RESUMEN
Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5' region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.
Asunto(s)
Chaperonas Moleculares/fisiología , Complejos Multiproteicos/fisiología , Extensión de la Cadena Peptídica de Translación/fisiología , Pliegue de Proteína , Proteostasis/fisiología , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Alelos , Mutación con Pérdida de Función , Chaperonas Moleculares/genética , Mutación Missense , Peptidil Transferasas/fisiología , Mutación Puntual , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/fisiología , Ribosomas/ultraestructura , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Bacterial cell shapes are manifestations of programs carried out by multi-protein machines that synthesize and remodel the resilient peptidoglycan (PG) mesh and other polymers surrounding cells. GpsB protein is conserved in low-GC Gram-positive bacteria and is not essential in rod-shaped Bacillus subtilis, where it plays a role in shuttling penicillin-binding proteins (PBPs) between septal and side-wall sites of PG synthesis. In contrast, we report here that GpsB is essential in ellipsoid-shaped, ovococcal Streptococcus pneumoniae (pneumococcus), and depletion of GpsB leads to formation of elongated, enlarged cells containing unsegregated nucleoids and multiple, unconstricted rings of fluorescent-vancomycin staining, and eventual lysis. These phenotypes are similar to those caused by selective inhibition of Pbp2x by methicillin that prevents septal PG synthesis. Dual-protein 2D and 3D-SIM (structured illumination) immunofluorescence microscopy (IFM) showed that GpsB and FtsZ have overlapping, but not identical, patterns of localization during cell division and that multiple, unconstricted rings of division proteins FtsZ, Pbp2x, Pbp1a and MreC are in elongated cells depleted of GpsB. These patterns suggest that GpsB, like Pbp2x, mediates septal ring closure. This first dual-protein 3D-SIM IFM analysis also revealed separate positioning of Pbp2x and Pbp1a in constricting septa, consistent with two separable PG synthesis machines.
Asunto(s)
Proteínas Bacterianas/fisiología , Peptidoglicano/metabolismo , Streptococcus pneumoniae/citología , Streptococcus pneumoniae/metabolismo , Factores de Virulencia/fisiología , Proteínas Bacterianas/metabolismo , División Celular , Proteínas del Citoesqueleto/metabolismo , Eliminación de Gen , Imagenología Tridimensional , Meticilina/farmacología , Microscopía Fluorescente , Proteínas de Unión a las Penicilinas/fisiología , Peptidil Transferasas/fisiología , Fenotipo , Transporte de Proteínas , Streptococcus pneumoniae/genética , Factores de Virulencia/metabolismoRESUMEN
In eukaryotes, permanent inhibition of the non-homologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non-essential Swi2/Snf2-related translocase and a Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere-telomere fusions. Uls1 requirement is alleviated by the absence of poly-SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly-SUMO conjugates. We propose that one of Uls1 functions is to clear non-functional poly-SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly-SUMOylated proteins on DNA in eukaryotes.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Helicasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Telómero/metabolismo , ADN Helicasas/metabolismo , Regulación hacia Abajo , Organismos Modificados Genéticamente , Peptidil Transferasas/metabolismo , Peptidil Transferasas/fisiología , Unión Proteica , Multimerización de Proteína/fisiología , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/fisiología , Sumoilación/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
The crystal structures of ribosomes that have been obtained since 2000 have transformed our understanding of protein synthesis. In addition to proving that RNA is responsible for catalyzing peptide bond formation, these structures have provided important insights into the mechanistic details of how the ribosome functions. This review emphasizes what has been learned about the mechanism of peptide bond formation, the antibiotics that inhibit ribosome function, and the fidelity of decoding.
Asunto(s)
Biosíntesis de Proteínas/genética , ARN/fisiología , Ribosomas/fisiología , Antibacterianos/farmacología , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Peptidil Transferasas/antagonistas & inhibidores , Peptidil Transferasas/fisiología , ARN/química , ARN Ribosómico/química , ARN Ribosómico/fisiología , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/químicaRESUMEN
Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation. The TS is formed simultaneously with the rotatory motion enabling the translocation of the A-site tRNA 3' end into the P site, and we estimated the magnitude of rotation angle between the A-site starting position and the place at which the TS occurs. The calculated TS activation energy, E(a), is 35.5 kcal (1 kcal = 4.18 kJ)/mol, and the increase in hydrogen bonding between the rotating A-site tRNA and ribosome nucleotides as the TS forms appears to stabilize it to a value qualitatively estimated to be approximately 18 kcal/mol. The optimized geometry corresponds to a structure in which the peptide bond is being formed as other bonds are being broken, in such a manner as to release the P-site tRNA so that it may exit as a free molecule and be replaced by the translocating A-site tRNA. At TS formation the 2' OH group of the P-site tRNA A76 forms a hydrogen bond with the oxygen atom of the carboxyl group of the amino acid attached to the A-site tRNA, which may be indicative of its catalytic role, consistent with recent biochemical experiments.
Asunto(s)
Péptidos/química , Péptidos/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Químicos , Modelos Moleculares , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , Peptidil Transferasas/fisiología , Unión Proteica , Conformación Proteica , Teoría Cuántica , ARN de Transferencia/química , ARN de Transferencia/metabolismo , RotaciónRESUMEN
The ribosomal peptidyl transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that peptidyl transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring ribozymes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accumulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.
Asunto(s)
Evolución Molecular , Peptidil Transferasas/química , Peptidil Transferasas/fisiología , Proteínas Ribosómicas/fisiología , Ribosomas/enzimología , Antibacterianos/farmacología , Catálisis , Modelos Moleculares , Peptidil Transferasas/antagonistas & inhibidores , ARN Catalítico/fisiología , Proteínas Ribosómicas/químicaRESUMEN
Ribosomes are ribozymes exerting substrate positioning and promoting substrate-mediated catalysis. Peptide-bonds are formed within a symmetrical region, thus suggesting that ribosomes evolved by gene-fusion. Remote interactions dominate substrate positioning at stereochemistry suitable for peptide-bond formation and elaborate architectural-design guides the processivity of the reaction by rotatory motion. Nascent proteins are directed into the exit tunnel at extended conformation, complying with the tunnel's narrow entrance. Tunnel dynamics facilitate its interactive participation in elongation, discrimination, cellular signaling and nascent-protein trafficking into the chaperon-aided folding site. Conformational alterations, induced by ribosomal-recycling factor, facilitate subunit dissociation. Remarkably, although antibiotics discrimination is determined by the identity of a single nucleotide, involved also in resistance, additional nucleotides dictate antibiotics effectiveness.
Asunto(s)
Peptidil Transferasas/química , Biosíntesis de Proteínas , Pliegue de Proteína , Ribosomas/química , Evolución Molecular , Estructura Molecular , Extensión de la Cadena Peptídica de Translación , Peptidil Transferasas/fisiología , Conformación Proteica , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/fisiologíaRESUMEN
The atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with substrates and antibiotics have provided insights into the way the 3000 nucleotide 23S rRNA folds into a compact, specific structure and interacts with 27 ribosomal proteins as well as the structural basis of the peptidyl transferase reaction and its inhibition by antibiotics. The structure shows that the ribosome is indeed a ribozyme.
Asunto(s)
Antibacterianos/química , Farmacorresistencia Microbiana , Biosíntesis de Proteínas , Ribosomas/química , Ribosomas/efectos de los fármacos , Antibacterianos/farmacología , Haloarcula marismortui/fisiología , Estructura Molecular , Conformación de Ácido Nucleico , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Extensión de la Cadena Peptídica de Translación/fisiología , Peptidil Transferasas/fisiología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Ribosómico 23S/metabolismo , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/fisiologíaRESUMEN
beta-lactams have a long history in the treatment of infectious diseases, though their use has been and continues to be confounded by the development of resistance in target organisms. beta-lactamases, particularly in Gram-negative pathogens, are a major determinant of this resistance, although alterations in the beta-lactam targets, the penicillin-binding proteins (PBPs), are also important, especially in Gram-positive pathogens. Mechanisms for the efflux and/or exclusion of these agents also contribute, though often in conjunction these other two. Approaches for overcoming these resistance mechanisms include the development of novel beta-lactamase-stable beta-lactams, beta-lactamase inhibitors to be employed with existing beta-lactams, beta-lactam compounds that bind strongly to low-affinity PBPs and agents that potentiate the activity of existing beta-lactams against low-affinity PBP-producing organisms.
Asunto(s)
Resistencia betalactámica , Antibacterianos/metabolismo , Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Resistencia a Múltiples Medicamentos , Hexosiltransferasas/fisiología , Muramoilpentapéptido Carboxipeptidasa/fisiología , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/fisiología , Permeabilidad , Plásmidos , beta-Lactamasas/metabolismoRESUMEN
We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all beta-lactams. Affinity for penicillin generally correlated with beta-lactam MICs for the mutants, but these associations were not strictly proportional.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Hexosiltransferasas/genética , Hexosiltransferasas/fisiología , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/fisiología , Mutación/genética , Mutación/fisiología , Peptidil Transferasas/genética , Peptidil Transferasas/fisiología , Resistencia betalactámica/genética , Resistencia a la Ampicilina/genética , Cristalografía por Rayos X , Vectores Genéticos/genética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Proteínas de Unión a las Penicilinas , Penicilinas/metabolismo , Plásmidos/genética , Unión ProteicaRESUMEN
Recent data highlight how eukaryotic ribosomes connect polypeptide synthesis to translational regulation and targeting. Information contained in nascent polypeptides can be transmitted by surprisingly diverse routes.
Asunto(s)
Péptidos/genética , Peptidil Transferasas/fisiología , Ribosomas/genética , Ribosomas/fisiología , Partícula de Reconocimiento de Señal/fisiología , Modelos Moleculares , Imitación Molecular/genética , Imitación Molecular/fisiología , Péptidos/fisiología , Peptidil Transferasas/genética , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Estructura Terciaria de Proteína , Partícula de Reconocimiento de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
This study examines the role of the penicillin-binding proteins (PBPs) of Bacteroides fragilis in the mechanism of resistance to different beta-lactam antibiotics. Six of the eight strains used were beta-lactamase-positive by the nitrocefin assay. These strains displayed reduced susceptibility to imipenem (MIC, 2-16 mg l(-1)) and some of them were resistant to the actions of ampicillin, cefuroxime, cephalexin, cefoxitin and piperacillin. When studying specific enzymic activity, the capacity to degrade cefuroxime was only detected in strains AK-4, R212 and 0423 and the capacity to degrade cephalexin was only detected in strains R212 and 2013E; no specific activity was detected on imipenem. Metallo-beta-lactamase activity was only detected in strains AK-2 and 119, despite the fact that the cfiA gene was identified in four strains (AK-2, 2013E, 119 and 7160). The cepA gene was detected in six of the eight strains studied. Three high-molecular-mass PBPs were detected in all strains; however, in some cases, PBP2Bfr and/or PBP3Bfr appeared as a faint band. PBP4Bfr and PBP5Bfr were detected in six strains. PBP6Bfr only was detected in B. fragilis strains AK-2, 0423, 119 and 7160. By analysis of the sequence of B. fragilis chromosomal DNA and comparison with genes that are known to encode PBPs in Escherichia coli, six genes that encode PBP-like proteins were detected in the former organism. The gene that encodes the PBP2 orthologue of E. coli (pbpABfr, PBP3Bfr) was sequenced in six of the eight strains and its implications for resistance were examined. Differences in the PBP3Bfr amino acid sequences of strains AK-2 and 119 and their production of beta-lactamases indicate that these differences are not involved in the mechanism of resistance to imipenem and/or cephalexin.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/química , Bacteroides fragilis/efectos de los fármacos , Proteínas Portadoras/química , Hexosiltransferasas/química , Muramoilpentapéptido Carboxipeptidasa/química , Peptidil Transferasas/química , beta-Lactamasas/química , beta-Lactamas/farmacología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Bacteroides fragilis/química , Bacteroides fragilis/enzimología , Bacteroides fragilis/genética , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Dermatoglifia del ADN , ADN Bacteriano/química , Farmacorresistencia Bacteriana , Hexosiltransferasas/genética , Hexosiltransferasas/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Peso Molecular , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/fisiología , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/genética , Peptidil Transferasas/fisiología , Reacción en Cadena de la Polimerasa , beta-Lactamasas/genética , beta-Lactamasas/fisiologíaRESUMEN
The penicillin-binding proteins (PBPs) polymerize and modify peptidoglycan, the stress-bearing component of the bacterial cell wall. As part of this process, the PBPs help to create the morphology of the peptidoglycan exoskeleton together with cytoskeleton proteins that regulate septum formation and cell shape. Genetic and microscopic studies reveal clear morphological responsibilities for class A and class B PBPs and suggest that the mechanism of shape determination involves differential protein localization and interactions with specific cell components. In addition, the low molecular weight PBPs, by varying the substrates on which other PBPs act, alter peptidoglycan synthesis or turnover, with profound effects on morphology.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Gramnegativas/fisiología , Hexosiltransferasas/fisiología , Muramoilpentapéptido Carboxipeptidasa/fisiología , Peptidil Transferasas/fisiología , Proteínas de Unión a las PenicilinasRESUMEN
Production of low-affinity forms of penicillin-binding proteins (PBPs), although essential, is not sufficient to protect pneumococci against the inhibitory action of penicillin. Resistance also requires the newly identified protein MurM which, together with MurN, is involved with the synthesis of short peptide branches in the pneumococcal cell wall. Cells in which murM was inactivated produced cell walls without branches and also completely lost penicillin resistance. To understand these surprising observations a 3D-model of MurM was constructed, which helped to put into structural context several of the biochemical and genetic observations made about this protein.
Asunto(s)
Bacterias/metabolismo , Resistencia a las Penicilinas , Péptido Sintasas/química , Péptido Sintasas/fisiología , Peptidoglicano/biosíntesis , Bacterias/efectos de los fármacos , Bacterias/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Pared Celular/química , Pared Celular/metabolismo , Pared Celular/ultraestructura , Hexosiltransferasas/fisiología , Modelos Moleculares , Muramoilpentapéptido Carboxipeptidasa/fisiología , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/fisiología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/ultraestructuraRESUMEN
In free-living eubacteria an external shell of peptidoglycan opposes internal hydrostatic pressure and prevents membrane rupture and death. At the same time, this wall imposes on each cell a shape. Because shape is both stable and heritable, as is the ability of many organisms to execute defined morphological transformations, cells must actively choose from among a large repertoire of available shapes. How they do so has been debated for decades, but recently experiment has begun to catch up with theory. Two discoveries are particularly informative. First, specific protein assemblies, nucleated by FtsZ, MreB or Mbl, appear to act as internal scaffolds that influence cell shape, perhaps by correctly localizing synthetic enzymes. Second, defects in cell shape are correlated with the presence of inappropriately placed, metabolically inert patches of peptidoglycan. When combined with what we know about mutants affecting cellular morphology, these observations suggest that bacteria may fabricate specific shapes by directing the synthesis of two kinds of cell wall: a long-lived, rigid framework that defines overall topology, and a metabolically plastic peptidoglycan whose shape is directed by internal scaffolds.
Asunto(s)
Bacterias/citología , Pared Celular/ultraestructura , Proteínas de Escherichia coli/fisiología , Peptidoglicano/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Bacterias/efectos de los fármacos , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular , División Celular , Pared Celular/efectos de los fármacos , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Replicación del ADN , ADN Bacteriano/biosíntesis , Proteínas de Escherichia coli/genética , Hexosiltransferasas/genética , Hexosiltransferasas/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Modelos Biológicos , Morfogénesis , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/fisiología , Presión Osmótica , Proteínas de Unión a las Penicilinas , Penicilinas/farmacología , Peptidoglicano/ultraestructura , Peptidil Transferasas/genética , Peptidil Transferasas/fisiología , Especificidad de la EspecieRESUMEN
The class B M1-V577 penicillin-binding protein (PBP) 3 of Escherichia coli consists of a M1-L39 membrane anchor (bearing a cytosolic tail) that is linked via a G40-S70 intervening peptide to an R71-I236 non-catalytic module (containing the conserved motifs 1-3) itself linked via motif 4 to a D237-V577 catalytic module (containing the conserved motifs 5-7 of the penicilloyl serine transferases superfamily). It has been proposed that during cell septation the peptidoglycan crosslinking activity of the acyl transferase module of PBP3 is regulated by the associated M1-I236 polypeptide itself in interaction with other components of the divisome. The fold adopted by the R71-V577 polypeptide of PBP3 has been modelled by reference to the corresponding R76-S634 polypeptide of the class B Streptococcus pneumoniae PBP2x. Based on these data and the results of site-directed mutagenesis of motifs 1-3 and of peptide segments of high amphiphilicity (identified from hydrophobic moment plots), the M1-I236 polypeptide of PBP3 appears to be precisely designed to work in the way proposed. The membrane anchor and the G40-S70 sequence (containing the G57-Q66 peptide segment) upstream from the non-catalytic module have the information ensuring that PBP3 undergoes proper insertion within the divisome at the cell septation site. Motif 1 and the I74-L82 overlapping peptide segment, motif 2 and the H160-G172 overlapping peptide segment, and the G188-D197 motif 3 are located at or close to the intermodule junction. They contain the information ensuring that PBP3 folds correctly and the acyl transferase catalytic centre adopts the active configuration. The E206-V217 peptide segment is exposed at the surface of the non-catalytic module. It has the information ensuring that PBP3 fulfils its cell septation activity within the fully complemented divisome.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras , Proteínas de Escherichia coli , Escherichia coli/química , Hexosiltransferasas/fisiología , Complejos Multienzimáticos/fisiología , Muramoilpentapéptido Carboxipeptidasa , Peptidoglicano Glicosiltransferasa , Peptidil Transferasas/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Hexosiltransferasas/química , Hexosiltransferasas/genética , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Proteínas de Unión a las Penicilinas , Péptidos/química , Péptidos/genética , Péptidos/fisiología , Peptidil Transferasas/química , Peptidil Transferasas/genética , Relación Estructura-ActividadRESUMEN
Ribosome, the ubiquitous organelle, is the site for protein synthesis in all types of cells. The consecutive peptide bonds are formed by the transpeptidation reaction between carboxyl group of peptidyl moiety and the amino group of the aminoacyl moiety. Both the moieties are attached to the appropiate tRNAs positioned on the ribosome at P and A sites, respectively, through codon-anticodon recognition directed by messenger RNA. The reaction seems to proceed by the nucleophillic attack of the amino group of the aminoacyl tRNA at the A site and on the carboxyl of the ester group of the tRNA at P-site of ribosome. The configuration of the carbon atom of the tetrahedral intermediate may be R or S depending on the direction of the nucleophillic attack. After selecting the favorable conformation of this tetrahedral intermediate quantum mechanical calculations have been carried out to determine the energy needed for its formation. A cyclic intermediate where 2'-OH of the ribose sugar of the P-site tRNA is a member of the ring can be formed from the tetrahedral intermediate. This cyclic intermediate produces a free tRNA and a tRNA attached to a planar peptide unit. Analysis of the energetics using semiempirical method for the formation of a cyclic intermediate indicates that the peptide bond formation through the tetrahedral intermediate in S configuration may not need assistance from any outside agent like an enzyme
Asunto(s)
Peptidil Transferasas/fisiología , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Péptidos/metabolismo , ARN de Transferencia/genéticaRESUMEN
The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/fisiología , Dipeptidasas , Proteínas de Escherichia coli , Escherichia coli/fisiología , Hexosiltransferasas/fisiología , Complejos Multienzimáticos/fisiología , Muramoilpentapéptido Carboxipeptidasa/fisiología , Mutación , Peptidoglicano/biosíntesis , Peptidil Transferasas/fisiología , beta-Lactamasas/fisiología , Bacteriólisis/genética , Endopeptidasas , Evolución Molecular , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Modelos Biológicos , Presión Osmótica , Proteínas de Unión a las PenicilinasRESUMEN
Histidine-constitutive (Hisc) strains of Salmonella typhimurium undergo cell division inhibition in the presence of high concentrations of a metabolizable carbon source. Filaments formed by Hisc strains show constrictions and contain evenly spaced nucleoids, suggesting a defect in septum formation. Inhibitors of penicillin-binding protein 3 (PBP3) induce a filamentation pattern identical to that of Hisc strains. However, the Hisc septation defect is caused neither by reduced PBP3 synthesis nor by reduced PBP3 activity. Gross modifications of peptidoglycan composition are also ruled out. D-Cycloserine, an inhibitor of the soluble pathway producing peptidoglycan precursors, causes phenotypic suppression of filamentation, suggesting that the septation defect of Hisc strains may be caused by scarcity of PBP3 substrate.