RESUMO
How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked dyskeratosis congenita (X-DC) and Hoyeraal-Hreidarsson (HH) syndrome. Here, we characterize ribosomes isolated from a yeast strain in which Cbf5p, the yeast homolog of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A and P sites as well as the cricket paralysis virus internal ribosome entry site (IRES), which interacts with both the P and the E sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification in ribosome-ligand interactions that are conserved in yeast, mouse, and humans.
Assuntos
Proteínas de Ciclo Celular/deficiência , Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Hidroliases/deficiência , Hidroliases/metabolismo , Deficiência Intelectual/genética , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Nucleares/deficiência , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Ribonucleoproteínas Nucleares Pequenas/deficiência , Saccharomyces cerevisiae/genética , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Disceratose Congênita/enzimologia , Retardo do Crescimento Fetal/enzimologia , Genes Reporter , Humanos , Hidroliases/genética , Deficiência Intelectual/enzimologia , Luciferases/análise , Camundongos , Microcefalia/enzimologia , Proteínas Associadas aos Microtúbulos/genética , Mutação , Proteínas Nucleares/genética , Plasmídeos , Biossíntese de Proteínas , RNA Ribossômico/química , RNA Ribossômico/genética , RNA de Transferência/química , RNA de Transferência/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transdução GenéticaRESUMO
The T box transcription antitermination regulatory system, found in Gram-positive bacteria, is dependent on a complex set of interactions between uncharged tRNA and the 5'-untranslated mRNA leader region of the regulated gene. One of these interactions involves the base pairing of the acceptor end of cognate tRNA with four bases in a 7 nt bulge of the antiterminator RNA. In vitro selection of randomized tRNA binding to Bacillus subtilis tyrS antiterminator model RNAs was used to determine what, if any, sequence trends there are for binding beyond the known base pair complementarity. The model antiterminator RNAs were selected for the wild-type tertiary fold of tRNA. While there were no obvious sequence correlations between the selected tRNAs, there were correlations between certain tertiary structural elements and binding efficiency to different antiterminator model RNAs. In addition, one antiterminator model selected primarily for a kissing tRNA T loop-antiterminator bulge interaction, while another antiterminator model resulted in no such selection. The selection results indicate that, at the level of tertiary structure, there are ideal matches between tRNAs and antiterminator model RNAs consistent with in vivo observations and that additional recognition features, beyond base pair complementarity, may play a role in the formation of the complex.
Assuntos
Regiões 5' não Traduzidas/química , Bacillus subtilis/genética , RNA de Transferência/química , Tirosina-tRNA Ligase/genética , Regiões 5' não Traduzidas/metabolismo , Bacillus subtilis/enzimologia , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Evolução Molecular Direcionada , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Transferência/metabolismo , RNA de Transferência de Tirosina/química , RNA de Transferência de Tirosina/metabolismoRESUMO
The study objective was to gain a better understanding of the level of literacy in information technology across the dental team within Scotland, thus allowing appropriate planning of education and training for effective use of information technology. In May 2004 a postal questionnaire survey was undertaken of all dentists (2679) and professionals complementary to dentistry (2861) within primary care in Scotland, in both general dental practice and the salaried dental service. Online reply was also an option. Results showed that 43 per cent of respondents considered their IT skills as 'moderate' with a further one-third reporting 'nil' or 'low' skill level. Only one quarter of respondents had accessed a learning programme by PC. The majority of IT competence was self-acquired. Hence 'upskilling' the dental team in IT may be required in order to take advantage of e-learning opportunities available now and in the future.
Assuntos
Atitude Frente aos Computadores , Computadores , Odontólogos , Internet , Adulto , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Pessoal de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , EscóciaRESUMO
OBJECTIVE: To gain a better understanding of the level of literacy in information technology (IT) across the dental team working within primary care in Scotland, thus allowing appropriate planning of education and training for effective use of IT. DESIGN: A postal questionnaire survey of all dentists and dental care professionals (DCPs) within primary care in Scotland; online reply was also an option. SETTING: General dental practice and the salaried dental service, May 2004. SUBJECTS AND METHODS: 2679 dentists and 2861 DCPs were surveyed. RESULTS: Forty-three percent of respondents considered their IT skills to be 'moderate', with a further one-third reporting 'nil' or 'low' skill level. Only a quarter of respondents had accessed a learning programme by computer. The majority of IT competence was self-acquired. CONCLUSIONS: 'Upskilling' the dental team in IT may be required in order to take advantage of e-learning opportunities available now and in the future.
Assuntos
Alfabetização Digital , Auxiliares de Odontologia , Serviços de Saúde Bucal , Odontólogos , Adulto , Capacitação de Usuário de Computador , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Atenção Primária à Saúde , EscóciaRESUMO
The T box antiterminator RNA element is an important component of the T box riboswitch that controls the transcription of vital genes in many Gram-positive bacteria. A series of 1,4-disubstituted 1,2,3-triazoles was screened in a fluorescence-monitored thermal denaturation assay to identify ligands that altered the stability of antiterminator model RNA. Several ligands were identified that significantly increased or decreased the melting temperature (T(m) ) of the RNA. The results indicate that this series of triazole ligands can alter the stability of antiterminator model RNA in a structure-dependent manner.