RESUMO
EF-P is a translation factor that facilitates the formation of peptide bonds between consecutive prolines. Using FRET between EF-P and ribosomal protein bL33, we studied dynamics and specificity of EF-P binding to the ribosome. Our findings reveal that EF-P rapidly scans for a free E site and can bind to any ribosome containing a P-site tRNA, regardless of the ribosome's functional state. The interaction with uL1 is essential for EF-P binding, while the ß-Lys modification of EF-P doubles the association rate. Specific interactions with the D-loop of tRNAPro or tRNAfMet and via the ß-Lys group with the tRNA in the peptidyl transferase center reduce the rate of EF-P dissociation from the ribosome, providing the specificity for complexes that need help in catalyzing peptide bond formation. The nature of the E-site codon has little effect on EF-P binding kinetics. Although EF-P dissociation is reduced upon recognizing its correct tRNA substrate, it remains sufficiently rapid compared to tRNA translocation and does not affect the translocation rate. These results highlight the importance of EF-P's scanning-engagement mechanism for dynamic substrate recognition during rapid translation.
Assuntos
Fatores de Alongamento de Peptídeos , Biossíntese de Proteínas , Proteínas Ribossômicas , Ribossomos , Ribossomos/metabolismo , Cinética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/química , RNA de Transferência/metabolismo , RNA de Transferência/química , Ligação Proteica , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Metionina/química , Transferência Ressonante de Energia de Fluorescência , RNA de Transferência de Prolina/metabolismo , RNA de Transferência de Prolina/genética , RNA de Transferência de Prolina/química , Códon/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Sítios de LigaçãoRESUMO
Mutations in 11ß-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) cause an extraordinarily rare autosomal recessive disorder, apparent mineralocorticoid excess (AME). AME is a form of low renin hypertension that is potentially fatal if untreated. Mutations in the HSD11B2 gene result either in severe AME or a milder phenotype (type 2 AME). To date, â¼40 causative mutations have been identified. As part of the International Consortium for Rare Steroid Disorders, we have diagnosed and followed the largest single worldwide cohort of 36 AME patients. Here, we present the genotype and clinical phenotype of these patients, prominently from consanguineous marriages in the Middle East, who display profound hypertension and hypokalemic alkalosis. To correlate mutations with phenotypic severity, we constructed a computational model of the HSD11B2 protein. Having used a similar strategy for the in silico evaluation of 150 mutations of CYP21A2, the disease-causing gene in congenital adrenal hyperplasia, we now provide a full structural explanation for the clinical severity of AME resulting from each known HSD11B2 missense mutation. We find that mutations that allow the formation of an inactive dimer, alter substrate/coenzyme binding, or impair structural stability of HSD11B2 yield severe AME. In contrast, mutations that cause an indirect disruption of substrate binding or mildly alter intramolecular interactions result in type 2 AME. A simple in silico evaluation of novel missense mutations could help predict the often-diverse phenotypes of an extremely rare monogenic disorder.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Genótipo , Síndrome de Excesso Aparente de Minerolocorticoides , Mutação de Sentido Incorreto , Multimerização Proteica/genética , Adolescente , Criança , Pré-Escolar , Simulação por Computador , Estabilidade Enzimática , Feminino , Humanos , Lactente , Masculino , Síndrome de Excesso Aparente de Minerolocorticoides/enzimologia , Síndrome de Excesso Aparente de Minerolocorticoides/genética , Síndrome de Excesso Aparente de Minerolocorticoides/patologiaRESUMO
Elongation factor P (EF-P) and its eukaryotic homolog eIF5A are auxiliary translation factors that facilitate peptide bond formation when several sequential proline (Pro) residues are incorporated into the nascent chain. EF-P and eIF5A bind to the exit (E) site of the ribosome and contribute to favorable entropy of the reaction by stabilizing tRNA binding in the peptidyl transferase center of the ribosome. In most organisms, EF-P and eIF5A carry a posttranslational modification that is crucial for catalysis. The chemical nature of the modification varies between different groups of bacteria and between pro- and eukaryotes, making the EF-P-modification enzymes promising targets for antibiotic development. In this review, we summarize our knowledge of the structure and function of EF-P and eIF5A, describe their modification enzymes, and present an approach for potential drug screening aimed at EarP, an enzyme that is essential for EF-P modification in several pathogenic bacteria.