RESUMO
Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome. This complex macromolecular machine is the target of important antimicrobial drugs, an integral line of defence against infectious diseases. Here, we describe how open access to cryo-electron microscopy facilities combined with bespoke user support enabled structural determination of the translating ribosome from Escherichia coli at 1.55 Å resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enable interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 Å resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.
Assuntos
Escherichia coli , Ribossomos , Microscopia Crioeletrônica/métodos , Escherichia coli/genética , Modelos Moleculares , Ribossomos/metabolismo , RNA Ribossômico/metabolismo , Bactérias/genéticaRESUMO
Many viruses, especially RNA viruses, utilize programmed ribosomal frameshifting and/or stop codon readthrough in their expression, and in the decoding of a few a UGA is dynamically redefined to specify selenocysteine. This recoding can effectively increase viral coding capacity and generate a set ratio of products with the same N-terminal domain(s) but different C-terminal domains. Recoding can also be regulatory or generate a product with the non-universal 21st directly encoded amino acid. Selection for translation speed in the expression of many viruses at the expense of fidelity creates host immune defensive opportunities. In contrast to host opportunism, certain viruses, including some persistent viruses, utilize recoding or adventitious frameshifting as part of their strategy to evade an immune response or specific drugs. Several instances of recoding in small intensively studied viruses escaped detection for many years and their identification resolved dilemmas. The fundamental importance of ribosome ratcheting is consistent with the initial strong view of invariant triplet decoding which however did not foresee the possibility of transitory anticodon:codon dissociation. Deep level dynamics and structural understanding of recoding is underway, and a high level structure relevant to the frameshifting required for expression of the SARS CoV-2 genome has just been determined.
Assuntos
Vírus de DNA/genética , Vírus de DNA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Evasão da Resposta Imune , Vírus de RNA/genética , Antivirais/farmacologia , Códon de Terminação , Vírus de DNA/efeitos dos fármacos , Mudança da Fase de Leitura do Gene Ribossômico , Antígenos de Histocompatibilidade Classe I/genética , Conformação de Ácido Nucleico , Peptídeos/imunologia , Biossíntese de Proteínas , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/imunologiaRESUMO
Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , RNA Viral/genética , Ribossomos/ultraestrutura , SARS-CoV-2/genética , Proteínas Virais/biossíntese , Animais , Antivirais/farmacologia , Códon de Terminação , RNA-Polimerase RNA-Dependente de Coronavírus/biossíntese , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Microscopia Crioeletrônica , Fluoroquinolonas/farmacologia , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , Genoma Viral , Humanos , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Dobramento de Proteína , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Mucoepidermoid carcinoma (MEC) of the lung is a rare subtype of non-small cell lung cancer. There is no consensus regarding optimal management for this disease. CASE REPORT: We present a case of MEC of the lung in a 75 year-old female with a history of superficial urothelial carcinoma of the bladder. The patient was found to have an asymptomatic lung mass. Initial biopsy suggested metastatic recurrence of urothelial carcinoma and therefore, cisplatin and gemcitabine chemotherapy was administered prior to surgical resection. Pathological analysis of the resected specimen confirmed a diagnosis of stage IIIA MEC with focal high-grade features including transitional cell-like areas. Adjuvant radiotherapy was administered due to a positive microscopic resection margin. No chemotherapy was given due to lack of supporting data. The patient developed widespread metastatic disease 3 months following completion of radiotherapy and died 1 month later. CONCLUSION: This case demonstrates the possibility of dual pathology in cases where metastatic disease is suspected. The use of small tissue samples may complicate diagnosis due to the heterogeneity of malignant tumours.
