RESUMEN
Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, PLP-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using E. coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated â¼7° closer to the EcSufS active site. EcSufE makes clear interactions with the α16 helix of EcSufS and site-directed mutants of several α16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces.
RESUMEN
Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, PLP-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using E. coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated ~7° closer to the EcSufS active site. EcSufE makes clear interactions with the α16 helix of EcSufS and site-directed mutants of several α16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces.
RESUMEN
Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 Å density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.
Asunto(s)
Proteínas Asociadas a CRISPR , ARN Guía de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas , ARN Bacteriano/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Asociadas a CRISPR/genéticaRESUMEN
Under oxidative stress and iron starvation conditions, Escherichia coli uses the Suf pathway to assemble iron-sulfur clusters. The Suf pathway mobilizes sulfur via SufS, a type II cysteine desulfurase. SufS is a pyridoxal-5'-phosphate-dependent enzyme that uses cysteine to generate alanine and an active-site persulfide (C364-S-S-). The SufS persulfide is protected from external oxidants/reductants and requires the transpersulfurase, SufE, to accept the persulfide to complete the SufS catalytic cycle. Recent reports on SufS identified a conserved "ß-latch" structural element that includes the α6 helix, a glycine-rich loop, a ß-hairpin, and a cis-proline residue. To identify a functional role for the ß-latch, we used site-directed mutagenesis to obtain the N99D and N99A SufS variants. N99 is a conserved residue that connects the α6 helix to the backbone of the glycine-rich loop via hydrogen bonds. Our x-ray crystal structures for N99A and N99D SufS show a distorted beta-hairpin and glycine-rich loop, respectively, along with changes in the dimer geometry. The structural disruption of the N99 variants allowed the external reductant TCEP to react with the active-site C364-persulfide intermediate to complete the SufS catalytic cycle in the absence of SufE. The substitutions also appear to disrupt formation of a high-affinity, close approach SufS-SufE complex as measured with fluorescence polarization. Collectively, these findings demonstrate that the ß-latch does not affect the chemistry of persulfide formation but does protect it from undesired reductants. The data also indicate the ß-latch plays an unexpected role in forming a close approach SufS-SufE complex to promote persulfide transfer.
Asunto(s)
Liasas de Carbono-Azufre , Proteínas de Escherichia coli , Proteínas Hierro-Azufre , Liasas , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Dominio Catalítico , Cisteína/metabolismo , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/metabolismo , Liasas/metabolismo , Sustancias Reductoras , Azufre/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
CRISPR systems elicit interference when a foreign nucleic acid is detected by its ability to base-pair to crRNA. Understanding what degree of complementarity between a foreign nucleic acid and crRNA is required for interference is a central question in the study of CRISPR systems. A clear description of which target-crRNA mismatches abrogate interference in type III, Cas10-containing, CRISPR systems has proved elusive due to the complexity of the system which utilizes three distinct interference activities. We characterized the effect of target-crRNA mismatches on in vitro cyclic oligoadenylate (cOA) synthesis and in vivo in an interference assay that depends on cOA synthesis. We found that sequence context affected whether a mismatched target was recognized by crRNA both in vitro and in vivo. We also investigated how the position of a mismatch within the target-crRNA duplex affected recognition by crRNA. Our data provide support for the hypothesis that a Cas10-activating region exists in the crRNA-target duplex, that the Cas10-proximal region of the duplex is the most critical in regulating cOA synthesis. Understanding the rules governing target recognition by type III CRISPR systems is critical: as one of the most prevalent CRISPR systems in nature, it plays an important role in the survival of many genera of bacteria. Recently, type III systems were re-purposed as a sensitive and accurate molecular diagnostic tool. Understanding the rules of target recognition in this system will be critical as it is engineered for biotechnology purposes.
Asunto(s)
Sistemas CRISPR-Cas , Ácidos Nucleicos , OligorribonucleótidosRESUMEN
6-Methyladenosine modification of DNA and RNA is widespread throughout the three domains of life and often accomplished by a Rossmann-fold methyltransferase domain which contains conserved sequence elements directing S-adenosylmethionine cofactor binding and placement of the target adenosine residue into the active site. Elaborations to the conserved Rossman-fold and appended domains direct methylation to diverse DNA and RNA sequences and structures. Recently, the first atomic-resolution structure of a ribosomal RNA adenine dimethylase (RRAD) family member bound to rRNA was solved, TFB1M bound to helix 45 of 12S rRNA. Since erythromycin resistance methyltransferases are also members of the RRAD family, and understanding how these enzymes recognize rRNA could be used to combat their role in antibiotic resistance, we constructed a model of ErmE bound to a 23S rRNA fragment based on the TFB1M-rRNA structure. We designed site-directed mutants of ErmE based on this model and assayed the mutants by in vivo phenotypic assays and in vitro assays with purified protein. Our results and additional bioinformatic analyses suggest our structural model captures key ErmE-rRNA interactions and indicate three regions of Erm proteins play a critical role in methylation: the target adenosine binding pocket, the basic ridge, and the α4-cleft.
Asunto(s)
Proteínas Bacterianas/química , Farmacorresistencia Microbiana/genética , Metiltransferasas/química , Procesamiento Postranscripcional del ARN , ARN Ribosómico/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Eritromicina/toxicidad , Metiltransferasas/genética , Metiltransferasas/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , ARN Ribosómico/metabolismoRESUMEN
Cysteine serves as the sulfur source for the biosynthesis of Fe-S clusters and thio-cofactors, molecules that are required for core metabolic processes in all organisms. Therefore, cysteine desulfurases, which mobilize sulfur for its incorporation into thio-cofactors by cleaving the Cα-S bond of cysteine, are ubiquitous in nature. SufS, a type 2 cysteine desulfurase that is present in plants and microorganisms, mobilizes sulfur from cysteine to the transpersulfurase SufE to initiate Fe-S biosynthesis. Here, a 1.5â Å resolution X-ray crystal structure of the Escherichia coli SufS homodimer is reported which adopts a state in which the two monomers are rotated relative to their resting state, displacing a ß-hairpin from its typical position blocking transpersulfurase access to the SufS active site. A global structure and sequence analysis of SufS family members indicates that the active-site ß-hairpin is likely to require adjacent structural elements to function as a ß-latch regulating access to the SufS active site.
Asunto(s)
Liasas de Carbono-Azufre/química , Dominio Catalítico , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dimerización , Proteínas de Escherichia coli/química , Azufre/metabolismoRESUMEN
Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m62A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity-binding assays. We demonstrate that residues in α4, α5, and the α5-α6 linker are essential for methyltransferase function, including an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine and basic residues in α5 and the α5-α6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.
Asunto(s)
Farmacorresistencia Microbiana/genética , Metiltransferasas/metabolismo , ARN Ribosómico 23S/metabolismo , Antibacterianos/farmacología , Eritromicina/farmacología , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Dominios ProteicosRESUMEN
Iron-sulfur (Fe-S) clusters are necessary for the proper functioning of numerous metalloproteins. Fe-S cluster (Isc) and sulfur utilization factor (Suf) pathways are the key biosynthetic routes responsible for generating these Fe-S cluster prosthetic groups in Escherichia coli Although Isc dominates under normal conditions, Suf takes over during periods of iron depletion and oxidative stress. Sulfur acquisition via these systems relies on the ability to remove sulfur from free cysteine using a cysteine desulfurase mechanism. In the Suf pathway, the dimeric SufS protein uses the cofactor pyridoxal 5'-phosphate (PLP) to abstract sulfur from free cysteine, resulting in the production of alanine and persulfide. Despite much progress, the stepwise mechanism by which this PLP-dependent enzyme operates remains unclear. Here, using rapid-mixing kinetics in conjunction with X-ray crystallography, we analyzed the pre-steady-state kinetics of this process while assigning early intermediates of the mechanism. We employed H123A and C364A SufS variants to trap Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction, enabling direct observations of these intermediates and associated conformational changes of the SufS active site. Of note, we propose that Cys-364 is essential for positioning the Cys-aldimine for Cα deprotonation, His-123 acts to protonate the Ala-enamine intermediate, and Arg-56 facilitates catalysis by hydrogen bonding with the sulfhydryl of Cys-aldimine. Our results, along with previous SufS structural findings, suggest a detailed model of the SufS-catalyzed reaction from Cys binding to C-S bond cleavage and indicate that Arg-56, His-123, and Cys-364 are critical SufS residues in this C-S bond cleavage pathway.
Asunto(s)
Escherichia coli/enzimología , Liasas/química , Modelos Moleculares , Sustitución de Aminoácidos , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/genética , Liasas/genética , Liasas/metabolismo , Mutación MissenseRESUMEN
CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that Type III CRISPR-Cas systems synthesize second messenger molecules previously unknown to exist in prokaryotes, cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal Type III systems, cOA generation and its regulation have not been explored for the Staphylococcus epidermidis Type III-A CRISPR-Cas system, a longstanding model for CRISPR-Cas function. Here, we demonstrate that this system performs Mg2+-dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest a natural mechanism to dampen immunity once the foreign RNA is destroyed.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Oligorribonucleótidos/metabolismo , ARN Bacteriano/metabolismo , Staphylococcus epidermidis/metabolismo , Nucleótidos de Adenina/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Magnesio/metabolismo , Modelos Moleculares , Oligorribonucleótidos/biosíntesis , Polimorfismo de Nucleótido Simple , Sistemas de Mensajero SecundarioRESUMEN
SufS is a type II cysteine desulfurase and acts as the initial step in the Suf Fe-S cluster assembly pathway. In Escherichia coli, this pathway is utilized under conditions of oxidative stress and is resistant to reactive oxygen species. Mechanistically, this means SufS must shift between protecting a covalent persulfide intermediate and making it available for transfer to the next protein partner in the pathway, SufE. Here, we report five X-ray crystal structures of SufS including a new structure of SufS containing an inward-facing persulfide intermediate on C364. Additional structures of SufS variants with substitutions at the dimer interface show changes in dimer geometry and suggest a conserved ß-hairpin structure plays a role in mediating interactions with SufE. These new structures, along with previous HDX-MS and biochemical data, identify an interaction network capable of communication between active-sites of the SufS dimer coordinating the shift between desulfurase and transpersulfurase activities.
Asunto(s)
Liasas de Carbono-Azufre/metabolismo , Proteínas de Escherichia coli/metabolismo , Sulfuros/metabolismo , Liasas de Carbono-Azufre/química , Liasas de Carbono-Azufre/genética , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/química , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mutación , Unión Proteica , Sulfuros/químicaRESUMEN
Accurate translation of the genetic code is critical to ensure expression of proteins with correct amino acid sequences. Certain tRNAs can cause a shift out of frame (i.e., frameshifting) due to imbalances in tRNA concentrations, lack of tRNA modifications or insertions or deletions in tRNAs (called frameshift suppressors). Here, we determined the structural basis for how frameshift-suppressor tRNASufA6 (a derivative of tRNAPro) reprograms the mRNA frame to translate a 4-nt codon when bound to the bacterial ribosome. After decoding at the aminoacyl (A) site, the crystal structure of the anticodon stem-loop of tRNASufA6 bound in the peptidyl (P) site reveals ASL conformational changes that allow for recoding into the +1 mRNA frame. Furthermore, a crystal structure of full-length tRNASufA6 programmed in the P site shows extensive conformational rearrangements of the 30S head and body domains similar to what is observed in a translocation intermediate state containing elongation factor G (EF-G). The 30S movement positions tRNASufA6 toward the 30S exit (E) site disrupting key 16S rRNA-mRNA interactions that typically define the mRNA frame. In summary, this tRNA-induced 30S domain change in the absence of EF-G causes the ribosome to lose its grip on the mRNA and uncouples the canonical forward movement of the tRNAs during elongation.
Asunto(s)
Mutación del Sistema de Lectura/genética , Sistema de Lectura Ribosómico/genética , ARN de Transferencia/genética , Ribosomas/genética , Bacterias/genética , Codón/genética , Factor G de Elongación Peptídica/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN Ribosómico 16S/genética , Sistemas de Lectura/genéticaRESUMEN
CRISPRCas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a prokaryotic immune system that destroys foreign nucleic acids in a sequence-specific manner using Cas nucleases guided by short RNAs (crRNAs). Staphylococcus epidermidis harbours a Type III-A CRISPRCas system that encodes the Cas10Csm interference complex and crRNAs that are subjected to multiple processing steps. The final step, called maturation, involves a concerted effort between Csm3, a ruler protein in Cas10Csm that measures six-nucleotide increments, and the activity of a nuclease(s) that remains unknown. Here, we elucidate the contributions of the Cas10Csm complex toward maturation and explore roles of non-Cas nucleases in this process. Using genetic and biochemical approaches, we show that charged residues in Csm3 facilitate its self-assembly and dictate the extent of maturation cleavage. Additionally, acidic residues in Csm5 are required for efficient maturation, but recombinant Csm5 fails to cleave crRNAs in vitro. However, we detected cellular nucleases that co-purify with Cas10Csm, and show that Csm5 regulates their activities through distinct mechanisms. Altogether, our results support roles for non-Cas nuclease(s) during crRNA maturation and establish a link between Type III-A CRISPRCas immunity and central nucleic acid metabolism.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ARN Bacteriano/metabolismo , Ribonucleasas/metabolismo , Proteínas Asociadas a CRISPR/química , Polirribonucleótido Nucleotidiltransferasa , Staphylococcus epidermidis/enzimología , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/metabolismoRESUMEN
Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosome-dependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. Here, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop to recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide.
Asunto(s)
Toxinas Bacterianas/metabolismo , ARN Mensajero/metabolismo , Sitios de Unión , Codón , Nucleótidos/metabolismoRESUMEN
Important viral and cellular gene products are regulated by stop codon readthrough and mRNA frameshifting, processes whereby the ribosome detours from the reading frame defined by three nucleotide codons after initiation of translation. In the last few years, rapid progress has been made in mechanistically characterizing both processes and also revealing that trans-acting factors play important regulatory roles in frameshifting. Here, we review recent biophysical studies that bring new molecular insights to stop codon readthrough and frameshifting. Lastly, we consider whether there may be common mechanistic themes in -1 and +1 frameshifting based on recent X-ray crystal structures of +1 frameshift-prone tRNAs bound to the ribosome.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Secuencia de Bases , Sistema de Lectura Ribosómico , Humanos , ARN Mensajero/genética , ARN de Transferencia/fisiología , Sistemas de Lectura , Ribosomas/fisiologíaRESUMEN
The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5' or 3' direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA(SufJ), a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA(SufJ) contains an insertion 5' to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL(SufJ) or tRNA(SufJ) does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL(SufJ) and ASL(Thr) bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34-37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL(SufJ) imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA(SufJ) during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.
Asunto(s)
Genes Supresores , Biosíntesis de Proteínas/genética , ARN Ribosómico 16S/ultraestructura , ARN de Transferencia/ultraestructura , Ribosomas/genética , Anticodón/genética , Anticodón/ultraestructura , Cristalografía por Rayos X , Escherichia coli , Conformación de Ácido Nucleico , Nucleótidos/química , Nucleótidos/genética , ARN Mensajero/genética , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Thermus thermophilus/genéticaRESUMEN
Maintenance of the correct reading frame on the ribosome is essential for accurate protein synthesis. Here, we report structures of the 70S ribosome bound to frameshift suppressor tRNA(SufA6) and N1-methylguanosine at position 37 (m(1)G37) modification-deficient anticodon stem loop(Pro), both of which cause the ribosome to decode 4 rather than 3 nucleotides, resulting in a +1 reading frame. Our results reveal that decoding at +1 suppressible codons causes suppressor tRNA(SufA6) to undergo a rearrangement of its 5' stem that destabilizes U32, thereby disrupting the conserved U32-A38 base pair. Unexpectedly, the removal of the m(1)G37 modification of tRNA(Pro) also disrupts U32-A38 pairing in a structurally analogous manner. The lack of U32-A38 pairing provides a structural correlation between the transition from canonical translation and a +1 reading of the mRNA. Our structures clarify the molecular mechanism behind suppressor tRNA-induced +1 frameshifting and advance our understanding of the role played by the ribosome in maintaining the correct translational reading frame.
Asunto(s)
Escherichia coli/genética , Sistema de Lectura Ribosómico/genética , ARN de Transferencia/genética , Ribosomas/genética , Salmonella typhimurium/genética , Thermus thermophilus/genética , Anticodón/química , Anticodón/genética , Cristalografía por Rayos X , Genes Supresores , Secuencias Invertidas Repetidas/genética , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genética , ARN de Transferencia/química , Ribosomas/químicaRESUMEN
Aminoglycosides are potent, broad spectrum, ribosome-targeting antibacterials whose clinical efficacy is seriously threatened by multiple resistance mechanisms. Here, we report the structural basis for 30S recognition by the novel plasmid-mediated aminoglycoside-resistance rRNA methyltransferase A (NpmA). These studies are supported by biochemical and functional assays that define the molecular features necessary for NpmA to catalyze m(1)A1408 modification and confer resistance. The requirement for the mature 30S as a substrate for NpmA is clearly explained by its recognition of four disparate 16S rRNA helices brought into proximity by 30S assembly. Our structure captures a "precatalytic state" in which multiple structural reorganizations orient functionally critical residues to flip A1408 from helix 44 and position it precisely in a remodeled active site for methylation. Our findings provide a new molecular framework for the activity of aminoglycoside-resistance rRNA methyltransferases that may serve as a functional paradigm for other modification enzymes acting late in 30S biogenesis.
Asunto(s)
Aminoglicósidos/farmacología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Metiltransferasas/química , Metiltransferasas/metabolismo , Modelos Moleculares , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/farmacología , Aminoácidos/metabolismo , Biocatálisis/efectos de los fármacos , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Transferencia de Gen Horizontal/efectos de los fármacos , Metilación/efectos de los fármacos , Nucleótidos/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Ribosómico 16S/química , Relación Estructura-ActividadRESUMEN
After four decades of research aimed at understanding tRNA selection on the ribosome, the mechanism by which ribosomal ambiguity (ram) mutations promote miscoding remains unclear. Here, we present two X-ray crystal structures of the Thermus thermophilus 70S ribosome containing 16S rRNA ram mutations, G347U and G299A. Each of these mutations causes miscoding in vivo and stimulates elongation factor thermo unstable (EF-Tu)-dependent GTP hydrolysis in vitro. Mutation G299A is located near the interface of ribosomal proteins S4 and S5 on the solvent side of the subunit, whereas G347U is located 77 Å distant, at intersubunit bridge B8, close to where EF-Tu engages the ribosome. Despite these disparate locations, both mutations induce almost identical structural rearrangements that disrupt the B8 bridge--namely, the interaction of h8/h14 with L14 and L19. This conformation most closely resembles that seen upon EF-Tu-GTP-aminoacyl-tRNA binding to the 70S ribosome. These data provide evidence that disruption and/or distortion of B8 is an important aspect of GTPase activation. We propose that, by destabilizing B8, G299A and G347U reduce the energetic cost of attaining the GTPase-activated state and thereby decrease the stringency of decoding. This previously unappreciated role for B8 in controlling the decoding process may hold relevance for many other ribosomal mutations known to influence translational fidelity.
Asunto(s)
Proteínas Bacterianas/genética , Mutación , Factor Tu de Elongación Peptídica/genética , ARN Ribosómico 16S/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Hidrólisis , Cinética , Modelos Moleculares , Conformación Molecular , Conformación de Ácido Nucleico , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/metabolismo , Unión Proteica , ARN Ribosómico 16S/química , ARN Ribosómico 16S/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/química , Subunidades Ribosómicas Grandes Bacterianas/genética , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Thermus thermophilus/genética , Thermus thermophilus/metabolismoRESUMEN
E. coli continues to serve as a key model for the structure and function of the ribosome, structures of ribosome from other organisms and domains of life have also greatly contributed to our knowledge of protein synthesis. Many structural models of the ribosome in a number of steps of the protein synthesis cycle have been solved by cryo-electron microscopy (cryo-EM) and x-ray crystallography. This chapter introduces the structure and dynamics of the ribosome based on these structures and ends with a brief discussion of the many questions that the structures leave unanswered. Protein synthesis is a multistep process, and the structural features of the ribosome along with the large number of cofactors reflect the complexity of translation. Numerous protein factors in addition to the ribosome contribute to translation in bacteria during the steps of initiation, elongation, termination, and recycling. These protein factors make intimate contacts to key regions of the ribosome, and this aspect is discussed in the chapter in light of our present understanding of the structure and function of the ribosome. The intact ribosome contains three binding sites for substrate tRNAs that are termed as the aminoacyl-tRNA site (A site), peptidyl-tRNA site (P site), and exit-tRNA site (E site). These three binding sites span the interface between the 30S and 50S subunits. The central activity of the ribosome is catalysis of peptide bond formation. The region of the ribosome responsible for catalyzing the reaction is called the peptidyl transferase center (PTC).