RESUMO
N-terminal acetylation is widespread in the eukaryotic proteome but in bacteria is restricted to a small number of proteins mainly involved in translation. It was long known that elongation factor Tu (EF-Tu) is N-terminally acetylated, whereas the enzyme responsible for this process was unclear. Here, we report that RimI acetyltransferase, known to modify ribosomal protein S18, is likewise responsible for N-acetylation of the EF-Tu. With the help of inducible tufA expression plasmid, we demonstrated that the acetylation does not alter the stability of EF-Tu. Binding of aminoacyl tRNA to the recombinant EF-Tu in vitro was found to be unaffected by the acetylation. At the same time, with the help of fast kinetics methods, we demonstrate that an acetylated variant of EF-Tu more efficiently accelerates A-site occupation by aminoacyl-tRNA, thus increasing the efficiency of in vitro translation. Finally, we show that a strain devoid of RimI has a reduced growth rate, expanded to an evolutionary timescale, and might potentially promote conservation of the acetylation mechanism of S18 and EF-Tu. This study increased our understanding of the modification of bacterial translation apparatus.
Assuntos
Acetiltransferases , Bactérias/metabolismo , Fator Tu de Elongação de Peptídeos , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Proteínas Ribossômicas , Ribossomos/metabolismoRESUMO
First triplets of mRNA coding region affect the yield of translation. We have applied the flowseq method to analyze >30 000 variants of the codons 2-11 of the fluorescent protein reporter to identify factors affecting the protein synthesis. While the negative influence of mRNA secondary structure on translation has been confirmed, a positive role of rare codons at the beginning of a coding sequence for gene expression has not been observed. The identity of triplets proximal to the start codon contributes more to the protein yield then more distant ones. Additional in-frame start codons enhance translation, while Shine-Dalgarno-like motifs downstream the initiation codon are inhibitory. The metabolic cost of amino acids affects the yield of protein in the poor medium. The most efficient translation was observed for variants with features resembling those of native Escherichia coli genes.
Assuntos
Códon de Iniciação/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/genética , Códon de Iniciação/ultraestrutura , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/ultraestrutura , Ribossomos/genética , Ribossomos/ultraestruturaRESUMO
Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5Î untranslated regions (5Î-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5Î-UTR sequences and introduced random and designed mutations into natural and artificially selected 5Î-UTRs. Several distinct properties could be ascribed to a group of 5Î-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5Î-UTRs.
Assuntos
Regiões 5' não Traduzidas , Escherichia coli/genética , Biossíntese de Proteínas , Sequências Reguladoras de Ácido Ribonucleico , Separação Celular , Citometria de Fluxo , Genes Reporter , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Conformação de Ácido Nucleico , Nucleotídeos/fisiologiaRESUMO
YciH is a bacterial protein, homologous to eukaryotic translation initiation factor eIF1. Preceding evidence obtained with the aid of in vitro translation initiation system suggested that it may play a role of a translation initiation factor, ensuring selection against suboptimal initiation complexes. Here we studied the effect of Escherichia coli yciH gene inactivation on translation of model mRNAs. Neither the translation efficiency of leaderless mRNAs, nor mRNAs with non AUG start codons, was found to be affected by YciH in vivo. Comparative proteome analysis revealed that yciH gene knockout leads to a more than fold2- increase in expression of 66 genes and a more than fold2- decrease in the expression of 20 genes. Analysis of these gene sets allowed us to suggest a role of YciH as an inhibitor of translation in a stress response rather than the role of a translation initiation factor.
Assuntos
Proteínas de Escherichia coli/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Biossíntese de Proteínas , ProteomaRESUMO
Regulation of gene expression at the level of translation accounts for up to three orders of magnitude in its efficiency. We systematically compared the impact of several mRNA features on translation initiation at the first gene in an operon with those for the second gene. Experiments were done in a system with internal control based on dual cerulean and red (CER/RFP) fluorescent proteins. We demonstrated significant differences in the efficiency of Shine Dalgarno sequences acting at the leading gene and at the following genes in an operon. The majority of frequent intercistronic arrangements possess medium SD dependence, medium dependence on the preceding cistron translation and efficient stimulation by A/U-rich sequences. The second cistron starting immediately after preceding cistron stop codon displays unusually high dependence on the SD sequence.
Assuntos
Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/química , Códon de Iniciação , Corantes Fluorescentes , Genes Reporter , Proteínas Luminescentes/genética , Conformação de Ácido Nucleico , Óperon , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteína Vermelha FluorescenteRESUMO
Non-invasive prenatal testing (NIPT) for aneuploidy on Chromosomes 21 (T21), 18 (T18) and 13 (T13) is actively used in clinical practice around the world. One of the limitations of the wider implementation of this test is the high cost of the analysis itself, as high-throughput sequencing is still relatively expensive. At the same time, there is an increasing trend in the length of reads yielded by sequencers. Since extracellular DNA is short, in the order of 140-160 bp, it is not possible to effectively use long reads. The authors used high-performance sequencing of cell-free DNA (cfDNA) libraries that went through additional stages of enzymatic fragmentation and random ligation of the resulting products to create long chimeric reads. The authors used a controlled set of samples to analyze a set of cfDNA samples from pregnant women with a high risk of fetus aneuploidy according to the results of the first trimester screening and confirmed by invasive karyotyping of the fetus using laboratory and analytical approaches developed by the authors. They evaluated the sensitivity, specificity, PPV (positive predictive value), and NPV (negative predictive value) of the results. The authors developed a technique for constructing long chimeric reads from short cfDNA fragments and validated the test using a control set of extracellular DNA samples obtained from pregnant women. The obtained sensitivity and specificity parameters of the NIPT developed by the authors corresponded to the approaches proposed earlier (99.93% and 99.14% for T21; 100% and 98.34% for T18; 100% and 99.17% for T13, respectively).
Assuntos
Aneuploidia , Ácidos Nucleicos Livres/sangue , Síndrome da Trissomia do Cromossomo 13/genética , Síndrome da Trissomía do Cromossomo 18/genética , Adulto , Ácidos Nucleicos Livres/genética , Quimera/genética , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 21/genética , Feminino , Humanos , Gravidez , Diagnóstico Pré-Natal , Síndrome da Trissomia do Cromossomo 13/sangue , Síndrome da Trissomia do Cromossomo 13/patologia , Síndrome da Trissomía do Cromossomo 18/sangue , Síndrome da Trissomía do Cromossomo 18/patologiaRESUMO
Ribosomal RNAs in all organisms are methylated. The functional role of the majority of modified nucleotides is unknown. We systematically questioned the influence of rRNA methylation in Escherichia coli on a number of characteristics of bacterial cells with the help of a set of rRNA methyltransferase (MT) gene knockout strains from the Keio collection. Analysis of ribosomal subunits sedimentation profiles of the knockout strains revealed a surprisingly small number of rRNA MT that significantly affected ribosome assembly. Accumulation of the assembly intermediates was observed only for the rlmE knockout strain whose growth was retarded most significantly among other rRNA MT knockout strains. Accumulation of the 17S rRNA precursor was observed for rsmA(ksgA) knockout cells as well as for cells devoid of functional rsmB and rlmC genes. Significant differences were found among the WT and the majority of rRNA MT knockout strains in their ability to sustain exogenous protein overexpression. While the majority of the rRNA MT knockout strains supported suboptimal reporter gene expression, the strain devoid of the rsmF gene demonstrated a moderate increase in the yield of ectopic gene expression. Comparative 2D protein gel analysis of rRNA MT knockout strains revealed only minor perturbations of the proteome.
RESUMO
Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine-Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next-generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine-Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100-fold range.
Assuntos
Escherichia coli , Biossíntese de Proteínas , Sequência de Bases , Códon de Iniciação , Escherichia coli/genética , Escherichia coli/metabolismo , RNA MensageiroRESUMO
During a cell's lifespan, DNA break formation is a common event, associated with many processes, from replication to apoptosis. Most of DNA breaks are readily repaired, but some are meant to persist in time, such as the chromosome ends, protected by telomeres. Besides them, eukaryotic genomes comprise shorter stretches of interstitial telomeric repeats. We assumed that the latter may also be associated with the formation of DNA breaks meant to persist in time. In zebrafish and mouse embryos, cells containing numerous breakage foci were identified. These breaks were not associated with apoptosis or replication, nor did they seem to activate DNA damage response machinery. Unlike short-living, accidental sparse breaks, the ones we found seem to be closely associated, forming discrete break foci. A PCR-based method was developed, allowing specific amplification of DNA regions located between inverted telomeric repeats associated with breaks. The cloning and sequencing of such DNA fragments were found to denote some specificity in their distribution for different tissue types and development stages.
Assuntos
Quebras de DNA de Cadeia Dupla , Sequências Repetitivas de Ácido Nucleico/genética , Telômero/genética , Animais , Apoptose , Cromatina/metabolismo , Fragmentação do DNA , Reparo do DNA , Replicação do DNA , Embrião de Mamíferos/metabolismo , Embrião não Mamífero/metabolismo , Histonas/metabolismo , Camundongos , Especificidade de Órgãos , Peixe-Zebra/embriologiaRESUMO
N6-methyladenosine (m(6)A) is ubiquitously present in the RNA of living organisms from Escherichia coli to humans. Methyltransferases that catalyze adenosine methylation are drastically different in specificity from modification of single residues in bacterial ribosomal or transfer RNA to modification of thousands of residues spread among eukaryotic mRNA. Interactions that are formed by m(6)A residues range from RNA-RNA tertiary contacts to RNA-protein recognition. Consequences of the modification loss might vary from nearly negligible to complete reprogramming of regulatory pathways and lethality. In this review, we summarized current knowledge on enzymes that introduce m(6)A modification, ways to detect m(6)A presence in RNA and the functional role of this modification everywhere it is present, from bacteria to humans.