RESUMO
Mycobacterial HflX confers resistance against macrolide antibiotics. However, the exact molecular mechanism is poorly understood. To gain further insights, we determined the cryo-EM structures of M. smegmatis (Msm) HflX-50S subunit and 50S subunit-erythromycin (ERY) complexes at a global resolution of approximately 3 Å. A conserved nucleotide A2286 at the gate of nascent peptide exit tunnel (NPET) adopts a swayed conformation in HflX-50S complex and interacts with a loop within the linker helical (LH) domain of MsmHflX that contains an additional 9 residues insertion. Interestingly, the swaying of this nucleotide, which is usually found in the non-swayed conformation, is induced by erythromycin binding. Furthermore, we observed that erythromycin decreases HflX's ribosome-dependent GTP hydrolysis, resulting in its enhanced binding and anti-association activity on the 50S subunit. Our findings reveal how mycobacterial HflX senses the presence of macrolides at the peptide tunnel entrance and confers antibiotic resistance in mycobacteria.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Farmacorresistência Bacteriana , Eritromicina , Modelos Moleculares , Mycobacterium smegmatis , Eritromicina/farmacologia , Eritromicina/química , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Antibacterianos/química , Ligação Proteica , Sítios de Ligação , Guanosina Trifosfato/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/químicaRESUMO
Ribosome biogenesis is a highly regulated cellular process that involves the control of numerous assembly factors. The small protein YjgA has been reported to play a role in the late stages of 50S assembly. However, the precise molecular mechanism underlying its function remains unclear. In this study, cryo-electron microscopy (cryo-EM) structures revealed that depletion of YjgA or its N-terminal loop in Escherichia coli both lead to the accumulation of immature 50S particles with structural abnormalities mainly in peptidyl transferase center (PTC) and H68/69 region. CryoDRGN analysis uncovered 8 and 6 distinct conformations of pre50S for ΔyjgA and YjgA-ΔNloop, respectively. These conformations highlighted the role of the N-terminal loop of YjgA in integrating uL16 and stabilizing H89 in PTC, which was further verified by the pull-down assays of YjgA and its mutants with uL16. Together with the function of undocking H68 through the binding of its C-terminal CTLH-like domain to the base of the L1 stalk, YjgA facilitates the maturation of PTC. This study identified critical domains of YjgA contributing to 50S assembly efficiency, providing a comprehensive understanding of the dual roles of YjgA in accelerating ribosome biogenesis and expanding our knowledge of the intricate processes governing cellular protein synthesis.
Assuntos
Microscopia Crioeletrônica , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Peptidil Transferases/metabolismo , Peptidil Transferases/genética , Ribossomos/metabolismo , Ribossomos/genética , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/química , Modelos Moleculares , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/química , Ligação ProteicaRESUMO
Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an Escherichia coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Re-introduction of the individual modification enzymes allows for the definition of their functions. The results demonstrate that in addition to previously known RlmE, also RlmB, RlmKL, RlmN and RluC facilitate large ribosome subunit assembly. RlmB and RlmKL have functions in ribosome assembly independent of their modification activities. While the assembly stage specificity of rRNA modification enzymes is well established, this study demonstrates that there is a mutual interdependence between the rRNA modification process and large ribosome subunit assembly.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , RNA Ribossômico , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Subunidades Ribossômicas Maiores/metabolismo , Subunidades Ribossômicas Maiores/genética , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/genética , Ribossomos/metabolismo , Ribossomos/genética , RNA Ribossômico/metabolismo , RNA Ribossômico/genética , RNA Ribossômico 23S/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/químicaRESUMO
During assembly, ribosomal particles in bacteria fold according to energy landscapes comprised of multiple parallel pathways. Cryo-electron microscopy studies have identified a critical maturation step that occurs during the late assembly stages of the 50S subunit in Bacillus subtilis. This step acts as a point of convergency for all the parallel assembly pathways of the subunit, where an assembly intermediate accumulates in a 'locked' state, causing maturation to pause. Assembly factors then act on this critical step to 'unlock' the last maturation steps involving the functional sites. Without these factors, the 50S subunit fails to complete its assembly, causing cells to die due to a lack of functional ribosomes to synthesize proteins. In this review, we analyze these findings in B. subtilis and examine other cryo-EM studies that have visualized assembly intermediates in different bacterial species, to determine if convergency points in the ribosome assembly process are a common theme among bacteria. There are still gaps in our knowledge, as these methodologies have not yet been applied to diverse species. However, identifying and characterizing these convergency points can reveal how different bacterial species implement unique mechanisms to regulate critical steps in the ribosome assembly process.
Assuntos
Bacillus subtilis , Subunidades Ribossômicas Maiores de Bactérias , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Modelos Moleculares , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Ribossomos/metabolismoRESUMO
During stress conditions such as heat shock and antibiotic exposure, ribosomes stall on messenger RNAs, leading to inhibition of protein synthesis. To remobilize ribosomes, bacteria use rescue factors such as HflXr, a homolog of the conserved housekeeping GTPase HflX that catalyzes the dissociation of translationally inactive ribosomes into individual subunits. Here we use time-resolved cryo-electron microscopy to elucidate the mechanism of ribosome recycling by Listeria monocytogenes HflXr. Within the 70S ribosome, HflXr displaces helix H69 of the 50S subunit and induces long-range movements of the platform domain of the 30S subunit, disrupting inter-subunit bridges B2b, B2c, B4, B7a and B7b. Our findings unveil a unique ribosome recycling strategy by HflXr which is distinct from that mediated by RRF and EF-G. The resemblance between HflXr and housekeeping HflX suggests that the alternative ribosome recycling mechanism reported here is universal in the prokaryotic kingdom.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Listeria monocytogenes , Ribossomos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Ribossomos/metabolismo , Listeria monocytogenes/metabolismo , Listeria monocytogenes/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/química , Biossíntese de Proteínas , Modelos Moleculares , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Fator G para Elongação de Peptídeos/metabolismo , Fator G para Elongação de Peptídeos/químicaRESUMO
In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fMet-tRNAfMet. Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entrance channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.
Assuntos
Bacteriófago lambda , Escherichia coli , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro , Proteínas Repressoras , Subunidades Ribossômicas Maiores de Bactérias , Proteínas Virais Reguladoras e Acessórias , Escherichia coli/genética , Escherichia coli/virologia , Proteínas Ribossômicas/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Proteínas Repressoras/genética , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic perturbations of the assembly process create bottlenecks where intermediates accumulate, facilitating structural characterization. We use cryo-electron microscopy, with iterative subclassification to identify intermediates in the assembly of the 50S ribosomal subunit from E. coli. The analysis of the ensemble of intermediates that spans the entire biogenesis pathway for the 50 S subunit was facilitated by a dimensionality reduction and cluster picking approach using PCA-UMAP-HDBSCAN. The identity of the cooperative folding units in the RNA with associated proteins is revealed, and the hierarchy of these units reveals a complete assembly map for all RNA and protein components. The assembly generally proceeds co-transcriptionally, with some flexibility in the landscape to ensure efficiency for this central cellular process under a variety of growth conditions.
Assuntos
Escherichia coli , Subunidades Ribossômicas Maiores de Bactérias , Microscopia Crioeletrônica , Escherichia coli/genética , Bactérias , RNARESUMO
RtcB is involved in transfer RNA (tRNA) splicing in archaeal and eukaryotic organisms. However, most RtcBs are found in bacteria, whose tRNAs have no introns. Because tRNAs are the substrates of archaeal and eukaryotic RtcB, it is assumed that bacterial RtcBs are for repair of damaged tRNAs. Here, we show that a subset of bacterial RtcB, denoted RtcB2 herein, specifically repair ribosomal damage in the decoding center. To access the damage site for repair, however, the damaged 70S ribosome needs to be dismantled first, and this is accomplished by bacterial PrfH. Peptide-release assays revealed that PrfH is only active with the damaged 70S ribosome but not with the intact one. A 2.55-Å cryo-electron microscopy structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into PrfH discriminating between the damaged and the intact ribosomes via specific recognition of the cleaved 3'-terminal nucleotide. RNA repair assays demonstrated that RtcB2 efficiently repairs the damaged 30S ribosomal subunit but not the damaged tRNAs. Cell-based assays showed that the RtcB2-PrfH pair reverse the damage inflicted by ribosome-specific ribotoxins in vivo. Thus, our combined biochemical, structural, and cell-based studies have uncovered a bacterial defense system specifically evolved to reverse the lethal ribosomal damage in the decoding center for cell survival.
Assuntos
Aminoacil-tRNA Sintetases , Proteínas de Escherichia coli , Subunidades Ribossômicas Maiores de Bactérias , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Microscopia Crioeletrônica , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Conformação Proteica , Splicing de RNA , RNA de Transferência/química , Subunidades Ribossômicas Maiores de Bactérias/efeitos dos fármacos , Subunidades Ribossômicas Maiores de Bactérias/metabolismoRESUMO
Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2'-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.
Assuntos
Proteínas de Bactérias , Metiltransferases , Mycobacterium tuberculosis , Subunidades Ribossômicas Maiores de Bactérias , Proteínas de Bactérias/química , Domínio Catalítico , Farmacorresistência Bacteriana/genética , Metiltransferases/química , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Conformação Proteica em alfa-Hélice , RNA Ribossômico 16S/química , RNA Ribossômico 23S/química , Subunidades Ribossômicas Maiores de Bactérias/químicaRESUMO
RbgA is an essential protein for the assembly of the 50S subunit in Bacillus subtilis. Depletion of RbgA leads to the accumulation of the 45S intermediate. A strain expressing a RbgA variant with reduced GTPase activity generates spontaneous suppressor mutations in uL6. Each suppressor strain accumulates a unique 44S intermediate. We reasoned that characterizing the structure of these mutant 44S intermediates may explain why RbgA is required to catalyze the folding of the 50S functional sites. We found that in the 44S particles, rRNA helices H42 and H97, near the binding site of uL6, adopt a flexible conformation and allow the central protuberance and functional sites in the mutant 44S particles to mature in any order. Instead, the wild-type 45S particles exhibit a stable H42-H97 interaction and their functional sites always mature last. The dependence on RbgA was also less pronounced in the 44S particles. We concluded that the binding of uL6 pauses the maturation of the functional sites, but the central protuberance continues to fold. RbgA exclusively binds intermediates with a formed central protuberance and licenses the folding of the functional sites. Through this mechanism, RbgA ensures that the functional sites of the 50S mature last.
Ribosomal subunits in bacteria assemble according to energy landscapes comprised of multiple parallel pathways. In this study, the authors identified a critical maturation step in the late assembly stages of the large 50S ribosomal subunit in bacteria. This step represents a merging point where all parallel assembly pathways of the ribosomal particles converge. At this critical step, the convergent assembly intermediate that accumulates in cells exists in a 'locked' state, and its maturation is paused. The RbgA protein acts on this critical step to 'unlock' the last maturation steps involving folding of the functional sites. Through this mechanism, RbgA ensures that the functional sites of the 50S mature last.
Assuntos
Proteínas Ribossômicas , Subunidades Ribossômicas Maiores de Bactérias , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Proteínas Ribossômicas/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , RNA Ribossômico/metabolismo , GTP Fosfo-Hidrolases/metabolismoRESUMO
Neisseria commensals are an indisputable source of resistance for their pathogenic relatives. However, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance (≥ 2 µg/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n = 7/16), we find mutations that reduce susceptibility to azithromycin converge on the locus encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through short tandem duplication events. Interestingly, one of the laboratory evolved mutations in rpmH is identical (7LKRTYQ12), and two nearly identical, to those recently reported to contribute to high-level azithromycin resistance in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Ultimately, studies like this will be critical for predicting commensal alleles that could rapidly disseminate into pathogen populations via allelic exchange across recombinogenic microbial genera.
Assuntos
Farmacorresistência Bacteriana/genética , Macrolídeos/farmacologia , Neisseria/genética , Antibacterianos/farmacologia , Azitromicina/farmacologia , Testes de Sensibilidade Microbiana , Microbiota/genética , Inibidores da Síntese de Proteínas , RNA Ribossômico 23S/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/genética , Ribossomos/genética , Deleção de Sequência/genéticaRESUMO
In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway.
Assuntos
Bacillus subtilis/genética , DNA Helicases/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Peptídeos/genética , Peptídeos/metabolismo , RNA de Transferência , Subunidades Ribossômicas Maiores de Bactérias/genéticaRESUMO
A series of novel pleuromutilin derivatives were designed and synthesized with 1,2,4-triazole as the linker connected to benzoyl chloride analogues under mild conditions. The in vitro antibacterial activities of the synthesized derivatives against four strains of Staphylococcus aureus (MRSA ATCC 43300, ATCC 29213, AD3 and 144) were tested by the broth dilution method. Most of the synthesized derivatives displayed potent activities, and 22-(3-amino-2-(4-methyl-benzoyl)-1,2,4-triazole-5-yl)-thioacetyl)-22-deoxypleuromutilin (compound 12) was found to be the most active antibacterial derivative against MRSA (MIC = 0.125 µg/mL). Furthermore, the time-kill curves showed compound 12 had a certain inhibitory effect against MRSA in vitro. The in vivo antibacterial activity of compound 12 was further evaluated using MRSA infected murine thigh model. Compound 12 exhibited superior antibacterial efficacy than tiamulin. It was also found that compound 12 had no significant inhibitory effect on the proliferation of RAW264.7 cells. Compound 12 was further evaluated in CYP450 inhibition assay and showed moderate inhibitory effect on CYP3A4 (IC50 = 3.95 µM). Moreover, seven candidate compounds showed different affinities with the 50S ribosome by SPR measurement. Subsequently, binding of compound 12 and 20 to the 50S ribosome was further investigated by molecular modeling. Three strong hydrogen bonds were formed through the interaction of compound 12 and 20 with 50S ribosome. The binding free energy of compound 12 and 20 with the ribosome was calculated to be -10.7 kcal/mol and -11.66 kcal/mol, respectively.
Assuntos
Antibacterianos/farmacologia , Diterpenos/farmacologia , Desenho de Fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Compostos Policíclicos/farmacologia , Subunidades Ribossômicas Maiores de Bactérias/efeitos dos fármacos , Animais , Antibacterianos/síntese química , Antibacterianos/química , Sobrevivência Celular/efeitos dos fármacos , Diterpenos/síntese química , Diterpenos/química , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Compostos Policíclicos/síntese química , Compostos Policíclicos/química , Células RAW 264.7 , Relação Estrutura-Atividade , PleuromutilinasRESUMO
Ribosome biogenesis is a fundamental multi-step cellular process that culminates in the formation of ribosomal subunits, whose production and modification are regulated by numerous biogenesis factors. In this study, we analyze physiologic prokaryotic ribosome biogenesis by isolating bona fide pre-50S subunits from an Escherichia coli strain with the biogenesis factor ObgE, affinity tagged at its native gene locus. Our integrative structural approach reveals a network of interacting biogenesis factors consisting of YjgA, RluD, RsfS, and ObgE on the immature pre-50S subunit. In addition, our study provides mechanistic insight into how the GTPase ObgE, in concert with other biogenesis factors, facilitates the maturation of the 50S functional core and reveals both conserved and divergent evolutionary features of ribosome biogenesis between prokaryotes and eukaryotes.
Assuntos
Proteínas de Escherichia coli , Evolução Molecular , Loci Gênicos , Hidroliases , Proteínas Monoméricas de Ligação ao GTP , Subunidades Ribossômicas Maiores de Bactérias , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hidroliases/química , Hidroliases/genética , Hidroliases/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/metabolismoRESUMO
Single particle cryo-EM excels in determining static structures of protein molecules, but existing 3D reconstruction methods have been ineffective in modelling flexible proteins. We introduce 3D variability analysis (3DVA), an algorithm that fits a linear subspace model of conformational change to cryo-EM data at high resolution. 3DVA enables the resolution and visualization of detailed molecular motions of both large and small proteins, revealing new biological insight from single particle cryo-EM data. Experimental results demonstrate the ability of 3DVA to resolve multiple flexible motions of α-helices in the sub-50 kDa transmembrane domain of a GPCR complex, bending modes of a sodium ion channel, five types of symmetric and symmetry-breaking flexibility in a proteasome, large motions in a spliceosome complex, and discrete conformational states of a ribosome assembly. 3DVA is implemented in the cryoSPARC software package.
Assuntos
Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Algoritmos , Proteínas Arqueais/química , Bases de Dados de Proteínas , Endopeptidases/química , Canal de Sódio Disparado por Voltagem NAV1.7/química , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Plasmodium falciparum/química , Receptores de Canabinoides/química , Subunidades Ribossômicas Maiores de Bactérias/química , Ribossomos/química , Razão Sinal-Ruído , Spliceossomos/químicaRESUMO
In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a 'closed' conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an 'open' conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Pentafosfato/biossíntese , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Acilação , Sítio Alostérico , Bacillus subtilis/genética , Domínio Catalítico , GTP Pirofosfoquinase/metabolismo , Hidrólise , Modelos Genéticos , Modelos Moleculares , Conformação Proteica , Processamento Pós-Transcricional do RNA , Subunidades Ribossômicas Maiores de Bactérias/metabolismoRESUMO
In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal "tails." How such tailing can occur without the regular suite of translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) of native complexes, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with RqcP, an Hsp15 family protein. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/ultraestruturaRESUMO
The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Precursores de RNA/metabolismo , Ribonucleases/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Sequência de Bases , Microscopia Crioeletrônica , Modelos Moleculares , Precursores de RNA/ultraestrutura , Ribonucleases/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Especificidade por SubstratoRESUMO
Prokaryotic messenger RNAs (mRNAs) are translated as they are transcribed. The lead ribosome potentially contacts RNA polymerase (RNAP) and forms a supramolecular complex known as the expressome. The basis of expressome assembly and its consequences for transcription and translation are poorly understood. Here, we present a series of structures representing uncoupled, coupled, and collided expressome states determined by cryo-electron microscopy. A bridge between the ribosome and RNAP can be formed by the transcription factor NusG, which stabilizes an otherwise-variable interaction interface. Shortening of the intervening mRNA causes a substantial rearrangement that aligns the ribosome entrance channel to the RNAP exit channel. In this collided complex, NusG linkage is no longer possible. These structures reveal mechanisms of coordination between transcription and translation and provide a framework for future study.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Fatores de Alongamento de Peptídeos/química , Biossíntese de Proteínas , Fatores de Transcrição/química , Transcrição Gênica , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica , Ligação Proteica , Conformação Proteica , RNA Mensageiro/química , Subunidades Ribossômicas Maiores de Bactérias/químicaRESUMO
Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of â¼20 aa/s.