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1.
Mol Biol (Mosk) ; 53(4): 561-573, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397432

RESUMO

The protein synthesis in cells occurs in ribosomes, with the involvement of protein translational factors. One of these translational factors is the elongation factor P (EF-P). EF-P is a three-domain protein that binds between the P and E sites of the ribosome, near the P-tRNA, the peptidyl transferase center, and E-site codon of the mRNA. The majority of studies showed that the EF-P helps the ribosome to synthesize stalling amino acid motifs, such as polyprolines. In the first part of this review, we inspect the general evolutionary variety of the EF-P in different organisms, the problems of the regulation provided by the EF-P, and its role in the sustainability of the protein balance in the cell in different physiological states. Although the functions of the EF-P have been well studied, there are still some problems that remain to be solved. The data from recent studies contradict the previous theories. Consequently, in the second part, we discuss the recent data that suggest the involvement of the EF-P in each translocation event, not only in those related to poly-proline synthesis. This activity contradicts some aspects of the known pathway of the removal of the E-tRNA during the translocation event. In addition, in the third part of this review, we tried to partly shift the interest from the antistalling activity of domain I of the EF-P to the action of domain III, the functions of which has not been closely studied. We expand on the idea about the involvement of domain III of the EF-P in preventing the frameshift and debate the EF-P's evolutionary history.


Assuntos
Evolução Molecular , Fatores de Alongamento de Peptídeos/metabolismo , Biossíntese de Proteínas , Animais , Humanos , Fatores de Alongamento de Peptídeos/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/metabolismo
2.
Arch Virol ; 164(10): 2627-2630, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31363923

RESUMO

A lytic bacteriophage, designated Vibrio phage vB_VpP_BA6, was isolated from sewage collected in Guangzhou, China. The double-stranded DNA genome of phage BA6 is composed of 50,520 bp with a G+C content of 41.77%. It possesses 64 open reading frames relating to phage structure, packaging, host lysis, DNA metabolism, and additional functions. Three tRNAs genes (encoding Pro, Ile and Trp) were detected. Comparison of its genomic features and phylogenetic analysis revealed that phage BA6 is a novel member of the family Podoviridae. This phage may represent a potential therapeutic agent against multidrug-resistant Vibrio parahaemolyticus.


Assuntos
Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Genoma Viral , Podoviridae/genética , Podoviridae/isolamento & purificação , Vibrio parahaemolyticus/virologia , Bacteriólise , Bacteriófagos/classificação , Bacteriófagos/crescimento & desenvolvimento , Composição de Bases , China , DNA/química , DNA/genética , Fases de Leitura Aberta , Filogenia , Podoviridae/classificação , Podoviridae/crescimento & desenvolvimento , RNA de Transferência/genética , Esgotos/virologia
3.
Genes Dev ; 33(13-14): 741-746, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171702

RESUMO

Site-specific 2'-O-ribose methylation of mammalian rRNAs and RNA polymerase II-synthesized spliceosomal small nuclear RNAs (snRNAs) is mediated by small nucleolar and small Cajal body (CB)-specific box C/D ribonucleoprotein particles (RNPs) in the nucleolus and the nucleoplasmic CBs, respectively. Here, we demonstrate that 2'-O-methylation of the C34 wobble cytidine of human elongator tRNAMet(CAT) is achieved by collaboration of a nucleolar and a CB-specific box C/D RNP carrying the SNORD97 and SCARNA97 box C/D 2'-O-methylation guide RNAs. Methylation of C34 prevents site-specific cleavage of tRNAMet(CAT) by the stress-induced endoribonuclease angiogenin, implicating box C/D guide RNPs in controlling stress-responsive production of putative regulatory tRNA fragments.


Assuntos
Nucléolo Celular/metabolismo , Corpos Enovelados/metabolismo , Citidina/metabolismo , RNA de Transferência/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Células HeLa , Humanos , Metilação , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/genética , Ribonuclease Pancreático/metabolismo , Ribonucleoproteínas/genética , Estresse Fisiológico
4.
BMC Evol Biol ; 19(1): 124, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31215393

RESUMO

BACKGROUND: Mycobacteria occupy various ecological niches and can be isolated from soil, tap water and ground water. Several cause diseases in humans and animals. To get deeper insight into our understanding of mycobacterial evolution focusing on tRNA and non-coding (nc)RNA, we conducted a comparative genome analysis of Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clade members. RESULTS: Genome sizes for Mmuc- and Mneo-clade members vary between 5.4 and 6.5 Mbps with the complete MmucT (type strain) genome encompassing 6.1 Mbp. The number of tRNA genes range between 46 and 79 (including one pseudo tRNA gene) with 39 tRNA genes common among the members of these clades, while additional tRNA genes were probably acquired through horizontal gene transfer. Selected tRNAs and ncRNAs (RNase P RNA, tmRNA, 4.5S RNA, Ms1 RNA and 6C RNA) are expressed, and the levels for several of these are higher in stationary phase compared to exponentially growing cells. The rare tRNAIleTAT isoacceptor and two for mycobacteria novel ncRNAs: the Lactobacillales-derived GOLLD RNA and a homolog to the antisense Salmonella typhimurium phage Sar RNA, were shown to be present and expressed in certain Mmuc-clade members. CONCLUSIONS: Phages, IS elements, horizontally transferred tRNA gene clusters, and phage-derived ncRNAs appears to have influenced the evolution of the Mmuc- and Mneo-clades. While the number of predicted coding sequences correlates with genome size, the number of tRNA coding genes does not. The majority of the tRNA genes in mycobacteria are transcribed mainly from single genes and the levels of certain ncRNAs, including RNase P RNA (essential for the processing of tRNAs), are higher at stationary phase compared to exponentially growing cells. We provide supporting evidence that Ms1 RNA represents a mycobacterial 6S RNA variant. The evolutionary routes for the ncRNAs RNase P RNA, tmRNA and Ms1 RNA are different from that of the core genes.


Assuntos
Genoma Bacteriano , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/genética , RNA Bacteriano/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Aminoacil-tRNA Sintetases/genética , Bacteriófagos/genética , Tamanho do Genoma , Genômica , Anotação de Sequência Molecular , Mycobacterium/classificação , Filogenia , Plasmídeos/genética , RNA não Traduzido/química , Ribonuclease P/genética , Inversão de Sequência
5.
DNA Cell Biol ; 38(8): 786-795, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31157984

RESUMO

The ambush hypothesis speculates that off-frame stop codons increase translational efficiency after ribosomal frameshifts by stopping early frameshifted translation. Some evidences fit this hypothesis: (1) synonymous codon usages increase with their potential contribution to off-frame stops; (2) the genetic code assigns frequent amino acids to codon families contributing to off-frame stops; (3) positive biases for off-frame stops (AT rich) occur despite adverse nucleotide (GC) biases; and (4) mitochondrial off-frame stop codon densities increase with ribosomal structural instability, potential proxy of frameshift frequencies. In this study, analyses of vertebrate mitogenes and tRNA synthetase genes from all superkingdoms and viruses test a new prediction of the ambush hypothesis: sequences immediately downstream of frameshift-inducing homopolymer codons (AAA, CCC, GGG, and TTT) are off-frame stop rich. Codons immediately downstream of homopolymer codons form more than average off-frame stops, biases are stronger than for corresponding upstream distances and for any other group of synonymous codons. Sequences downstream of that high-density region are off-frame stop depleted. This decrease suggests that off-frame stops, combined with suppressor tRNAs regulate translation of overlapping coding sequences. Results show the predictive power of the ambush hypothesis, from macroevolutionary (genetic code structure) to detailed gene sequence anatomy.


Assuntos
Códon de Terminação , Código Genético , Mitocôndrias/genética , RNA de Transferência/genética , Animais , Archaea/genética , Bactérias/genética , Códon , Humanos , Camundongos , Fases de Leitura Aberta , Vírus/genética
6.
Gene ; 710: 59-65, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31039434

RESUMO

Mitochondrial DNA is typically passed to offspring through maternal inheritance. However, in mussels, two kinds of mitochondrial DNA exist: F and M type, which are referred to as doubly uniparental inheritance (DUI). Studies have shown that DUI may be related to gender determination. In this study, we obtained the first complete F-type mitochondrial genome of Lamprotula scripta and Lamprotula caveata which were 16,250 bp and 16,641 bp in length, respectively, and had 13 protein coding genes (PCGs), 22 transfer RNAs, 2 ribosomal RNAs and 27 non-coding (NC) regions. The largest NC region of L. scripta was 639 bp and located between ND5 and tRNAGln. The largest NC of L. caveata was 1046 bp and also located between ND5 and tRNAGln. The overall AT content of L. scripta and L. caveata was 58.95% and 58.66%, respectively, which were lower than Lamprotula leai, Lamprotula gottschei and Lamprotula tortuosa. We next compared F and M mitochondrial genomic data on freshwater mussels and established a phylogenetic tree based on amino acid sequences of 13 PCGs and COII gene. Our results showed that F- and M-type mitochondria were significantly separated into two branches, and the basic structure of phylogenetic trees were divided into four distinct groups: Unioninae, Anodontini, Gonideinae and Ambleminae. Relatives of Gonideinae and Ambleminae were more closely related than Unioninae and Anodontini, indicating significant differences in mtDNA between the two mitogenome types. Moreover, we revealed that L. scripta and L. caveata are closely relatives, suggesting that they are both subordinates of the Gonideinae subfamily. Consequently, we speculate that the formation of DUI hinders their disappearance, which provides a basis for further studies into the mechanisms and genetic diversities of DUI formation.


Assuntos
Genoma Mitocondrial , Unionidae/classificação , Unionidae/genética , Animais , Evolução Molecular , Tamanho do Genoma , Herança Materna , Mitocôndrias/genética , Fases de Leitura Aberta , Filogenia , RNA de Transferência/genética , RNA não Traduzido/genética
7.
Gene ; 706: 146-153, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31077734

RESUMO

Necrophagous Dermestes species have high forensic importance in relation to the estimation of elapsed time since death or death season. To further supplement the genome-level features for related species, the complete mitochondrial genome (mitogenome) of Dermestes species D. essellatocollis, D. frischii and D. coarctatus are amplified, sequenced, annotated, analyzed, and compared with other twelve species of the infraorder Bostrichoidea. The mitochondrial genomes were typical circular molecules with 16,218, 15,873 and 15,873 bp in length, respectively. They included 13 protein coding genes, two rRNAs, and 22 tRNAs, as well as the putative control region. The gene orders and orientations are identical to those of other recorded bostrichiformian species and had the ancestral insect gene composition. Furthermore, phylogenetic analyses based on all the mitochondrial protein coding genes for 13 Bostrichoidea and 16 outgroup taxa were performed using Bayesian and Maximum Likelihood analyses. The inferred trees indicate that the genus Dermestes is monophyletic. The monophyly of infraorder Bostrichiformia is not supported. This study provides genomic data for mitochondrial genome library of the genus Dermestes to investigate evolutionary and systematic studies.


Assuntos
Besouros/genética , Genoma Mitocondrial/genética , Mitocôndrias/genética , Animais , Composição de Bases/genética , Sequência de Bases , Ciências Forenses/métodos , Ordem dos Genes/genética , Filogenia , RNA Ribossômico/genética , RNA de Transferência/genética
8.
Mem Inst Oswaldo Cruz ; 114: e180443, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31090860

RESUMO

The presence of tRNA array, a region with high tRNA gene number and density, has been demonstrated in Mycobacterium genus. However, a recent phylogenomic study revealed the existence of five distinct monophyletic groups (genera) within this genus. Considering this new scenario, and based on in-silico analyses, we have identified and characterised the abundance and diversity of tRNA array units within Mycobacterium, Mycolicibacterium gen. nov., Mycolicibacillus gen. nov., and Mycobacteroides gen. nov. The occurrence and prevalence of tRNA arrays among the genera belonging to Actinobacteria indicate their possible role in the organismal fitness.


Assuntos
Técnicas de Tipagem Bacteriana , Mycobacterium/genética , RNA de Transferência/genética , Mycobacterium/classificação , Filogenia
9.
Microbiol Immunol ; 63(7): 243-250, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31136000

RESUMO

Lancefield group C Streptococcus dysgalactiae causes infections in farmed fish. Here, the genome of S. dysgalactiae strain kdys0611, isolated from farmed amberjack (Seriola dumerili) was sequenced. The complete genome sequence of kdys0611 consists of a single chromosome and five plasmids. The chromosome is 2,142,780 bp long and has a GC content of 40%. It possesses 2061 coding sequences and 67 tRNA and 6 rRNA operons. One clustered regularly interspaced short palindromic repeat, 125 insertion sequences, and four predicted prophage elements were identified. Phylogenetic analysis based on 126 core genes suggested that the kdys0611 strain is more closely related to S. dysgalactiae subsp. dysgalactiae than to S. dysgalactiae subsp. equisimilis. The genome of kdys0611 harbors 87 genes with sequence similarity to putative virulence-associated genes identified in other bacteria, of which 57 exhibit amino acid identity (>52%) to genes of the S. dysgalactiae subsp. equisimilis GGS124 human clinical isolate. Four putative virulence genes, emm5 (FGCSD_0256), spg_2 (FGCSD_1961), skc (FGCSD_1012), and cna (FGCSD_0159), in kdys0611 did not show significant homology with any deposited S. dysgalactiae genes. The chromosomal sequence of kdys0611 has been deposited in GenBank under Accession No. AP018726. This is the first report of the complete genome sequence of S. dysgalactiae isolated from fish.


Assuntos
Peixes/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma , Animais , Composição de Bases , Sistemas CRISPR-Cas , Genoma Bacteriano , Humanos , Filogenia , RNA Ribossômico/genética , RNA de Transferência/genética , Análise de Sequência de DNA , Infecções Estreptocócicas/microbiologia , Streptococcus/classificação , Streptococcus/isolamento & purificação , Virulência/genética
10.
Mol Biol (Mosk) ; 53(2): 349-352, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31099785

RESUMO

Despite protein biosynthesis being studied for decades, some major questions concerning this process are still to be addressed. We elucidate a close connection between proofreading of the emerging amino acid sequence during its normal, elongation factor-dependent ribosomal biosynthesis and the existence of the factor-free synthesis of a polypeptide chain on a ribosome. In this factor-free process, the biological role of proofreading is played by a process opposite to the factor-free attachment of Aa-tRNA to the ribosome, namely, the removal via the same pathway of that Aa-tRNA, which is not complementary to the mRNA codon exhibited by the ribosome.


Assuntos
Biossíntese de Proteínas , Ribossomos/metabolismo , Códon/genética , RNA Mensageiro/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Termodinâmica
11.
Retrovirology ; 16(1): 11, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30947720

RESUMO

BACKGROUND: Reverse transcription (RT) of HIV and SIV is initiated by the binding of the acceptor stem of tRNALys3 to the primer binding site (PBS) of the viral RNA genome. Previous studies have suggested that this tRNALys3 is not the only molecule capable of priming reverse transcription, and that at least one other lysyl tRNA, tRNALys5, which has an acceptor stem sequence varying from tRNALys3 by only a single transition mutation resulting in the integration of a thymine (T) at position 8 of the PBS in the viral genome, can prime reverse transcription. RESULTS: We undertook an unbiased approach, evaluating the primer binding site by deep-sequencing of HIV and SIV directly from the plasma of 15 humans and 11 macaques. We found that in humans there are low but measurable levels of viral RNA genomes harboring a PBS containing the noncanonical T at position 8 (PBS-Lys5) corresponding to the tRNAlys5 sequence and representing an average of 0.52% (range 0.07-1.6%) of the total viral population. This value is remarkably consistent with the proportion of PBS-Lys5 we identified in a cross-sectional assessment of the LANL HIV database (0.51%). In macaques chronically infected with SIVmac239, the PBS-Lys5 was also detected but at a frequency 1-log less than seen for HIV, with an average of 0.056% (range 0.01-0.09%). At this proportion, PBS-Lys5 was comparable to other transition mutations, making it impossible to determine whether the mutation observed is a result of use of tRNALys5 as an RT primer at very low levels or merely the product of in vitro cDNA synthesis/PCR error. We also identified two novel PBS sequences in HIV and SIV at low levels in vivo corresponding to tRNALys6 and tRNALys1,2, suggesting that these tRNAs may rarely also be used to prime RT. In vivo reversion of the PBS-Lys5 found in SIVmac239 was rapid and reached background levels by 30 days post-infection. CONCLUSIONS: We conclude that while alternative tRNAs can initiate reverse transcription of HIV and SIV in vivo, their overall contributions to the replicating viral population are small.


Assuntos
HIV-1/genética , RNA de Transferência/genética , Transcrição Reversa , Vírus da Imunodeficiência Símia/genética , Animais , Sítios de Ligação , Estudos Transversais , DNA Viral/genética , Feminino , Genoma Viral , HIV-1/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Macaca/virologia , Masculino , RNA Viral/sangue , Vírus da Imunodeficiência Símia/fisiologia , Transcrição Genética , Replicação Viral
12.
Genome Biol Evol ; 11(5): 1398-1416, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980669

RESUMO

The evolution of mitochondrial genomes and their population-genetic environment among unicellular eukaryotes are understudied. Ciliate mitochondrial genomes exhibit a unique combination of characteristics, including a linear organization and the presence of multiple genes with no known function or detectable homologs in other eukaryotes. Here we study the variation of ciliate mitochondrial genomes both within and across 13 highly diverged Paramecium species, including multiple species from the P. aurelia species complex, with four outgroup species: P. caudatum, P. multimicronucleatum, and two strains that may represent novel related species. We observe extraordinary conservation of gene order and protein-coding content in Paramecium mitochondria across species. In contrast, significant differences are observed in tRNA content and copy number, which is highly conserved in species belonging to the P. aurelia complex but variable among and even within the other Paramecium species. There is an increase in GC content from ∼20% to ∼40% on the branch leading to the P. aurelia complex. Patterns of polymorphism in population-genomic data and mutation-accumulation experiments suggest that the increase in GC content is primarily due to changes in the mutation spectra in the P. aurelia species. Finally, we find no evidence of recombination in Paramecium mitochondria and find that the mitochondrial genome appears to experience either similar or stronger efficacy of purifying selection than the nucleus.


Assuntos
Evolução Molecular , Genoma Mitocondrial , Paramecium/genética , Recombinação Genética , Seleção Genética , Composição de Bases , Mutação , Polimorfismo de Nucleotídeo Único , RNA de Transferência/genética , Especificidade da Espécie
13.
Genome Biol Evol ; 11(4): 1258-1274, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30937434

RESUMO

Both direct repeats (DR) and inverted repeats (IR) are documented in the published plastomes of Selaginella species indicating the unusual and diverse plastome structure in the family Selaginellaceae. In this study, we newly sequenced complete plastomes of seven species from five main lineages of Selaginellaceae and also resequenced three species (Selaginella tamariscina, Selaginella uncinata, and Selaginella moellendorffii) to explore the evolutionary trajectory of Selaginellaceae plastomes. Our results showed that the plastomes of Selaginellaceae vary remarkably in size, gene contents, gene order, and GC contents. Notably, both DR and IR structures existed in the plastomes of Selaginellaceae with DR structure being an ancestral state. The occurrence of DR structure was at ∼257 Ma and remained in most subgenera of Selaginellaceae, whereas IR structure only reoccurred in Selaginella sect. Lepidophyllae (∼143 Ma) and Selaginella subg. Heterostachys (∼19 Ma). The presence of a pair of large repeats psbK-trnQ, together with DR/IR region in Selaginella bisulcata, Selaginella pennata, S. uncinata, and Selaginella hainanensis, could frequently mediate diverse homologous recombination and create approximately equal stoichiometric isomers (IR/DR-coexisting) and subgenomes. High proportion of repeats is presumably responsible for the dynamic IR/DR-coexisting plastomes, which possess a lower synonymous substitution rate (dS) compared with DR-possessing and IR-possessing plastomes. We propose that the occurrence of DR structure, together with few repeats, is possibly selected to keep the stability of plastomes and the IR/DR-coexisting plastomes also reached an equilibrium in plastome organization through highly efficient homologous recombination to maintain stability.


Assuntos
Evolução Molecular , Genomas de Plastídeos , Selaginellaceae/genética , Deleção de Genes , Rearranjo Gênico , Íntrons , Sequências Repetidas Invertidas , Filogenia , RNA de Transferência/genética
14.
Mol Cancer ; 18(1): 74, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940133

RESUMO

tRNA-derived small RNA (tsRNA) is a novel regulatory small non-coding RNA and participates in diverse physiological and pathological processes. However, the presence of tsRNAs in exosome and their diagnostic potential remain unclear. In this study, we took advantage of small RNA-seq technology to profile exosomal tsRNAs from cell culture medium and plasma, and found ubiquitous presence of tsRNAs in exosome. To explore the potential value of tsRNA for cancer diagnosis, we compared exosomal tsRNA levels between liver cancer patients and healthy donors, revealing that tsRNAs were dramatically increased in plasma exosomes of liver cancer patients. Importantly, patients with liver cancer exhibited significantly higher levels of four tsRNAs (tRNA-ValTAC-3, tRNA-GlyTCC-5, tRNA-ValAAC-5 and tRNA-GluCTC-5) in plasma exosome, demonstrating that plasma exosomal tsRNA could serve as a novel diagnostic biomarker. Taken together, our results not only expand non-coding RNA species in exosome, but also highlight the potential of tsRNAs as a promising biomarker for cancer diagnosis.


Assuntos
Exossomos/genética , Neoplasias/diagnóstico , RNA de Transferência/genética , RNA não Traduzido/genética , Biomarcadores Tumorais/genética , Técnicas de Cultura de Células , Detecção Precoce de Câncer , Humanos , Neoplasias/genética , Análise de Sequência de RNA , Células Tumorais Cultivadas
15.
Curr Microbiol ; 76(6): 732-737, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30993398

RESUMO

Several bioprocessing technologies, such as separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and consolidated bioprocessing (CBP), have been highlighted to produce bio-based fuels and chemicals from lignocellulosic biomass. Successful CBP, an efficient and economical lignocellulosic biorefinery process compared with other processes, requires microorganisms with sufficient cellulolytic activity and biofuel/chemical-producing ability. Here, we report the complete genome of Paenibacillus sp. CAA11, a newly isolated promising microbial host for CBP-producing ethanol and organic acids from cellulose. The genome of Paenibacillus sp. CAA11 comprises one 4,888,410 bp chromosome with a G + C content of 48.68% containing 4418 protein-coding genes, 102 tRNA genes, and 39 rRNA genes. The functionally active cellulase, encoded by CAA_GH5 was identified to belong to glycosyl hydrolase family 5 (GH5) and consisted of a catalytic domain and a cellulose-binding domain 3 (CBM3). When cellulolytic activity of CAA_GH5 was assayed through Congo red method by measuring the size of halo zone, the recombinant Bacillus subtilis RIK1285 expressing CAA_GH5 showed a comparable cellulolytic activity to B. subtilis RIK1285 expressing Cel5, a previously verified powerful bacterial cellulase. This study demonstrates the potential of Paenibacillus sp. CAA11 as a CBP-enabling microbe for cost-effective biofuels/chemicals production from lignocellulosic biomass.


Assuntos
Genoma Bacteriano , Paenibacillus/genética , Análise de Sequência de DNA , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Composição de Bases , Biotransformação , Ácidos Carboxílicos/metabolismo , Vermelho Congo/metabolismo , Etanol/metabolismo , Genes Bacterianos , Lignina/genética , Lignina/metabolismo , RNA Ribossômico/genética , RNA de Transferência/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Int J Mol Sci ; 20(8)2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31010123

RESUMO

In the past two decades, tRNA molecules and their corresponding aminoacyl-tRNA synthetases (aaRS) have been extensively used in synthetic biology to genetically encode post-translationally modified and unnatural amino acids. In this review, we briefly examine one fundamental requirement for the successful application of tRNA/aaRS pairs for expanding the genetic code. This requirement is known as "orthogonality"-the ability of a tRNA and its corresponding aaRS to interact exclusively with each other and avoid cross-reactions with additional types of tRNAs and aaRSs in a given organism.


Assuntos
Aminoacil-tRNA Sintetases/genética , Código Genético , RNA de Transferência/genética , Aminoacil-tRNA Sintetases/química , Engenharia Genética , RNA de Transferência/química , Biologia Sintética
17.
Nucleic Acids Res ; 47(6): 3072-3085, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30952159

RESUMO

Alanyl-tRNA synthetases (AlaRSs) from three domains of life predominantly rely on a single wobble base pair, G3-U70, of tRNAAla as a major determinant. However, this base pair is divergent in human mitochondrial tRNAAla, but instead with a translocated G5-U68. How human mitochondrial AlaRS (hmtAlaRS) recognizes tRNAAla, in particular, in the acceptor stem region, remains unknown. In the present study, we found that hmtAlaRS is a monomer and recognizes mitochondrial tRNAAla in a G3-U70-independent manner, requiring several elements in the acceptor stem. In addition, we found that hmtAlaRS misactivates noncognate Gly and catalyzes strong transfer RNA (tRNA)-independent pre-transfer editing for Gly. A completely conserved residue outside of the editing active site, Arg663, likely functions as a tRNA translocation determinant to facilitate tRNA entry into the editing domain during editing. Finally, we investigated the effects of the severe infantile-onset cardiomyopathy-associated R592W mutation of hmtAlaRS on the canonical enzymatic activities of hmtAlaRS. Overall, our results provide fundamental information about tRNA recognition and deepen our understanding of translational quality control mechanisms by hmtAlaRS.


Assuntos
Conformação de Ácido Nucleico , RNA Mitocondrial/genética , RNA de Transferência de Alanina/genética , RNA de Transferência/genética , Alanina-tRNA Ligase/genética , Pareamento de Bases/genética , Domínio Catalítico , Escherichia coli/genética , Humanos , Cinética , Modelos Moleculares , Especificidade por Substrato
18.
Gene ; 705: 95-102, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30940527

RESUMO

Small circular RNAs occur in many cells. Theoretical considerations designed to mimick primordial RNAs designed 25 theoretical, minimal 22-nucleotide long circular RNAs (RNA rings) forming stem-loop hairpins, including one codon per amino acid, a start and a stop codon. These RNA rings resemble consensus tRNAs, whose predicted anticodons assign to each RNA ring a potential cognate amino acid. Assuming dual translator and messenger roles, three consecutive translation rounds produce 21-residue-long peptides, seven codons at each round. In these conditions, steric hindrances between tRNA(-like) translators competing for partially overlapping nucleotide triplets can be reduced if none of the seven codons produces by circular permutation (position 1 → 3, or position 3 → 1) any of the six others. This non-permutability of codon sets is one of the conditions that define potential circular codes regulating translational frame. A near-universal maximal self-complementary circular code exists in reading frames of natural genes, and permutations of this circular code define maximal circular codes in each +1, +2 gene frames. Chronologically scaling RNA rings according to the genetic code inclusion order of their cognate amino acid, codon numbers belonging to the natural frame +1 circular code X1 decrease with cognate amino acid inclusion order, those belonging to the natural frame 0 circular code X0 increase. RNA rings with early cognates apparently reflect pre-(tRNA-like)-adaptor translation by direct codon-amino acid affinity with partially overlapping consecutive codons where X1 regulated translation. Translation of non-overlapping consecutive codons evolved in parallel with RNA rings' cognate inclusion in the genetic code and with X0. Hence the complex "multi-frame" natural circular codes potentially evolved spontaneously from small coding circular RNAs mimicked by theoretical minimal RNA rings. Modern reading frames evolved from earlier reading frames corresponding to modern +1 frames.


Assuntos
Biologia Computacional/métodos , RNA/química , RNA/genética , Evolução Molecular , Regulação da Expressão Gênica , Código Genético , Humanos , Modelos Genéticos , Modelos Moleculares , Biossíntese de Proteínas , RNA de Transferência/química , RNA de Transferência/genética
19.
Nat Commun ; 10(1): 1490, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940799

RESUMO

Spatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here, to address this limitation, we present a screening platform which allows simultaneous measurements of the promoter strength, 5', and 3' processing efficiencies across a library of tRNA variants. This analysis reveals that the sequence determinants underlying these activities, while overlapping, are dissociable. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enables highly specific guide RNA production from a Pol-II promoter. When benchmarked against other reported systems this tRNA scaffold is superior to most alternatives, and is equivalent in function to an optimized version of the Csy4-based guide RNA release system. The results and methods described in this manuscript enable avenues of research both in genome engineering and basic tRNA biology.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , RNA Polimerase II/genética , RNA Guia/genética , RNA de Transferência/genética , Edição de Genes , Regulação da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Guia/química , RNA Guia/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo
20.
Gene ; 703: 145-152, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-30940526

RESUMO

We developed a novel assay system to quantitatively detect amber codon suppression by tRNAs expressed in plant cells. The assay was based on recovery of the expression of the green fluorescent protein (GFP) as a reporter, in which a fourth Lys codon (AAG) was changed to a premature amber codon TAG, designated as GFP/amber. Plasmids carrying GFP/amber, suppressor tRNA, and red fluorescent protein (RFF) as an internal control, respectively, were introduced into onion epidermal cells to monitor cell numbers with GFP and RFP fluorescence. First, an amber suppressor tRNASer from tobacco (NtS2) to suppress a TAG codon in GFP mRNA was examined, leading to the recovery of GFP fluorescence. Second, we used two different tRNAs (i.e., AtY3II-am and AtY3II-amiG7), both of which are intron-containing amber suppressor tRNAsTyr, the former impaired precursor-tRNA splicing but the latter did not, as confirmed previously using two different approaches (Szeykowska-Kulinska and Beier, 1991; Akama and Beier, 2003). As expected, coexpression of GFP/amber with AtY3II-am gave no green fluorescence, but significant fluorescence was observed with AtY3II-amiG7. Then, we applied this system for the analysis of 5'-regulatory sequences of the tRNAGln gene family from Arabidopsis. A 5'-flanking sequence of each of the 17 tRNAGln genes was fused to a coding region of an amber suppressor tRNASer gene (NtS2/amber) and its 3'-flanking sequence. Chimeric tRNASer gene, GFP/amber, and RFP were coexpressed, and the GFP or RFP fluorescence intensity was determined in cells using laser-scanning microscopy. In parallel, 17 kinds of original Arabidopsis tRNAGln genes and their chimeric genes with NtS2/amber were all analyzed in cell-free nuclear extract (Yukawa et al., 1997). Comparison of in vitro and in vivo expression of these chimeric tRNA genes displayed generally similar results, accompanied by a wide range of variance in the expression of each gene. Nevertheless, the expression patterns of several genes were clearly the opposite of each other comparing between the two different system, demonstrating the importance of in vivo systems in the study on tRNA expression in plants.


Assuntos
Proteínas de Fluorescência Verde/genética , Plantas/genética , RNA de Transferência/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/metabolismo , Cebolas/genética , Cebolas/crescimento & desenvolvimento , RNA de Plantas/genética
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