RESUMO
The proper functioning and assembly of the sperm flagella structures contribute significantly to spermatozoa motility and overall male fertility. However, the fine mechanisms of assembly steps are poorly studied due to the high diversity of cell types, low solubility of the corresponding protein structures, and high tissue and cell specificity. One of the open questions for investigation is the attachment of longitudinal columns to the doublets 3 and 8 of axonemal microtubules through the outer dense fibers. A number of mutations affecting the assembly of flagella in model organisms are known. Additionally, evolutionary genomics data and comparative analysis of flagella morphology are available for a set of non-model species. This review is devoted to the analysis of diverse ultrastructures of sperm flagellum of Metazoa combined with an overview of the evolutionary distribution and function of the mammalian fibrous sheath proteins.
Assuntos
Cauda do Espermatozoide , Espermatozoides , Masculino , Animais , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Espermatozoides/fisiologia , Cauda do Espermatozoide/ultraestrutura , Cauda do Espermatozoide/metabolismo , Humanos , Axonema/ultraestrutura , Axonema/metabolismo , Motilidade dos Espermatozoides/fisiologiaRESUMO
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
In bacteria, rapid adaptation to changing environmental conditions depends on the interplay between housekeeping and alternative σ factors, responsible for transcription of specific regulons by RNA polymerase (RNAP). In comparison with alternative σ factors, primary σs contain poorly conserved region 1.1, whose functions in transcription are only partially understood. We found that a single mutation in region 1.1 in Escherichia coli σ70 rewires transcription regulation during cell growth resulting in profound phenotypic changes. Despite its destabilizing effect on promoter complexes, this mutation increases the activity of rRNA promoters and also decreases RNAP sensitivity to the major regulator of stringent response DksA. Using total RNA sequencing combined with single-cell analysis of gene expression we showed that changes in region 1.1 disrupt the balance between the "greed" and "fear" strategies thus making the cells more susceptible to environmental threats and antibiotics. Our results reveal an unexpected role of σ region 1.1 in growth-dependent transcription regulation and suggest that changes in this region may facilitate rapid switching of RNAP properties in evolving bacterial populations.
Assuntos
Divisão Celular , Regulação Bacteriana da Expressão Gênica , Fator sigma/genética , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação Puntual , Domínios Proteicos , Fator sigma/química , Fator sigma/metabolismo , Transcrição GênicaRESUMO
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
Ribosomal protein S6 in Escherichia coli is modified by ATP-dependent glutamate ligase RimK. Up to four glutamate residues are added to the C-terminus of S6 protein. In this work we demonstrated that unlike the majority of ribosome modifications in E. coli, oligoglutamylation of S6 protein is regulated and happens only in the stationary phase of bacterial culture. Only S6 protein incorporated into assembled small ribosomal subunits, but not newly made free S6 protein is a substrate for RimK protein. Overexpression of the rimK gene leads to the modification of S6 protein even in the exponential phase of bacterial culture. Thus, it is unlikely that any stationary phase specific factor is needed for the modification. We propose a model that S6 modification is regulated solely via the rate of ribosome biosynthesis at limiting concentration of RimK enzyme.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Peptídeo Sintases/metabolismo , Proteína S6 Ribossômica/metabolismo , Proteínas Ribossômicas/metabolismo , Escherichia coli/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismoRESUMO
Genes coding for small peptides have been frequently misannotated as long noncoding RNA (lncRNA) genes. Here we have demonstrated that one such transcript is translated into a 56-amino-acid-long peptide conserved in chordates, corroborating the work published while this manuscript was under review. The Mtln peptide could be detected in mitochondria of mouse cell lines and tissues. In line with its mitochondrial localization, lack of the Mtln decreases the activity of mitochondrial respiratory chain complex I. Unlike the integral components and assembly factors of NADH:ubiquinone oxidoreductase, Mtln does not alter its enzymatic activity directly. Interaction of Mtln with NADH-dependent cytochrome b5 reductase stimulates complex I functioning most likely by providing a favorable lipid composition of the membrane. Study of Mtln illuminates the importance of small peptides, whose genes might frequently be misannotated as lncRNAs, for the control of vitally important cellular processes.
Assuntos
Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Peptídeos/metabolismo , RNA Longo não Codificante/metabolismo , Sequência de Aminoácidos , Animais , Respiração Celular , Citosol/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Camundongos , NAD/metabolismo , Células NIH 3T3 , Consumo de Oxigênio , Fosfolipídeos/metabolismo , RNA Longo não Codificante/genética , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Adenosina/análogos & derivados , Adenosina/metabolismo , Escherichia coli/metabolismo , RNA/metabolismo , Humanos , Metilação , Metiltransferases/metabolismoRESUMO
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.