RESUMO
The role of post-transcriptional RNA modification is of growing interest. One example is the addition of non-templated uridine residues to the 3' end of transcripts. In mammalian systems, uridylation is integral to cell cycle control of histone mRNA levels. This regulatory mechanism is dependent on the nonsense-mediated decay (NMD) component, Upf1, which promotes histone mRNA uridylation and degradation in response to the arrest of DNA synthesis. We have identified a similar system in Aspergillus nidulans, where Upf1 is required for the regulation of histone mRNA levels. However, other NMD components are also implicated, distinguishing it from the mammalian system. As in human cells, 3' uridylation of histone mRNA is induced upon replication arrest. Disruption of this 3' tagging has a significant but limited effect on histone transcript regulation, consistent with multiple mechanisms acting to regulate mRNA levels. Interestingly, 3' end degraded transcripts are also subject to re-adenylation. Both mRNA pyrimidine tagging and re-adenylation are dependent on the same terminal-nucleotidyltransferases, CutA, and CutB, and we show this is consistent with the in vitro activities of both enzymes. Based on these data we argue that mRNA 3' tagging has diverse and distinct roles associated with transcript degradation, functionality and regulation.
Assuntos
Aspergillus nidulans/genética , Histonas/genética , RNA Mensageiro/genética , Regiões 3' não Traduzidas/genética , Replicação do DNA/fisiologia , Glutationa/análogos & derivados , Glutationa/genética , Glutationa/metabolismo , Histonas/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/metabolismo , Processamento Pós-Transcricional do RNA/genética , Processamento Pós-Transcricional do RNA/fisiologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Uridina/químicaRESUMO
Recent reports indicate that the Type six secretion system exported effector 8 (Tse8) is a cytoactive effector secreted by the Type VI secretion system (T6SS) of the human pathogen Pseudomonas aeruginosa. The T6SS is a nanomachine that assembles inside of the bacteria and injects effectors/toxins into target cells, providing a fitness advantage over competing bacteria and facilitating host colonisation. Here we present the first crystal structure of Tse8 revealing that it conserves the architecture of the catalytic triad Lys84-transSer162-Ser186 that characterises members of the Amidase Signature superfamily. Furthermore, using binding affinity experiments, we show that the interaction of phenylmethylsulfonyl fluoride (PMSF) to Tse8 is dependent on the putative catalytic residue Ser186, providing support for its nucleophilic reactivity. This work thus demonstrates that Tse8 belongs to the Amidase Signature (AS) superfamily. Furthermore, it highlights Tse8 similarity to two family members: the Stenotrophomonas maltophilia Peptide Amidase and the Glutamyl-tRNAGln amidotransferase subunit A from Staphylococcus aureus.
Assuntos
Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos/química , Pseudomonas aeruginosa/química , Sistemas de Secreção Tipo VI/química , Amidoidrolases/química , RNA de Transferência/químicaRESUMO
In bacteria, the start site and the reading frame of the messenger RNA are selected by the small ribosomal subunit (30S) when the start codon, typically an AUG, is decoded in the P-site by the initiator tRNA in a process guided and controlled by three initiation factors. This process can be efficiently inhibited by GE81112, a natural tetrapeptide antibiotic that is highly specific toward bacteria. Here GE81112 was used to stabilize the 30S pre-initiation complex and obtain its structure by cryo-electron microscopy. The results obtained reveal the occurrence of changes in both the ribosome conformation and initiator tRNA position that may play a critical role in controlling translational fidelity. Furthermore, the structure highlights similarities with the early steps of initiation in eukaryotes suggesting that shared structural features guide initiation in all kingdoms of life.
Assuntos
Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , RNA de Transferência de Metionina/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Células Eucarióticas/metabolismo , Modelos Moleculares , Conformação Molecular , Fatores de Iniciação em Procariotos/química , Fatores de Iniciação em Procariotos/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/químicaRESUMO
During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, namely, kinetically trapped particles. Among the assembly factors, the circularly permuted GTPase, RsgA, plays a crucial role in the maturation of the 30S decoding center. Here, directed hydroxyl radical probing and single particle cryo-EM are employed to elucidate RsgAÎs mechanism of action. Our results show that RsgA destabilizes the 30S structure, including late binding r-proteins, providing a structural basis for avoiding kinetically trapped assembly intermediates. Moreover, RsgA exploits its distinct GTPase pocket and specific interactions with the 30S to coordinate GTPase activation with the maturation state of the 30S subunit. This coordination validates the architecture of the decoding center and facilitates the timely release of RsgA to control the progression of 30S biogenesis.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , GTP Fosfo-Hidrolases/química , Domínio Catalítico , Microscopia Crioeletrônica , Ativação Enzimática , Proteínas de Escherichia coli/fisiologia , GTP Fosfo-Hidrolases/fisiologia , Guanosina Trifosfato/química , Ligação de Hidrogênio , Hidrólise , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Ribossômicas Menores de BactériasRESUMO
In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.
Assuntos
Antibacterianos/química , Escherichia coli/química , Iniciação Traducional da Cadeia Peptídica , RNA Bacteriano/química , RNA Ribossômico 16S/química , RNA de Transferência/química , Subunidades Ribossômicas Menores de Bactérias/química , Conformação de Ácido NucleicoRESUMO
Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.
Assuntos
Cinamatos/química , Cinamatos/farmacologia , Higromicina B/análogos & derivados , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/farmacologia , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/efeitos dos fármacos , Sítios de Ligação , Cinamatos/metabolismo , Cristalografia por Raios X , Higromicina B/química , Higromicina B/metabolismo , Higromicina B/farmacologia , Modelos Moleculares , Peptidil Transferases/química , Peptidil Transferases/efeitos dos fármacos , Inibidores da Síntese de Proteínas/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/enzimologia , Subunidades Ribossômicas Maiores de Bactérias/metabolismoRESUMO
The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. Despite the availability of structures of various EF-G-ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G-ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit 'pe/E' hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the 'missing link' in terms of tRNA intermediates involved in the universally conserved translocation process.
Assuntos
Movimento , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Modelos Moleculares , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA de Transferência/química , RNA de Transferência/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Thermus thermophilus/químicaRESUMO
The thiopeptide class of antibiotics targets the GTPase-associated center (GAC) of the ribosome to inhibit translation factor function. Using X-ray crystallography, we have determined the binding sites of thiostrepton (Thio), nosiheptide (Nosi), and micrococcin (Micro), on the Deinococcus radiodurans large ribosomal subunit. The thiopeptides, by binding within a cleft located between the ribosomal protein L11 and helices 43 and 44 of the 23S rRNA, overlap with the position of domain V of EF-G, thus explaining how this class of drugs perturbs translation factor binding to the ribosome. The presence of Micro leads to additional density for the C-terminal domain (CTD) of L7, adjacent to and interacting with L11. The results suggest that L11 acts as a molecular switch to control L7 binding and plays a pivotal role in positioning one L7-CTD monomer on the G' subdomain of EF-G to regulate EF-G turnover during protein synthesis.
Assuntos
Bacteriocinas , Regulação da Expressão Gênica , Peptídeos , Biossíntese de Proteínas , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos , Tioestreptona , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacteriocinas/química , Bacteriocinas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Deinococcus/química , Deinococcus/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Ribossômicas/genética , Ribossomos/química , Ribossomos/metabolismo , Tiazóis/química , Tiazóis/metabolismo , Tioestreptona/química , Tioestreptona/metabolismoRESUMO
Although both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in both in vitro and in vivo activity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to the tert-butyl-glycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins.
Assuntos
Minociclina/análogos & derivados , Ribossomos/metabolismo , Cristalografia por Raios X , Minociclina/metabolismo , Minociclina/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , RNA Ribossômico 16S/metabolismo , Thermus thermophilus/efeitos dos fármacos , Thermus thermophilus/metabolismo , TigeciclinaRESUMO
The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800 kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo- and heteronuclear (13)C and (15)N NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of ~25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexes.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Ribossomos/química , Congelamento , Modelos Moleculares , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular/métodosRESUMO
EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the tRNAs on the ribosome, thereby reverting the canonical translocation reaction. In the current work, EF4 was directly visualized in the process of back-translocating tRNAs by single-particle cryo-EM. Using flexible fitting methods, we built a model of ribosome-bound EF4 based on the cryo-EM map and a recently published unbound EF4 X-ray structure. The cryo-EM map establishes EF4 as a noncanonical elongation factor that interacts not only with the elongating ribosome, but also with the back-translocated tRNA in the A-site region, which is present in a previously unseen, intermediate state and deviates markedly from the position of a canonical A-tRNA. Our results, therefore, provide insight into the underlying structural principles governing back-translocation.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Transporte Biológico Ativo , Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Substâncias Macromoleculares , Modelos Moleculares , Fatores de Iniciação de PeptídeosRESUMO
The ribosomal stalk complex plays a crucial role in delivering translation factors to the catalytic site of the ribosome. It has a very similar architecture in all cells, although the protein components in bacteria are unrelated to those in archaea and eukaryotes. Here we used mass spectrometry to investigate ribosomal stalk complexes from bacteria, eukaryotes, and archaea in situ on the ribosome. Specifically we targeted ribosomes with different optimal growth temperatures. Our results showed that for the mesophilic bacterial ribosomes we investigated the stalk complexes are exclusively pentameric or entirely heptameric in the case of thermophilic bacteria, whereas we observed only pentameric stalk complexes in eukaryotic species. We also found the surprising result that for mesophilic archaea, Methanococcus vannielii, Methanococcus maripaludis, and Methanosarcina barkeri, both pentameric and heptameric stoichiometries are present simultaneously within a population of ribosomes. Moreover the ratio of pentameric to heptameric stalk complexes changed during the course of cell growth. We consider these differences in stoichiometry within ribosomal stalk complexes in the context of convergent evolution.
Assuntos
Filogenia , Ribossomos/química , Ribossomos/genética , Espectrometria de Massas em Tandem , Animais , Archaea/metabolismo , Eucariotos , Peso Molecular , Proteínas Ribossômicas/química , Temperatura , Thermus thermophilus/crescimento & desenvolvimento , Thermus thermophilus/metabolismoRESUMO
Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Plantas/metabolismo , Plastídeos/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas/química , Subunidades Ribossômicas/metabolismo , Subunidades Ribossômicas/ultraestrutura , Ribossomos/química , Ribossomos/ultraestrutura , Homologia de Sequência de Aminoácidos , Spinacia oleracea/genética , Spinacia oleracea/metabolismoRESUMO
Kasugamycin (Ksg) specifically inhibits translation initiation of canonical but not of leaderless messenger RNAs. Ksg inhibition is thought to occur by direct competition with initiator transfer RNA. The 3.35-A structure of Ksg bound to the 30S ribosomal subunit presented here provides a structural description of two Ksg-binding sites as well as a basis for understanding Ksg resistance. Notably, neither binding position overlaps with P-site tRNA; instead, Ksg mimics codon nucleotides at the P and E sites by binding within the path of the mRNA. Coupled with biochemical experiments, our results suggest that Ksg indirectly inhibits P-site tRNA binding through perturbation of the mRNA-tRNA codon-anticodon interaction during 30S canonical initiation. In contrast, for 70S-type initiation on leaderless mRNA, the overlap between mRNA and Ksg is reduced and the binding of tRNA is further stabilized by the presence of the 50S subunit, minimizing Ksg efficacy.
Assuntos
Aminoglicosídeos/farmacologia , Escherichia coli/química , Iniciação Traducional da Cadeia Peptídica , RNA Bacteriano/química , RNA Mensageiro/química , RNA de Transferência/metabolismo , Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Antibacterianos/química , Sítios de Ligação , Códon , Modelos Moleculares , Nucleotídeos/química , Estrutura Terciária de Proteína , RNA de Transferência/química , Relação Estrutura-AtividadeRESUMO
The oxazolidinones represent the first new class of antibiotics to enter into clinical usage within the past 30 years, but their binding site and mechanism of action has not been fully characterized. We have determined the crystal structure of the oxazolidinone linezolid bound to the Deinococcus radiodurans 50S ribosomal subunit. Linezolid binds in the A site pocket at the peptidyltransferase center of the ribosome overlapping the aminoacyl moiety of an A-site bound tRNA as well as many clinically important antibiotics. Binding of linezolid stabilizes a distinct conformation of the universally conserved 23S rRNA nucleotide U2585 that would be nonproductive for peptide bond formation. In conjunction with available biochemical data, we present a model whereby oxazolidinones impart their inhibitory effect by perturbing the correct positioning of tRNAs on the ribosome.
Assuntos
Antibacterianos/química , Oxazolidinonas/química , Peptidil Transferases/química , Peptidil Transferases/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/enzimologia , Antibacterianos/farmacologia , Sítios de Ligação , Deinococcus/efeitos dos fármacos , Deinococcus/enzimologia , Modelos Moleculares , Conformação de Ácido Nucleico , Oxazolidinonas/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Ribossomos/efeitos dos fármacosRESUMO
While a structural description of the molecular mechanisms guiding ribosome assembly in eukaryotic systems is emerging, bacteria use an unrelated core set of assembly factors for which high-resolution structural information is still missing. To address this, we used single-particle cryo-electron microscopy to visualize the effects of bacterial ribosome assembly factors RimP, RbfA, RsmA, and RsgA on the conformational landscape of the 30S ribosomal subunit and obtained eight snapshots representing late steps in the folding of the decoding center. Analysis of these structures identifies a conserved secondary structure switch in the 16S ribosomal RNA central to decoding site maturation and suggests both a sequential order of action and molecular mechanisms for the assembly factors in coordinating and controlling this switch. Structural and mechanistic parallels between bacterial and eukaryotic systems indicate common folding features inherent to all ribosomes.
Assuntos
Subunidades Ribossômicas Menores de Bactérias , Ribossomos , Microscopia Crioeletrônica , RNA Ribossômico 16S/genética , Subunidades Ribossômicas MenoresRESUMO
Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a three-dimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes. Fitting of the conserved portions of the x-ray crystallographic structure of the bacterial 70S ribosome into our cryo-EM map of the chloro-ribosome reveals the positions of PRP extensions and the locations of the PSRPs. Surprisingly, PSRP1 binds in the decoding region of the small (30S) ribosomal subunit, in a manner that would preclude the binding of messenger and transfer RNAs to the ribosome, suggesting that PSRP1 is a translation factor rather than a ribosomal protein. PSRP2 and PSRP3 appear to structurally compensate for missing segments of the 16S rRNA within the 30S subunit, whereas PSRP4 occupies a position buried within the head of the 30S subunit. One of the two PSRPs in the large (50S) ribosomal subunit lies near the tRNA exit site. Furthermore, we find a mass of density corresponding to chloro-ribosome recycling factor; domain II of this factor appears to interact with the flexible C-terminal domain of PSRP1. Our study provides evolutionary insights into the structural and functional roles that the PSRPs play during protein synthesis in chloroplasts.
Assuntos
Cloroplastos/química , Proteínas de Plantas/química , Proteínas Ribossômicas/química , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/química , Cloroplastos/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Evolução Molecular , Proteínas de Plantas/metabolismo , Plastídeos/química , Plastídeos/ultraestrutura , Conformação Proteica , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Spinacia oleracea/metabolismoRESUMO
RbfA (ribosome binding factor A; 15.2 kDa) is a protein involved in ribosome biogenesis and has been shown to be important for growth at low temperatures and to act as a suppressor for a cold-sensitive mutation (C23U) in the ribosomal RNA of the small 30S ribosomal subunit. The 3D structure of isolated RbfA has been determined from several organisms showing that RbfA has type-II KH-domain fold topology similar to the KH domain of another assembly factor, Era, whose overexpression can compensate for the deletion of rbfA, suppressing both the cold sensitivity and abnormal accumulation of 17S rRNA in rbfA knockout stains. Interestingly, a RbfAΔ25 variant used in previous NMR studies, truncated at the C-terminal domain to remove 25 unstructured residues causing aggregation at room temperature, was biologically active in the sense that it could complement a knock-out of wildtype RbfA, although it did not act as a suppressor for a 16S cold-sensitive mutation (C23U), nor did it interact stably with the 30S subunit. To complement this work, we report the 1H, 13C, and 15 N backbone and sidechain NMR resonance assignments of full length RbfA from Escherichia coli measured under physiological conditions (pH 7.6). This construct contains seven additional C-terminal residues from the cloning (i.e. one alanine and six residues from the HRV 3C cleavage site) and no aggregation issues were observed over a 1-week period at 293 K. The assignment data has been deposited in the BMRB data bank under Accession No. 27857.
Assuntos
Proteínas de Escherichia coli/análise , Escherichia coli/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Ribossômicas/análise , Ribossomos/metabolismo , Sequência de Aminoácidos , Proteínas de Escherichia coli/química , Estrutura Secundária de Proteína , Proteínas Ribossômicas/químicaRESUMO
Ribosome biogenesis is an energetically expensive and complex cellular process that involves the coordinated folding of the ribosomal RNA and dozens of ribosomal proteins. It proceeds along multiple parallel pathways and is guided by trans-acting factors called ribosome assembly factors. Although this process has been studied for decades, there are still many open questions regarding the role of the ribosome assembly factors in directing the folding of ribosome biogenesis intermediates. RimP is one of the early acting factors and guides the assembly of the small 30S ribosomal subunit by facilitating the binding of ribosomal proteins uS5 and uS12. Here we report the virtually complete 1H, 15N, and 13C chemical shift assignment of RimP from Escherichia coli. The NMR chemical shift data, deposited in the BMRB data bank under Accession No. 28014, indicates a widely folded protein composed of three alpha helices and eight beta strands.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Ribossômicas/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Isótopos de Nitrogênio , Estrutura Secundária de ProteínaRESUMO
We report here the use of methyl NMR spectroscopy with a selective-excitation pulsing scheme to extract structural information about a ribosome-bound nascent chain, a complex with a molecular weight of more than 2 MDa and a sample concentration in the micromolar range. The carbon chemical shifts of methyl groups are particularly sensitive to the development of the tertiary structure of a protein it folds, and crucially for systems that are at the limit of acceptable signal-to-noise-ratios, methyl group spectroscopy has higher sensitivity than does backbone amide group-based NMR spectroscopy. Comparison of the side-chain methyl correlations of the ribosome-bound nascent chain to previously obtained backbone amide correlations reveals dynamical perturbations within the hydrophobic core of the folded domain, which are attributed to motional restriction of the nascent chain as a result of ribosome attachment. Methyl NMR spectroscopy therefore provides improved spectral quality and complementary structural information to that of the amide groups and hence promises to provide a greatly enhanced understanding of the molecular basis of cotranslational folding at atomic resolution.