ABSTRACT
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 (â¼95%) and sparsely populated excited (â¼5%) states with an exchange lifetime of â¼3 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.
Subject(s)
Escherichia coli Proteins , Protein Folding , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Nuclear Magnetic Resonance, Biomolecular , Peptide Elongation Factors/metabolism , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/genetics , Trans-Activators/metabolism , Trans-Activators/chemistry , Trans-Activators/genetics , Models, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , DNA-Directed RNA Polymerases/metabolism , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/genetics , Protein ConformationABSTRACT
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.
Subject(s)
Amino Acids , Peptide Elongation Factors , Ribosomes , Ribosomes/metabolism , Amino Acids/chemistry , Amino Acids/metabolism , Methylation , Peptide Elongation Factors/metabolism , Peptide Elongation Factors/chemistry , Peptides/chemistry , Peptides/metabolismABSTRACT
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.
Subject(s)
Bacterial Proteins , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Protein Biosynthesis , Peptide Elongation Factors/metabolism , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/genetics , Antitubercular Agents/pharmacology , Antitubercular Agents/chemistry , Ribosomes/metabolism , Models, Molecular , Tuberculosis/drug therapy , Tuberculosis/microbiology , Tuberculosis/metabolism , Protein ConformationABSTRACT
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 (Pâ <â 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 Pâ <â 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.
Subject(s)
Antiviral Agents , Hepatitis B virus , Virus Replication , Humans , Antiviral Agents/pharmacology , DNA, Viral , Guanine/analogs & derivatives , Hep G2 Cells , Hepatitis B e Antigens/metabolism , Hepatitis B Surface Antigens/metabolism , Hepatitis B virus/drug effects , Virus Replication/drug effects , Indoles/chemistry , Indoles/pharmacology , Pentanoic Acids/chemistry , Pentanoic Acids/pharmacology , Peptide Elongation Factors/antagonists & inhibitors , Peptide Elongation Factors/metabolism , Ribonucleoprotein, U5 Small Nuclear/antagonists & inhibitors , Ribonucleoprotein, U5 Small Nuclear/metabolismABSTRACT
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.
Subject(s)
Intellectual Disability , Mandibulofacial Dysostosis , Microcephaly , Humans , Microcephaly/pathology , Mandibulofacial Dysostosis/genetics , Mandibulofacial Dysostosis/pathology , Phenotype , Mutation , Intellectual Disability/genetics , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Ribonucleoprotein, U5 Small Nuclear/genetics , Ribonucleoprotein, U5 Small Nuclear/metabolismABSTRACT
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.
Subject(s)
Escherichia coli Proteins , Transcription Factors , Transcription Factors/metabolism , Transcription, Genetic , Trans-Activators/metabolism , Escherichia coli Proteins/metabolism , Peptide Elongation Factors/metabolism , DNA-Directed RNA Polymerases/metabolism , DNAABSTRACT
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.
Subject(s)
Alzheimer Disease , Cardiomyopathies , Neoplasms , Humans , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Reactive Oxygen Species/metabolism , Aspartic Acid Endopeptidases/genetics , Alzheimer Disease/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Neoplasms/metabolism , Cardiomyopathies/metabolism , Peptide Elongation Factors/metabolism , Adenosine Triphosphate , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolismABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters , Escherichia coli Proteins , Escherichia coli , Peptides , Amino Acid Sequence , ATP-Binding Cassette Transporters/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/chemistry , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Peptides/metabolism , Peptides/chemistry , Peptides/genetics , Protein Biosynthesis , Ribosomes/metabolism , Ribosomes/geneticsABSTRACT
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.
Subject(s)
Escherichia coli , Peptide Elongation Factors , Peptide Elongation Factors/metabolism , Peptide Elongation Factors/genetics , Escherichia coli/metabolism , Escherichia coli/genetics , Protein Biosynthesis , Protein Processing, Post-Translational , Ribosomes/metabolism , Amino Acid SequenceABSTRACT
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.
Subject(s)
Acyl Coenzyme A , Dynamins , GTP Phosphohydrolases , Mitochondria , Mitochondrial Dynamics , Mitochondrial Proteins , Mitochondrial Dynamics/drug effects , Dynamins/metabolism , Dynamins/genetics , Humans , Mitochondria/metabolism , Mitochondria/drug effects , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/genetics , Acyl Coenzyme A/metabolism , Protein Multimerization , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/genetics , Animals , Protein Binding , HeLa Cells , HEK293 Cells , Oleic Acid/pharmacology , Oleic Acid/metabolism , Membrane Proteins , Peptide Elongation FactorsABSTRACT
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.
Subject(s)
Bacterial Proteins , Drug Resistance, Bacterial , Evolution, Molecular , Genetic Fitness , Mycobacterium tuberculosis , Rifampin , Humans , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Conserved Sequence , DNA-Directed RNA Polymerases/antagonists & inhibitors , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Drug Resistance, Bacterial/drug effects , Drug Resistance, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Genomics , Mutation , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Rifampin/pharmacology , Rifampin/therapeutic use , Transcription Factors/genetics , Transcription Factors/metabolism , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/microbiologyABSTRACT
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.
Subject(s)
Protein Serine-Threonine Kinases , Animals , Humans , Mice , Amino Acids/metabolism , Homeostasis , Peptide Elongation Factors/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA-Binding Proteins/metabolism , Trans-Activators/metabolismABSTRACT
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.
Subject(s)
Peptide Elongation Factors , Staphylococcal Infections , Staphylococcus aureus , Humans , Staphylococcus aureus/genetics , Bacterial Proteins/metabolism , Transcription Factors/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Gene Expression Regulation, BacterialABSTRACT
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.
Subject(s)
Eukaryotic Initiation Factor-2 , Exoribonucleases , Peptide Elongation Factors , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Amino Acids/metabolism , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Mammals/metabolism , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolismABSTRACT
During transcription elongation, NusG aids RNA polymerase by inhibiting pausing, promoting anti-termination on rRNA operons, coupling transcription with translation on mRNA genes, and facilitating Rho-dependent termination. Despite extensive work, the in vivo functional allocation and spatial distribution of NusG remain unknown. Using single-molecule tracking and super-resolution imaging in live E. coli cells, we found NusG predominantly in a chromosome-associated population (binding to RNA polymerase in elongation complexes) and a slowly diffusing population complexed with the 30S ribosomal subunit; the latter provides a "30S-guided" path for NusG into transcription elongation. Only â¼10% of NusG is fast diffusing, with its mobility suggesting non-specific interactions with DNA for >50% of the time. Antibiotic treatments and deletion mutants revealed that chromosome-associated NusG participates mainly in rrn anti-termination within phase-separated transcriptional condensates and in transcription-translation coupling. This study illuminates the multiple roles of NusG and offers a guide on dissecting multi-functional machines via in vivo imaging.
Subject(s)
Escherichia coli Proteins , Transcription Factors , Transcription Factors/genetics , Transcription Factors/chemistry , Transcription, Genetic , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/chemistry , Peptide Elongation Factors/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Bacterial Proteins/geneticsABSTRACT
Alternative splicing (AS), found in approximately 95 % of human genes, significantly amplifies protein diversity and is implicated in disease pathogenesis when dysregulated. However, the precise involvement of AS in the toxic mechanisms induced by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) remains incompletely elucidated. This study conducted a thorough global AS analysis in six human cell lines following TCDD exposure. Our findings revealed that environmentally relevant concentration (0.1 nM) of TCDD significantly suppressed AS events in all cell types, notably inhibiting diverse splicing events and reducing transcript diversity, potentially attributed to modifications in the splicing patterns of the inhibitory factor family, particularly hnRNP. And we identified 151 genes with substantial AS alterations shared among these cell types, particularly enriched in immune and metabolic pathways. Moreover, TCDD induced cell-specific changes in splicing patterns and transcript levels, with increased sensitivity notably in THP-1 monocyte, potentially linked to aberrant expression of pivotal genes within the spliceosome pathway (DDX5, EFTUD2, PUF60, RBM25, SRSF1, and CRNKL1). This study extends our understanding of disrupted alternative splicing and its relation to the multisystem toxicity of TCDD. It sheds light on how environmental toxins affect post-transcriptional regulatory processes, offering a fresh perspective for toxicology and disease etiology investigations.
Subject(s)
Polychlorinated Dibenzodioxins , Humans , Polychlorinated Dibenzodioxins/toxicity , Alternative Splicing , Serine-Arginine Splicing Factors , Peptide Elongation Factors , Ribonucleoprotein, U5 Small NuclearABSTRACT
Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, ß'1 and ß'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spanning approximately half of the ß'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP in the absence of iNTP bound at the active site and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG, or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms and organelle.
Subject(s)
Cyanobacteria , Escherichia coli Proteins , Transcription, Genetic , Escherichia coli/genetics , DNA-Directed RNA Polymerases/metabolism , Cyanobacteria/genetics , Cyanobacteria/metabolism , DNA/metabolism , Peptide Elongation Factors/metabolism , Transcription Factors/metabolism , Escherichia coli Proteins/metabolismABSTRACT
Protein synthesis is performed by the ribosome and a host of highly conserved elongation factors. Elongation factor P (EF-P) prevents ribosome stalling at difficult-to-translate sequences, such as polyproline tracts. In bacteria, phenotypes associated with efp deletion range from modest to lethal, suggesting that some species encode an additional translation factor that has similar function to EF-P. Here we identify YfmR as a translation factor that is essential in the absence of EF-P in Bacillus subtilis. YfmR is an ABCF ATPase that is closely related to both Uup and EttA, ABCFs that bind the ribosomal E-site and are conserved in more than 50% of bacterial genomes. We show that YfmR associates with actively translating ribosomes and that depleting YfmR from Δefp cells causes severe ribosome stalling at a polyproline tract in vivo. YfmR depletion from Δefp cells was lethal and caused reduced levels of actively translating ribosomes. Our results therefore identify YfmR as an important translation factor that is essential in B. subtilis in the absence of EF-P.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Escherichia coli/genetics , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Protein Biosynthesis , Cell Death , ATP-Binding Cassette Transporters/metabolism , Escherichia coli Proteins/metabolismABSTRACT
Targeting translational factor proteins (TFPs) presents significant promise for the development of innovative antitubercular drugs. Previous insights from antibiotic binding mechanisms and recently solved 3D crystal structures of Mycobacterium tuberculosis (Mtb) elongation factor thermo unstable-GDP (EF-Tu-GDP), elongation factor thermo stable-EF-Tu (EF-Ts-EF-Tu), and elongation factor G-GDP (EF-G-GDP) have opened up new avenues for the design and development of potent antituberculosis (anti-TB) therapies.
Subject(s)
Antitubercular Agents , Peptide Elongation Factor Tu , Guanosine Diphosphate/chemistry , Guanosine Diphosphate/metabolism , Peptide Elongation Factor Tu/chemistry , Peptide Elongation Factor Tu/metabolism , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/metabolism , Proteins/metabolismABSTRACT
RNA polymerase II (Pol II) transcription is regulated by many elongation factors. Among these factors, TFIIF, PAF-RTF1, ELL and Elongin stimulate mRNA chain elongation by Pol II. Cryo-EM structures of Pol II complexes with these elongation factors now reveal some general principles on how elongation factors bind Pol II and how they stimulate transcription. All four elongation factors contact Pol II at domains external 2 and protrusion, whereas TFIIF and ELL additionally bind the Pol II lobe. All factors apparently stabilize cleft-flanking elements, whereas RTF1 and Elongin additionally approach the active site with a latch element and may influence catalysis or translocation. Due to the shared binding sites on Pol II, factor binding is mutually exclusive, and thus it remains to be studied what determines which elongation factors bind at a certain gene and under which condition.