RESUMEN

RNA polymerase (RNAP), the central enzyme of transcription, intermittently pauses during the elongation stage of RNA synthesis. Pausing provides an opportunity for regulatory events such as nascent RNA folding or the recruitment of transregulators. NusG (Spt5 in eukaryotes and archaea) regulates RNAP pausing and is the only transcription factor conserved across all cellular life. NusG is a multifunctional protein: its N-terminal domain (NGN) binds to RNAP, and its C-terminal KOW domain in bacteria interacts with transcription regulators such as ribosomes and termination factors. In Escherichia coli, NusG acts as an antipausing factor. However, recent studies have revealed that NusG has distinct transcriptional regulatory roles specific to bacterial clades with clinical implications. Here, we focus on NusG's dual roles in the regulation of pausing.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli , Factores de Elongación de Péptidos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Factores de Elongación de Péptidos/metabolismo , Factores de Elongación de Péptidos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Regulación Bacteriana de la Expresión Génica , Transcripción Genética , Bacterias/genética , Bacterias/metabolismo

RESUMEN

BACKGROUND: Achondroplasia and mandibulofacial dysostosis with microcephaly (MFDM) are rare monogenic, dominant disorders, caused by gain-of-function fibroblast growth factor receptor 3 (FGFR3) gene variants and loss-of-function elongation factor Tu GTP binding domain-containing 2 (EFTUD2) gene variants, respectively. The coexistence of two distinct Mendelian disorders in a single individual is uncommon and challenges the traditional paradigm of a single genetic disorder explaining a patient's symptoms, opening new avenues for diagnosis and management. CASE PRESENTATION: We present a case of a female patient initially diagnosed with achondroplasia due to a maternally inherited pathogenic FGFR3 variant. She was referred to our genetic department due to her unusually small head circumference and short stature, which were both significantly below the expected range for achondroplasia. Additional features included distinctive facial characteristics, significant speech delay, conductive hearing loss, and epilepsy. Given the complexity of her phenotype, she was recruited to the DDD (Deciphering Developmental Disorders) study and the 100,000 Genomes project for further investigation. Subsequent identification of a complex EFTUD2 intragenic rearrangement confirmed an additional diagnosis of mandibulofacial dysostosis with microcephaly (MFDM). CONCLUSION: This report presents the first case of a dual molecular diagnosis of achondroplasia and mandibulofacial dysostosis with microcephaly in the same patient. This case underscores the complexity of genetic diagnoses and the potential for coexistence of multiple genetic syndromes in a single patient. This case expands our understanding of the molecular basis of dual Mendelian disorders and highlights the importance of considering the possibility of dual molecular diagnoses in patients with phenotypic features that are not fully accounted for by their primary diagnosis.

Asunto(s)

Acondroplasia , Disostosis Mandibulofacial , Microcefalia , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Ribonucleoproteína Nuclear Pequeña U5 , Humanos , Microcefalia/genética , Microcefalia/diagnóstico , Microcefalia/complicaciones , Femenino , Disostosis Mandibulofacial/genética , Acondroplasia/genética , Acondroplasia/complicaciones , Ribonucleoproteína Nuclear Pequeña U5/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Factores de Elongación de Péptidos/genética , Fenotipo

RESUMEN

RfaH is a two-domain transcription factor in which the C-terminal domain switches fold from an α-helical hairpin to a ß-roll upon binding the ops-paused RNA polymerase. To ascertain the presence of a sparsely populated excited state that may prime the autoinhibited resting state of RfaH for binding ops-paused RNA polymerase, we carried out a series of NMR-based exchange experiments to probe for conformational exchange on the millisecond time scale. Quantitative analysis of these data reveals exchange between major ground (∼95%) and sparsely populated excited (∼5%) states with an exchange lifetime of ∼3 ms involving residues at the interface between the N-terminal and C-terminal domains formed by the ß3/ß4 hairpin and helix α3 of the N-terminal domain and helices α4 and α5 of the C-terminal domain. The largest 15N backbone chemical shift differences are associated with the ß3/ß4 hairpin, leading us to suggest that the excited state may involve a rigid body lateral displacement/rotation away from the C-terminal domain to adopt a position similar to that seen in the active RNA polymerase-bound state. Such a rigid body reorientation would result in a reduction in the interface between the N- and C-terminal domains with the possible introduction of a cavity or cavities. This hypothesis is supported by the observation that the population of the excited species and the exchange rate of interconversion between ground and excited states are reduced at a high (2.5 kbar) pressure. Mechanistic implications for fold switching of the C-terminal domain in the context of RNA polymerase binding are discussed.

Asunto(s)

Proteínas de Escherichia coli , Pliegue de Proteína , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Resonancia Magnética Nuclear Biomolecular , Factores de Elongación de Péptidos/metabolismo , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/genética , Transactivadores/metabolismo , Transactivadores/química , Transactivadores/genética , Modelos Moleculares , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Conformación Proteica

RESUMEN

Prolines cause ribosomes to stall during translation due to their rigid structure. This phenomenon occurs in all domains of life and is exacerbated at polyproline motifs. Such stalling can be eased by the elongation factor P (EF-P) in bacteria. We discovered a potential connection between the loss of ancestral EF-P, the appearance of horizontally transferred EF-P variants, and genomic signs of EF-P dysfunction. Horizontal transfer of the efp gene has occurred several times among bacteria and is associated with the loss of highly conserved polyproline motifs. In this study, we pinpoint cases of horizontal EF-P transfer among a diverse set of bacteria and examine genomic features associated with these events in the phyla Thermotogota and Planctomycetes. In these phyla, horizontal EF-P transfer is also associated with the loss of entire polyproline motif-containing proteins, whose expression is likely dependent on EF-P. In particular, three proteases (Lon, ClpC, and FtsH) and three tRNA synthetases (ValS, IleS1, and IleS2) appear highly sensitive to EF-P transfer. The conserved polyproline motifs within these proteins all reside within close proximity to ATP-binding-regions, some of which are crucial for their function. Our work shows that an ancient EF-P dysfunction has left genomic traces that persist to this day, although it remains unclear whether this dysfunction was strictly due to loss of ancestral EF-P or was related to the appearance of an exogenous variant. The latter possibility would imply that the process of "domesticating" a horizontally transferred efp gene can perturb the overall function of EF-P.

Asunto(s)

Evolución Molecular , Transferencia de Gen Horizontal , Factores de Elongación de Péptidos , Péptidos , Proteoma , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Péptidos/metabolismo , Péptidos/genética , Bacterias/genética , Bacterias/metabolismo , Filogenia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo

RESUMEN

After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli (E. coli) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 (σ70) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α-C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ-independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation.

Asunto(s)

ARN Polimerasas Dirigidas por ADN , Proteínas de Escherichia coli , Escherichia coli , Regiones Promotoras Genéticas , Factor sigma , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Factor sigma/metabolismo , Factor sigma/genética , Factor sigma/química , Transcripción Genética , Factores de Transcripción/metabolismo , Dominios Proteicos , Factores de Elongación de Péptidos/metabolismo , Factores de Elongación de Péptidos/genética , ADN Bacteriano/metabolismo , ADN Bacteriano/genética , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/química , Represoras Lac/metabolismo , Represoras Lac/genética

RESUMEN

The prokaryotic translation elongation factor P (EF-P) and the eukaryotic/archaeal counterparts eIF5A/aIF5A are proteins that serve a crucial role in mitigating ribosomal stalling during the translation of specific sequences, notably those containing consecutive proline residues (1,2). Although mitochondrial DNA-encoded proteins synthesized by mitochondrial ribosomes also contain polyproline stretches, an EF-P/eIF5A mitochondrial counterpart remains unidentified. Here, we show that the missing factor is TACO1, a protein causative of a juvenile form of neurodegenerative Leigh's syndrome associated with cytochrome c oxidase deficiency, until now believed to be a translational activator of COX1 mRNA. By using a combination of metabolic labeling, puromycin release and mitoribosome profiling experiments, we show that TACO1 is required for the rapid synthesis of the polyproline-rich COX1 and COX3 cytochrome c oxidase subunits, while its requirement is negligible for other mitochondrial DNA-encoded proteins. In agreement with a role in translation efficiency regulation, we show that TACO1 cooperates with the N-terminal extension of the large ribosomal subunit bL27m to provide stability to the peptidyl-transferase center during elongation. This study illuminates the translation elongation dynamics within human mitochondria, a TACO1-mediated biological mechanism in place to mitigate mitoribosome stalling at polyproline stretches during protein synthesis, and the pathological implications of its malfunction.

Asunto(s)

Complejo IV de Transporte de Electrones , Proteínas Mitocondriales , Ribosomas Mitocondriales , Péptidos , Biosíntesis de Proteínas , Humanos , Complejo IV de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Péptidos/metabolismo , Péptidos/genética , Ribosomas Mitocondriales/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Factores de Elongación de Péptidos/genética , Células HEK293 , Ciclooxigenasa 1

RESUMEN

Efficiency of protein synthesis on the ribosome is strongly affected by the amino acid composition of the assembled amino acid chain. Challenging sequences include proline-rich motifs as well as highly positively and negatively charged amino acid stretches. Members of the F subfamily of ABC ATPases (ABCFs) have been long hypothesised to promote translation of such problematic motifs. In this study we have applied genetics and reporter-based assays to characterise the four housekeeping ABCF ATPases of Bacillus subtilis: YdiF, YfmM, YfmR/Uup and YkpA/YbiT. We show that YfmR cooperates with the translation factor EF-P that promotes translation of Pro-rich motifs. Simultaneous loss of both YfmR and EF-P results in a dramatic growth defect. Surprisingly, this growth defect can be largely suppressed though overexpression of an EF-P variant lacking the otherwise crucial 5-amino-pentanolylated residue K32. Using in vivo reporter assays, we show that overexpression of YfmR can alleviate ribosomal stalling on Asp-Pro motifs. Finally, we demonstrate that YkpA/YbiT promotes translation of positively and negatively charged motifs but is inactive in resolving ribosomal stalls on proline-rich stretches. Collectively, our results provide insights into the function of ABCF translation factors in modulating protein synthesis in B. subtilis.

Asunto(s)

Bacillus subtilis , Proteínas Bacterianas , Biosíntesis de Proteínas , Ribosomas , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Ribosomas/metabolismo , Ribosomas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Factores de Elongación de Péptidos/metabolismo , Factores de Elongación de Péptidos/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencias de Aminoácidos

RESUMEN

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Asunto(s)

Proteínas Bacterianas , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Biosíntesis de Proteínas , Factores de Elongación de Péptidos/metabolismo , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/genética , Antituberculosos/farmacología , Antituberculosos/química , Ribosomas/metabolismo , Modelos Moleculares , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Tuberculosis/metabolismo , Conformación Proteica

RESUMEN

N-Methylated amino acids are constituents of natural bioactive peptides and proteins. Nα-methylated amino acids appear abundantly in natural cyclic peptides, likely due to their constraint of peptide conformation and contribution to peptide stability. Peptides containing Nα-methylated amino acids have long been prepared by chemical synthesis. While such natural peptides are not produced ribosomally, recent ribosomal strategies have afforded Nα-methylated peptides. Presently, we define new strategies for the ribosomal incorporation of Nα-methylated amino acids into peptides and proteins. First, we identify modified ribosomes capable of facilitating the incorporation of six N-methylated amino acids into antibacterial scorpion peptide IsCT. Also synthesized analogously was a protein domain (RRM1) from hnRNP LL; improved yields were observed for nearly all tested N-methylated amino acids. Computational modeling of the ribosomal assembly illustrated how the distortion imposed by N-methylation could be compensated by altering the nucleotides in key 23S rRNA positions. Finally, it is known that incorporation of multiple prolines (an N-alkylated amino acid) ribosomally can be facilitated by bacterial elongation factor P. We report that supplementing endogenous EF-P during IsCT peptide and RRM1 protein synthesis gave improved yields for most of the N-methylated amino acids studied.

Asunto(s)

Aminoácidos , Factores de Elongación de Péptidos , Ribosomas , Ribosomas/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Metilación , Factores de Elongación de Péptidos/metabolismo , Factores de Elongación de Péptidos/química , Péptidos/química , Péptidos/metabolismo

RESUMEN

Objective: To explore the antiviral activity of the small-molecule compound AM679 in hepatitis B virus (HBV) replication and infection cell models. Methods: The positive regulatory effect of AM679 on EFTUD2 expression was validated by qPCR and Western blotting. HepAD38 and HepG2-NTCP cells were treated with AM679 (0.5, 1, and 2 nmol/L). Negative control, positive control, and AM679 combined with the entecavir group were set up. HBV DNA intra-and extracellularly, as well as the expression levels of intracellular HBV total RNAs and 3.5kb-RNA changes, were detected with qPCR. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured in the cell supernatant by an enzyme-linked immunosorbent assay (ELISA). The t-test method was used for the statistical analysis of the mean difference between groups. Results: EFTUD2 mRNA and protein expression levels were significantly increased in HepAD38 and HepG2-NTCP cells following AM679 treatment, with a statistically significant difference (P < 0.001). Intra-and extracellular indicators such as HBV DNA, HBV RNAs, HBV 3.5kb-RNA, HBsAg, and HBeAg were decreased to varying degrees in both cell models, and the decrease in these indicators was more pronounced with the increase in AM679 concentration and prolonged treatment duration, while the combined use of AM679 and entecavir had a more significant antiviral effect. The HBV DNA inhibition rates in the supernatant of HepAD38 cells with the use of 2 nmol/L AM679 were 21% and 48% on days three and nine, respectively. The AM679 combined with the ETV treatment group had the most significant inhibitory effect (62%), with a P < 0.01. More active HBV replication was observed after silencing EFTUD2, while the antiviral activity of AM679 was significantly weakened. Conclusion: AM679 exerts anti-HBV activity in vitro by targeting the regulation of EFTUD2 expression.

Asunto(s)

Antivirales , Virus de la Hepatitis B , Replicación Viral , Humanos , Antivirales/farmacología , ADN Viral , Guanina/análogos & derivados , Células Hep G2 , Antígenos e de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Indoles/química , Indoles/farmacología , Ácidos Pentanoicos/química , Ácidos Pentanoicos/farmacología , Factores de Elongación de Péptidos/antagonistas & inhibidores , Factores de Elongación de Péptidos/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/antagonistas & inhibidores , Ribonucleoproteína Nuclear Pequeña U5/metabolismo

RESUMEN

Mitochondrial fission occurs in many cellular processes, but the regulation of fission is poorly understood. We show that long-chain acyl-coenzyme A (LCACA) activates two related mitochondrial fission proteins, MiD49 and MiD51, by inducing their oligomerization, which activates their ability to stimulate the DRP1 GTPase. The 1:1 stoichiometry of LCACA:MiD in the oligomer suggests interaction in the previously identified nucleotide-binding pocket, and a point mutation in this pocket reduces LCACA binding and LCACA-induced oligomerization for MiD51. In cells, this LCACA binding mutant does not assemble into puncta on mitochondria or rescue MiD49/51 knockdown effects on mitochondrial length and DRP1 recruitment. Furthermore, cellular treatment with BSA-bound oleic acid, which causes increased LCACA, promotes mitochondrial fission in an MiD49/51-dependent manner. These results suggest that LCACA is an endogenous ligand for MiDs, inducing mitochondrial fission and providing a potential mechanism for fatty-acid-induced mitochondrial division. Finally, MiD49 or MiD51 oligomers synergize with Mff, but not with actin filaments, in DRP1 activation, suggesting distinct pathways for DRP1 activation.

Asunto(s)

Acilcoenzima A , Dinaminas , GTP Fosfohidrolasas , Mitocondrias , Dinámicas Mitocondriales , Proteínas Mitocondriales , Dinámicas Mitocondriales/efectos de los fármacos , Dinaminas/metabolismo , Dinaminas/genética , Humanos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Acilcoenzima A/metabolismo , Multimerización de Proteína , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Animales , Unión Proteica , Células HeLa , Células HEK293 , Ácido Oléico/farmacología , Ácido Oléico/metabolismo , Proteínas de la Membrana , Factores de Elongación de Péptidos

RESUMEN

Organisms possess a wide variety of proteins with diverse amino acid sequences, and their synthesis relies on the ribosome. Empirical observations have led to the misconception that ribosomes are robust protein factories, but in reality, they have several weaknesses. For instance, ribosomes stall during the translation of the proline-rich sequences, but the elongation factor EF-P assists in synthesizing proteins containing the poly-proline sequences. Thus, living organisms have evolved to expand the translation capability of ribosomes through the acquisition of translation elongation factors. In this study, we have revealed that Escherichia coli ATP-Binding Cassette family-F (ABCF) proteins, YheS, YbiT, EttA and Uup, individually cope with various problematic nascent peptide sequences within the exit tunnel. The correspondence between noncanonical translations and ABCFs was YheS for the translational arrest by nascent SecM, YbiT for poly-basic sequence-dependent stalling and poly-acidic sequence-dependent intrinsic ribosome destabilization (IRD), EttA for IRD at the early stage of elongation, and Uup for poly-proline-dependent stalling. Our results suggest that ATP hydrolysis-coupled structural rearrangement and the interdomain linker sequence are pivotal for handling 'hard-to-translate' nascent peptides. Our study highlights a new aspect of ABCF proteins to reduce the potential risks that are encoded within the nascent peptide sequences.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Proteínas de Escherichia coli , Escherichia coli , Péptidos , Secuencia de Aminoácidos , Transportadoras de Casetes de Unión a ATP/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Péptidos/metabolismo , Péptidos/química , Péptidos/genética , Biosíntesis de Proteínas , Ribosomas/metabolismo , Ribosomas/genética

RESUMEN

Bacteria overcome ribosome stalling by employing translation elongation factor P (EF-P), which requires post-translational modification (PTM) for its full activity. However, EF-Ps of the PGKGP subfamily are unmodified. The mechanism behind the ability to avoid PTM while retaining active EF-P requires further examination. Here, we investigate the design principles governing the functionality of unmodified EF-Ps in Escherichia coli. We screen for naturally unmodified EF-Ps with activity in E. coli and discover that the EF-P from Rhodomicrobium vannielii rescues growth defects of a mutant lacking the modification enzyme EF-P-(R)-ß-lysine ligase. We identify amino acids in unmodified EF-P that modulate its activity. Ultimately, we find that substitution of these amino acids in other marginally active EF-Ps of the PGKGP subfamily leads to fully functional variants in E. coli. These results provide strategies to improve heterologous expression of proteins with polyproline motifs in E. coli and give insights into cellular adaptations to optimize protein synthesis.

Asunto(s)

Escherichia coli , Factores de Elongación de Péptidos , Factores de Elongación de Péptidos/metabolismo , Factores de Elongación de Péptidos/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Ribosomas/metabolismo , Secuencia de Aminoácidos

RESUMEN

RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an α-helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a ß-barrel, which binds the ribosome. Here, we report structures of ops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH, which reveal swiveled, pre-translocated pause states stabilized by an ops hairpin in the non-template DNA. Autoinhibited RfaH binds and twists the ops hairpin, expanding the RNA:DNA hybrid to 11 base pairs and triggering the KOW release. Once activated, RfaH hyper-stabilizes the pause, which thus requires anti-backtracking factors for escape. Our results suggest that the entire RfaH cycle is solely determined by the ops and RfaH sequences and provide insights into mechanisms of recruitment and metamorphosis of NusG homologs across all life.

Asunto(s)

Proteínas de Escherichia coli , Factores de Transcripción , Factores de Transcripción/metabolismo , Transcripción Genética , Transactivadores/metabolismo , Proteínas de Escherichia coli/metabolismo , Factores de Elongación de Péptidos/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , ADN

RESUMEN

BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM, OMIM# 610536) is a rare monogenic disease that is caused by a mutation in the elongation factor Tu GTP binding domain containing 2 gene (EFTUD2, OMIM* 603892). It is characterized by mandibulofacial dysplasia, microcephaly, malformed ears, cleft palate, growth and intellectual disability. MFDM can be easily misdiagnosed due to its phenotypic overlap with other craniofacial dysostosis syndromes. The clinical presentation of MFDM is highly variable among patients. METHODS: A patient with craniofacial anomalies was enrolled and evaluated by a multidisciplinary team. To make a definitive diagnosis, whole-exome sequencing was performed, followed by validation by Sanger sequencing. RESULTS: The patient presented with extensive facial bone dysostosis, upward slanting palpebral fissures, outer and middle ear malformation, a previously unreported orbit anomaly, and spina bifida occulta. A novel, pathogenic insertion mutation (c.215_216insT: p.Tyr73Valfs*4) in EFTUD2 was identified as the likely cause of the disease. CONCLUSIONS: We diagnosed this atypical case of MFDM by the detection of a novel pathogenetic mutation in EFTUD2. We also observed previously unreported features. These findings enrich both the genotypic and phenotypic spectrum of MFDM.

Asunto(s)

Discapacidad Intelectual , Disostosis Mandibulofacial , Microcefalia , Humanos , Microcefalia/patología , Disostosis Mandibulofacial/genética , Disostosis Mandibulofacial/patología , Fenotipo , Mutación , Discapacidad Intelectual/genética , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/genética , Ribonucleoproteína Nuclear Pequeña U5/metabolismo

RESUMEN

BACKGROUND: Mitochondrial Ts translation elongation factor (TSFM) is an enzyme that catalyzes exchange of guanine nucleotides. By forming a complex with mitochondrial Tu translation elongation factor (TUFM), TSFM participates in mitochondrial protein translation. We have previously reported that TUFM regulates translation of beta-site APP cleaving enzyme 1 (BACE1) via ROS (reactive oxygen species)-dependent mechanism, suggesting a potential role in amyloid precursor protein (APP) processing associated with Alzheimer's disease (AD), which led to the speculation that TSFM may regulate APP processing in a similar way to TUFM. METHODS AND RESULTS: Here, we report that in cultured cells, knockdown or overexpression TSFM did not change protein levels in BACE1 and APP. Besides, the levels of cytoplasmic ROS and mitochondrial superoxide, in addition to ATP level, cell viability and mitochondrial membrane potential were not significantly altered by TSFM knockdown in the short term. Further transcriptome analysis revealed that expression of majority of mitochondrial genes were not remarkably changed by TSFM silencing. The possibility of TSFM involved in cardiomyopathy and cancer development was uncovered using bioinformatics analysis. CONCLUSIONS: Collectively, short-term regulation of TSFM level in cultured cells does not cause a significant change in proteins involved in APP processing, levels in ROS and ATP associated with mitochondrial function. Whereas our study could contribute to comprehend certain clinical features of TSFM mutations, the roles of TSFM in cardiomyopathy and cancer development might deserve further investigation.

Asunto(s)

Enfermedad de Alzheimer , Cardiomiopatías , Neoplasias , Humanos , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ácido Aspártico Endopeptidasas/genética , Enfermedad de Alzheimer/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias/metabolismo , Cardiomiopatías/metabolismo , Factores de Elongación de Péptidos/metabolismo , Adenosina Trifosfato , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo

RESUMEN

The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.

Asunto(s)

Factor 2 Eucariótico de Iniciación , Exorribonucleasas , Factores de Elongación de Péptidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Aminoácidos/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Mamíferos/metabolismo , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo

RESUMEN

Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1-8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG-RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.

Asunto(s)

Proteínas Bacterianas , Farmacorresistencia Bacteriana , Evolución Molecular , Aptitud Genética , Mycobacterium tuberculosis , Rifampin , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia Conservada , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genómica , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Rifampin/farmacología , Rifampin/uso terapéutico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología

RESUMEN

GCN1 is recognized as a factor that is essential for the activation of GCN2, which is a sensor of amino acid starvation. This function is evolutionarily conserved from yeast to higher eukaryotes. However, recent studies have revealed non-canonical functions of GCN1 that are independent of GCN2, such as its participation in cell proliferation, apoptosis, and the immune response, beyond the borders of species. Although it is known that GCN1 and GCN2 interact with ribosomes to accomplish amino acid starvation sensing, recent studies have reported that GCN1 binds to disomes (i.e., ribosomes that collide each other), thereby regulating both the co-translational quality control and stress response. We propose that GCN1 regulates ribosome-mediated signaling by dynamically changing its partners among RWD domain-possessing proteins via unknown mechanisms. We recently demonstrated that GCN1 is essential for cell proliferation and whole-body energy regulation in mice. However, the manner in which ribosome-initiated signaling via GCN1 is related to various physiological functions warrants clarification. GCN1-mediated mechanisms and its interaction with other quality control and stress response signals should be important for proteostasis during aging and neurodegenerative diseases, and may be targeted for drug development.

Asunto(s)

Proteínas Serina-Treonina Quinasas , Animales , Humanos , Ratones , Aminoácidos/metabolismo , Homeostasis , Factores de Elongación de Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismo

RESUMEN

Translation elongation factor P, expressed by the efp gene, is a conserved protein closely related to bacterial virulence and environmental stress regulation responses, however, little is known about the efp gene expression regulations. Here, the strain of Staphylococcus aureus subsp. aureus NCTC 8325 was taken as the research object and cultured under different conditions, including different culture temperatures, pH, and antibiotics, to study the expression of the efp gene in S. aureus by qRT-PCR, the results showed that the expression of the efp gene is upregulated under high temperature (40 °C), acidic (pH 5.4) or alkaline (pH 9.4) culture conditions, but upregulated early and downregulated later under the conditions of 0.5 MIC antibiotics (chloramphenicol at the final concentration of 2 µg/mL and vancomycin at the final concentration of 0.25 µg/mL), indicating that the efp promoter in S. aureus is inducible. The efp promoter sequence and structure in S. aureus were predicted by bioinformatics methods, and the predicted promoter was validated by constructing a promoter-probe vector and a series of promoter mutants, the results showed that the efp promoter sequence in S. aureus, named Pro, located in 1,548,179-1,548,250 of the S. aureus genome (NC_007795.1), and the sequence of - 10 element is CCTTATAGT, - 35 element is TTTACT. The results above could lay a foundation for screening transcription factors involved in the expression of the efp gene and then exploring the transcriptional regulation mechanism of EF-P in S. aureus.

Asunto(s)

Factores de Elongación de Péptidos , Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/genética , Proteínas Bacterianas/metabolismo , Factores de Transcripción/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Regulación Bacteriana de la Expresión Génica