so challenging to prove.


Assuntos
Uso do Códon , Códon/genética , Biossíntese de Proteínas , Dobramento de Proteína , Proteínas/genética , Proteínas/metabolismo , Biossíntese de Proteínas/genética , Proteínas/química
17.
BMC Biol ; 17(1): 105, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842858

RESUMO

BACKGROUND: Single nucleotide substitutions in protein-coding genes can be divided into synonymous (S), with little fitness effect, and non-synonymous (N) ones that alter amino acids and thus generally have a greater effect. Most of the N substitutions are affected by purifying selection that eliminates them from evolving populations. However, additional mutations of nearby bases potentially could alleviate the deleterious effect of single substitutions, making them subject to positive selection. To elucidate the effects of selection on double substitutions in all codons, it is critical to differentiate selection from mutational biases. RESULTS: We addressed the evolutionary regimes of within-codon double substitutions in 37 groups of closely related prokaryotic genomes from diverse phyla by comparing the fractions of double substitutions within codons to those of the equivalent double S substitutions in adjacent codons. Under the assumption that substitutions occur one at a time, all within-codon double substitutions can be represented as "ancestral-intermediate-final" sequences (where "intermediate" refers to the first single substitution and "final" refers to the second substitution) and can be partitioned into four classes: (1) SS, S intermediate-S final; (2) SN, S intermediate-N final; (3) NS, N intermediate-S final; and (4) NN, N intermediate-N final. We found that the selective pressure on the second substitution markedly differs among these classes of double substitutions. Analogous to single S (synonymous) substitutions, SS double substitutions evolve neutrally, whereas analogous to single N (non-synonymous) substitutions, SN double substitutions are subject to purifying selection. In contrast, NS show positive selection on the second step because the original amino acid is recovered. The NN double substitutions are heterogeneous and can be subject to either purifying or positive selection, or evolve neutrally, depending on the amino acid similarity between the final or intermediate and the ancestral states. CONCLUSIONS: The results of the present, comprehensive analysis of the evolutionary landscape of within-codon double substitutions reaffirm the largely conservative regime of protein evolution. However, the second step of a double substitution can be subject to positive selection when the first step is deleterious. Such positive selection can result in frequent crossing of valleys on the fitness landscape.


Assuntos
Códon/genética , Evolução Molecular , Mutação , Células Procarióticas/fisiologia , Seleção Genética
18.
Genes (Basel) ; 10(11)2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31698749

RESUMO

Codon usage bias (CUB)-preferential use of one of the synonymous codons, has been described in a wide range of organisms from bacteria to mammals, but it has not yet been studied in marine phytoplankton. CUB is thought to be caused by weak selection for translational accuracy and efficiency. Weak selection can overpower genetic drift only in species with large effective population sizes, such as Drosophila that has relatively strong CUB, while organisms with smaller population sizes (e.g., mammals) have weak CUB. Marine plankton species tend to have extremely large populations, suggesting that CUB should be very strong. Here we test this prediction and describe the patterns of codon usage in a wide range of diatom species belonging to 35 genera from 4 classes. We report that most of the diatom species studied have surprisingly modest CUB (mean Effective Number of Codons, ENC = 56), with some exceptions showing stronger codon bias (ENC = 44). Modest codon bias in most studied diatom species may reflect extreme disparity between astronomically large census and modest effective population size (Ne), with fluctuations in population size and linked selection limiting long-term Ne and rendering selection for optimal codons less efficient. For example, genetic diversity (pi ~0.02 at silent sites) in Skeletonema marinoi corresponds to Ne of about 10 million individuals, which is likely many orders of magnitude lower than its census size. Still, Ne ~107 should be large enough to make selection for optimal codons efficient. Thus, we propose that an alternative process-frequent changes of preferred codons, may be a more plausible reason for low CUB despite highly efficient selection for preferred codons in diatom populations. The shifts in the set of optimal codons should result in the changes of the direction of selection for codon usage, so the actual codon usage never catches up with the moving target of the optimal set of codons and the species never develop strong CUB. Indeed, we detected strong shifts in preferential codon usage within some diatom genera, with switches between preferentially GC-rich and AT-rich 3rd codon positions (GC3). For example, GC3 ranges from 0.6 to 1 in most Chaetoceros species, while for Chaetoceros dichaeta GC3 = 0.1. Both variation in selection intensity and mutation spectrum may drive such shifts in codon usage and limit the observed CUB. Our study represents the first genome-wide analysis of CUB in diatoms and the first such analysis for a major phytoplankton group.


Assuntos
Uso do Códon/genética , Códon/genética , Diatomáceas/genética , Evolução Biológica , Evolução Molecular , Mutação , Seleção Genética/genética
19.
Proc Natl Acad Sci U S A ; 116(46): 23068-23074, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31672910

RESUMO

Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Ψ), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with Ψ in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the Thermus thermophilus 70S ribosome with a tRNAPhe bound to a ΨUU codon in the A site supports these findings. We also find that the presence of Ψ can promote the low-level synthesis of multiple peptide products from a single mRNA sequence in the reconstituted translation system as well as human cells, and increases the rate of near-cognate Val-tRNAVal reacting on a ΨUU codon. The vast majority of Ψ moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Ψ can be to modestly alter both translation speed and mRNA decoding.


Assuntos
Biossíntese de Proteínas , Pseudouridina/metabolismo , RNA Bacteriano/genética , RNA Mensageiro/genética , Thermus thermophilus/genética , Códon/genética , Códon/metabolismo , Fases de Leitura Aberta , Elongação Traducional da Cadeia Peptídica , Pseudouridina/genética , Processamento Pós-Transcricional do RNA , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Thermus thermophilus/metabolismo , Uridina/metabolismo
20.
Genetics ; 213(4): 1531-1544, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31653677

RESUMO

Targeted identification and purging of deleterious genetic variants has been proposed as a novel approach to animal and plant breeding. This strategy is motivated, in part, by the observation that demographic events and strong selection associated with cultivated species pose a "cost of domestication." This includes an increase in the proportion of genetic variants that are likely to reduce fitness. Recent advances in DNA resequencing and sequence constraint-based approaches to predict the functional impact of a mutation permit the identification of putatively deleterious SNPs (dSNPs) on a genome-wide scale. Using exome capture resequencing of 21 barley lines, we identified 3855 dSNPs among 497,754 total SNPs. We generated whole-genome resequencing data of Hordeum murinum ssp. glaucum as a phylogenetic outgroup to polarize SNPs as ancestral vs. derived. We also observed a higher proportion of dSNPs per synonymous SNPs (sSNPs) in low-recombination regions of the genome. Using 5215 progeny from a genomic prediction experiment, we examined the fate of dSNPs over three breeding cycles. Adjusting for initial frequency, derived alleles at dSNPs reduced in frequency or were lost more often than other classes of SNPs. The highest-yielding lines in the experiment, as chosen by standard genomic prediction approaches, carried fewer homozygous dSNPs than randomly sampled lines from the same progeny cycle. In the final cycle of the experiment, progeny selected by genomic prediction had a mean of 5.6% fewer homozygous dSNPs relative to randomly chosen progeny from the same cycle.


Assuntos
Variação Genética , Genômica , Hordeum/genética , Variação Biológica da População , Códon/genética , Exoma/genética , Frequência do Gene/genética , Genética Populacional , Genótipo , Homozigoto , Melhoramento Vegetal , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA
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