RESUMO
Protein-protein and protein-rRNA interactions at the interface between ribosomal proteins uS4 and uS5 are thought to maintain the accuracy of protein synthesis by increasing selection of cognate aminoacyl-tRNAs. Selection involves a major conformational change-domain closure-that stabilizes aminoacyl-tRNA in the ribosomal acceptor (A) site. This has been thought a constitutive function of the ribosome ensuring consistent accuracy. Recently, the Saccharomyces cerevisiae Ctk1 cyclin-dependent kinase was demonstrated to ensure translational accuracy and Ser238 of uS5 proposed as its target. Surprisingly, Ser238 is outside the uS4-uS5 interface and no obvious mechanism has been proposed to explain its role. We show that the true target of Ctk1 regulation is another uS5 residue, Ser176, which lies in the interface opposite to Arg57 of uS4. Based on site specific mutagenesis, we propose that phospho-Ser176 forms a salt bridge with Arg57, which should increase selectivity by strengthening the interface. Genetic data show that Ctk1 regulates accuracy indirectly; the data suggest that the kinase Ypk2 directly phosphorylates Ser176. A second kinase pathway involving TORC1 and Pkc1 can inhibit this effect. The level of accuracy appears to depend on competitive action of these two pathways to regulate the level of Ser176 phosphorylation.
Assuntos
Arginina , Fosfosserina , Biossíntese de Proteínas , Proteínas Quinases , Proteínas Ribossômicas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/química , Arginina/metabolismo , Arginina/química , Fosfosserina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , Fosforilação , Evolução MolecularRESUMO
Protein synthesis on the ribosome involves successive rapid recruitment of cognate aminoacyl-tRNAs and rejection of the much more numerous incorrect near- or non-cognates. The principal feature of translation elongation is that at every step, many incorrect aa-tRNAs unsuccessfully enter the A site for each cognate accepted. Normal levels of translational accuracy require that cognate tRNAs have relatively similar acceptance rates by the ribosome. To achieve that, tRNAs evolved to compensate for differences in amino acid properties and codon-anticodon strength that affect acceptance. Part of that response involved tRNA posttranscriptional modifications, which can affect tRNA decoding efficiency, accuracy, and structural stability. The most intensively modified regions of the tRNA are the anticodon loop and structural core of the tRNA. Anticodon loop modifications directly affect codon-anticodon pairing and therefore modulate accuracy. Core modifications have been thought to ensure consistent decoding rates principally by stabilizing tRNA structure to avoid degradation; however, degradation due to instability appears to only be a significant issue above normal growth temperatures. We suspected that the greater role of modification at normal temperatures might be to tune tRNAs to maintain consistent intrinsic rates of acceptance and peptide transfer and that hypomodification by altering these rates might degrade the process of discrimination, leading to increased translational errors. Here, we present evidence that most tRNA core modifications do modulate the frequency of misreading errors, suggesting that the need to maintain accuracy explains their deep evolutionary conservation.
Assuntos
Anticódon , RNA de Transferência , Anticódon/genética , Anticódon/metabolismo , RNA de Transferência/química , Biossíntese de Proteínas , Códon/genética , Códon/metabolismo , Ribossomos/metabolismoRESUMO
OBJECTIVES: Correctional settings (prisons, jails, detention facilities) provide a unique opportunity to screen for sexually transmitted infections (STIs) among correctional populations with a high prevalence of infection. Immigrant detainees are a distinct and poorly described correctional population. The main objective of this study was to determine the feasibility of a national STI screening program for immigrant detainees. METHODS AND MATERIALS: We developed an opt-out STI testing program that included electronic health record integration, patient education, and staff member training. We piloted this program from June 22 through August 19, 2018, at 2 detention facilities with different operational requirements and detainee demographic characteristics. We assessed STI test positivity rates, treatment outcomes, estimated cost to conduct testing and counseling, and staff member perceptions of program value and challenges to implementation. RESULTS: Of 1041 immigrant detainees approached for testing, 526 (50.5%) declined. Of 494 detainees who were tested, 42 (8.5%) tested positive for at least 1 STI; the percentage positivity rates were 6.7% (n = 33) for chlamydia, 0.8% (n = 4) for syphilis, 0.8% (n = 4) for gonorrhea, 0.6% (n = 3) for hepatitis B, and 0.2% (n = 1) for HIV. The estimated cost to detect any STI ranged from $500 to $961; the estimated cost to identify 1 person infected with HIV ranged from $22â 497 to $43 244. Forty of 42 persons who tested positive began treatment before release from custody. Medical staff members had positive views of the program but had concerns about workload. PRACTICE IMPLICATIONS: STIs are prevalent among immigrant detainees. A routine screening program is feasible if operational aspects are carefully considered and would provide counseling, education, and treatment for this vulnerable population.
Assuntos
Emigrantes e Imigrantes/estatística & dados numéricos , Programas de Rastreamento/estatística & dados numéricos , Infecções Sexualmente Transmissíveis/diagnóstico , Recusa do Paciente ao Tratamento/estatística & dados numéricos , Adolescente , Adulto , Idoso , Feminino , Nível de Saúde , Humanos , Masculino , Programas de Rastreamento/economia , Pessoa de Meia-Idade , Infecções Sexualmente Transmissíveis/tratamento farmacológico , Infecções Sexualmente Transmissíveis/etnologia , Fatores Socioeconômicos , Transtornos Relacionados ao Uso de Substâncias/etnologia , Adulto JovemRESUMO
Saccharomyces cerevisiae has been an important model for determining the frequency of translational misreading events, those in which a tRNA pairs incorrectly to the mRNA and inserts an amino acid not specified by the codon in the mRNA. Misreading errors have been quantified in vivo using reporter protein systems or mass spectrometry with both approaches converging on a simple model for most misreading. The available data show that misreading tRNAs must form stereotypical base mismatches that correspond to those that can mimic Watson-Crick base pairs when formed in the ribosomal A site. Errors involving other mismatches occur significantly less frequently. This work debunks the idea of an average misreading frequency of 5 × 10-4 per codon that extends across the genetic code. Instead, errors come in two distinct classes-high frequency and low frequency events-with most errors being of the low frequency type. A comparison of misreading errors in S. cerevisiae and Escherichia coli suggests the existence of a mechanism that reduces misreading frequency in yeast; this mechanism may operate in eukaryotes generally.
Assuntos
Códon , Código Genético , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Escherichia coli/genética , Mutação , RNA Mensageiro/genéticaRESUMO
Protein synthesis requires both high speed and accuracy to ensure a healthy cellular environment. Estimates of errors during protein synthesis in Saccharomyces cerevisiae have varied from 10-3 to 10-4 errors per codon. Here, we show that errors made by ${\rm{tRNA}}^{\rm Glu}_{\rm UUC}$ in yeast can vary 100-fold, from 10-6 to 10-4 errors per codon. The most frequent errors require a Gâ¢U mismatch at the second position for the near cognate codon GGA (Gly). We also show, contrary to our previous results, that yeast tRNAs can make errors involving mismatches at the wobble position but with low efficiency. We have also assessed the effect on misreading frequency of post-transcriptional modifications of tRNAs, which are known to regulate cognate codon decoding in yeast. We tested the roles of mcm5s2U34 and t6A37 and show that their effects depend on details of the codon anticodon interaction including the position of the modification with respect to the base mismatch and the nature of that mismatch. Both mcm5 and s2 modification of wobble uridine strongly stabilizes G2â¢U35 mismatches when ${\rm{tRNA}}^{\rm Glu}_{\rm UUC}$ misreads the GGA Gly codon but has weaker effects on other mismatches. By contrast, t6A37 destabilizes U1â¢U36 mismatches when ${\rm{tRNA}}^{\rm Lys}_{\rm UUU}$ misreads UAA or UAG but stabilizes mismatches at the second and wobble positions.
Assuntos
Anticódon/genética , Códon/genética , Biossíntese de Proteínas , RNA de Transferência/genética , Código Genético , Glutamina/genética , Processamento de Proteína Pós-Traducional/genética , Processamento Pós-Transcricional do RNA/genética , Saccharomyces cerevisiae/genética , Uridina/genéticaRESUMO
BACKGROUND: UMBC, a diverse public research university, "builds" upon its reputation in producing highly capable undergraduate scholars to create a comprehensive new model, STEM BUILD at UMBC. This program is designed to help more students develop the skills, experience and motivation to excel in science, technology, engineering, and mathematics (STEM). This article provides an in-depth description of STEM BUILD at UMBC and provides the context of this initiative within UMBC's vision and mission. KEY HIGHLIGHTS: The STEM BUILD model targets promising STEM students who enter as freshmen or transfer students and do not qualify for significant university or other scholarship support. Of primary importance to this initiative are capacity, scalability, and institutional sustainability, as we distill the advantages and opportunities of UMBC's successful scholars programs and expand their application to more students. The general approach is to infuse the mentoring and training process into the fabric of the undergraduate experience while fostering community, scientific identity, and resilience. At the heart of STEM BUILD at UMBC is the development of BUILD Group Research (BGR), a sequence of experiences designed to overcome the challenges that undergraduates without programmatic support often encounter (e.g., limited internship opportunities, mentorships, and research positions for which top STEM students are favored). BUILD Training Program (BTP) Trainees serve as pioneers in this initiative, which is potentially a national model for universities as they address the call to retain and graduate more students in STEM disciplines - especially those from underrepresented groups. As such, BTP is a research study using random assignment trial methodology that focuses on the scalability and eventual incorporation of successful measures into the traditional format of the academy. IMPLICATIONS: Critical measures to transform institutional culture include establishing an extensive STEM Living and Learning Community to increase undergraduate retention, expanding the adoption of "active learning" pedagogies to increase the efficiency of learning, and developing programs to train researchers to effectively mentor a greater portion of the student population. The overarching goal of STEM BUILD at UMBC is to retain students in STEM majors and better prepare them for post baccalaureate, graduate, or professional programs as well as careers in biomedical and behavioral research.
RESUMO
[This corrects the article DOI: 10.1186/s13100-015-0053-5.].
RESUMO
Cellular health and growth requires protein synthesis to be both efficient to ensure sufficient production, and accurate to avoid producing defective or unstable proteins. The background of misreading error frequency by individual tRNAs is as low as 2 × 10(-6) per codon but is codon-specific with some error frequencies above 10(-3) per codon. Here we test the effect on error frequency of blocking post-transcriptional modifications of the anticodon loops of four tRNAs in Escherichia coli. We find two types of responses to removing modification. Blocking modification of tRNA(UUC)(Glu) and tRNA(QUC)(Asp) increases errors, suggesting that the modifications act at least in part to maintain accuracy. Blocking even identical modifications of tRNA(UUU)(Lys) and tRNA(QUA)(Tyr) has the opposite effect of decreasing errors. One explanation could be that the modifications play opposite roles in modulating misreading by the two classes of tRNAs. Given available evidence that modifications help preorder the anticodon to allow it to recognize the codons, however, the simpler explanation is that unmodified 'weak' tRNAs decode too inefficiently to compete against cognate tRNAs that normally decode target codons, which would reduce the frequency of misreading.
Assuntos
Anticódon/genética , Códon/genética , Biossíntese de Proteínas , RNA de Transferência/genética , Escherichia coli , Mutação , Processamento Pós-Transcricional do RNA/genéticaRESUMO
BACKGROUND: A large number of Saccharomyces cerevisiae cellular factors modulate the movement of the retrovirus-like transposon Ty1. Surprisingly, a significant number of chromosomal genes required for Ty1 transposition encode components of the translational machinery, including ribosomal proteins, ribosomal biogenesis factors, protein trafficking proteins and protein or RNA modification enzymes. RESULTS: To assess the mechanistic connection between Ty1 mobility and the translation machinery, we have determined the effect of these mutations on ribosome biogenesis and Ty1 transcriptional and post-transcriptional regulation. Lack of genes encoding ribosomal proteins or ribosome assembly factors causes reduced accumulation of the ribosomal subunit with which they are associated. In addition, these mutations cause decreased Ty1 + 1 programmed translational frameshifting, and reduced Gag protein accumulation despite at least normal levels of Ty1 mRNA. Several ribosome subunit mutations increase the level of both an internally initiated Ty1 transcript and its encoded truncated Gag-p22 protein, which inhibits transposition. CONCLUSIONS: Together, our results suggest that this large class of cellular genes modulate Ty1 transposition through multiple pathways. The effects are largely post-transcriptional acting at a variety of levels that may include translation initiation, protein stability and subcellular protein localization.
RESUMO
The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome's central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA.
Assuntos
Bactérias/crescimento & desenvolvimento , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Letais , Modelos Moleculares , Mutação , Estrutura Secundária de Proteína , RNA Bacteriano/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismoRESUMO
The translation of genes into functional proteins involves error. Mistranslation is a known cause of disease, but, surprisingly, recent studies suggest that certain organisms from all domains of life have evolved diverse pathways that increase their tolerance of translational error. Although the reason for these high error rates are not yet clear, evidence suggests that increased mistranslation may have a role in the generation of diversity within the proteome and other adaptive functions. Error rates are regulated, and there appears to be an optimal mistranslation rate that varies by organism and environmental condition. Advances in unbiased interrogation of error types and experiments involving wild organisms may help our understanding of the potentially adaptive roles for protein translation errors.
Assuntos
Evolução Molecular , Variação Genética , Biossíntese de Proteínas , Proteínas/genética , Proteoma/biossíntese , Edição de RNA/genética , Animais , HumanosRESUMO
Protein synthesis must rapidly and repeatedly discriminate between a single correct and many incorrect aminoacyl-tRNAs. We have attempted to measure the frequencies of all possible missense errors by tRNA , tRNA and tRNA . The most frequent errors involve three types of mismatched nucleotide pairs, Uâ¢U, Uâ¢C, or Uâ¢G, all of which can form a noncanonical base pair with geometry similar to that of the canonical Uâ¢A or Câ¢G Watson-Crick pairs. Our system is sensitive enough to measure errors at other potential mismatches that occur at frequencies as low as 1 in 500,000 codons. The ribosome appears to discriminate this efficiently against any pair with non-Watson-Crick geometry. This extreme accuracy may be necessary to allow discrimination against the errors involving near Watson-Crick pairing.
Assuntos
Pareamento Incorreto de Bases/fisiologia , Mutação de Sentido Incorreto , Biossíntese de Proteínas/fisiologia , Ribossomos/fisiologia , Substituição de Aminoácidos , Pareamento de Bases/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Mutagênese/fisiologia , Mutação de Sentido Incorreto/fisiologia , Conformação de Ácido Nucleico , Organismos Geneticamente Modificados , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Ácido Glutâmico/metabolismo , RNA de Transferência de Tirosina/metabolismo , beta-Galactosidase/química , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
tRNA isopentenyltransferases (Tit1) modify tRNA position 37, adjacent to the anticodon, to N6-isopentenyladenosine (i6A37) in all cells, yet the tRNA subsets selected for modification vary among species, and their relevance to phenotypes is unknown. We examined the function of i6A37 in Schizosaccharomyces pombe tit1+ and tit1-Δ cells by using a ß-galactosidase codon-swap reporter whose catalytic activity is sensitive to accurate decoding of codon 503. i6A37 increased the activity of tRNACys at a cognate codon and that of tRNATyr at a near-cognate codon, suggesting that i6A37 promotes decoding activity generally and increases fidelity at cognate codons while decreasing fidelity at noncognate codons. S. pombe cells lacking tit1+ exhibit slow growth in glycerol or rapamycin. While existing data link wobble base U34 modifications to translation of functionally related mRNAs, whether this might extend to the anticodon-adjacent position 37 was unknown. Indeed, we found a biased presence of i6A37-cognate codons in high-abundance mRNAs for ribosome subunits and energy metabolism, congruent with the observed phenotypes and the idea that i6A37 promotes translational efficiency. Polysome profiles confirmed the decreased translational efficiency of mRNAs in tit1-Δ cells. Because subsets of i6A37-tRNAs differ among species, as do their cognate codon-sensitive mRNAs, these genomic variables may underlie associated phenotypic differences.
Assuntos
Regulação Fúngica da Expressão Gênica , Isopenteniladenosina/genética , RNA Fúngico/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Schizosaccharomyces/genética , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Códon/genética , Códon/metabolismo , Deleção de Genes , Isopenteniladenosina/metabolismo , Biossíntese de Proteínas , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismoRESUMO
Ty3 elements of S. cerevisiae contain two overlapping coding regions, GAG3 and POL3, which are functional homologues of retroviral gag and pol genes, respectively. Pol3 is translated as a Gag3-Pol3 fusion protein dependent on a +1 programmed frameshift at a site with the overlap between the two genes. We show that the Ty3 frameshift frequency varies up to 10-fold in S. cerevisiae cells depending on carbon source. Frameshift efficiency is significantly lower in cells growing on glucose as carbon source than in cells growing on poor alternative carbon sources (glycerol/lactate or galactose). Our results indicate that Ty3 programmed ribosomal frameshift efficiency in response to glucose signalling requires two protein kinases: Snf1p and cAMP-dependent protein kinase A (PKA). Increased frameshifting on alternative carbon sources also appears to require cytoplasmic localization of Snf1p, mediated by the Sip2p protein. In addition to the two required protein kinases, our results implicate that Stm1p, a ribosome-associated protein involved in nutrient sensing, is essential for the carbon source-dependent regulation of Ty3 frameshifting. These data indicate that Ty3 programmed ribosomal frameshift is not a constitutive process but that it is regulated in response to the glucose-signalling pathway.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Glucose/metabolismo , Retroelementos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis of possible errors. We recently used a highly quantitative system to measure the frequency of all types of misreading errors by a single tRNA in E. coli. That study found a wide variation in error frequencies among codons; a major factor causing that variation is competition between the correct (cognate) and incorrect (near-cognate) aminoacyl-tRNAs for the mutant codon. Here we extend that analysis to measure the frequency of missense errors by two tRNAs in a eukaryote, the yeast Saccharomyces cerevisiae. The data show that in yeast errors vary by codon from a low of 4 x 10(-5) to a high of 6.9 x 10(-4) per codon and that error frequency is in general about threefold lower than in E. coli, which may suggest that yeast has additional mechanisms that reduce missense errors. Error rate again is strongly influenced by tRNA competition. Surprisingly, missense errors involving wobble position mispairing were much less frequent in S. cerevisiae than in E. coli. Furthermore, the error-inducing aminoglycoside antibiotic, paromomycin, which stimulates errors on all error-prone codons in E. coli, has a more codon-specific effect in yeast.
Assuntos
Códon , Mutação de Sentido Incorreto , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Escherichia coli/genética , Paromomicina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA de Transferência de Arginina/genética , RNA de Transferência de Arginina/metabolismo , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
A variety of cellular factors affect the movement of the retrovirus-like transposon Ty1. To identify genes involved in Ty1 virus-like particle (VLP) function, the level of the major capsid protein (Gag-p45) and its proteolytic precursor (Gag-p49p) was monitored in a subset of Ty1 cofactor mutants. Twenty-nine of 87 mutants contained alterations in the level of Gag; however, only bud22Delta showed a striking defect in Gag processing. BUD22 affected the +1 translational frameshifting event required to express the Pol proteins protease, integrase, and reverse transcriptase. Therefore, it is possible that the bud22Delta mutant may not produce enough functional Ty1 protease to completely process Gag-p49 to p45. Furthermore, BUD22 is required for 18S rRNA processing and 40S subunit biogenesis and influences polysome density. Together our results suggest that BUD22 is involved in a step in ribosome biogenesis that not only affects general translation, but also may alter the frameshifting efficiency of ribosomes, an event central to Ty1 retrotransposition.
Assuntos
Retroelementos/genética , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Northern Blotting , Western Blotting , Mudança da Fase de Leitura do Gene Ribossômico , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Modelos Genéticos , Mutagênese Insercional , Mutação , Polirribossomos/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
During the process of translation, an aminoacyl tRNA is selected in the A site of the decoding center of the small subunit based on the correct codon-anticodon base pairing. Though selection is usually accurate, mutations in the ribosomal RNA and proteins and the presence of some antibiotics like streptomycin alter translational accuracy. Recent crystallographic structures of the ribosome suggest that cognate tRNAs induce a "closed conformation" of the small subunit that stabilizes the codon-anticodon interactions at the A site. During formation of the closed conformation, the protein interface between rpS4 and rpS5 is broken while new contacts form with rpS12. Mutations in rpS12 confer streptomycin resistance or dependence and show a hyperaccurate phenotype. Mutations reversing streptomycin dependence affect rpS4 and rpS5. The canonical rpS4 and rpS5 streptomycin independent mutations increase translational errors and were called ribosomal ambiguity mutations (ram). The mutations in these proteins are proposed to affect formation of the closed complex by breaking the rpS4-rpS5 interface, which reduces the cost of domain closure and thus increases translational errors. We used a yeast two-hybrid system to study the interactions between the small subunit ribosomal proteins rpS4 and rpS5 and to test the effect of ram mutations on the stability of the interface. We found no correlation between ram phenotype and disruption of the interface.
Assuntos
Mutação , Proteínas Ribossômicas/genética , Modelos Moleculares , Fenótipo , Conformação Proteica , RNA de Transferência/metabolismo , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Ciliated protozoa of the genus Euplotes have undergone genetic code reassignment, redefining the termination codon UGA to encode cysteine. In addition, Euplotes spp. genes very frequently employ shifty stop frameshifting. Both of these phenomena involve noncanonical events at a termination codon, suggesting they might have a common cause. We recently demonstrated that Euplotes octocarinatus peptide release factor eRF1 ignores UGA termination codons while continuing to recognize UAA and UAG. Here we show that both the Tetrahymena thermophila and E. octocarinatus eRF1 factors allow efficient frameshifting at all three termination codons, suggesting that UGA redefinition also impaired UAA/UAG recognition. Mutations of the Euplotes factor restoring a phylogenetically conserved motif in eRF1 (TASNIKS) reduced programmed frameshifting at all three termination codons. Mutation of another conserved residue, Cys124, strongly reduces frameshifting at UGA while actually increasing frameshifting at UAA/UAG. We will discuss these results in light of recent biochemical characterization of these mutations.
Assuntos
Códon de Terminação , Euplotes/genética , Mudança da Fase de Leitura do Gene Ribossômico , Tetrahymena thermophila/genética , Animais , Código Genético , Humanos , Modelos Moleculares , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismoRESUMO
Errors during the process of translating mRNA information into protein products occur infrequently. Frameshift errors occur less frequently than other types of errors, suggesting that the translational machinery has more robust mechanisms for precluding that kind of error. Despite these mechanisms, mRNA sequences have evolved that increase the frequency up to 10,000-fold. These sequences, termed programmed frameshift sites, usually consist of a heptameric nucleotide sequence, at which the change in frames occurs along with additional sequences that stimulate the efficiency of frameshifting. One such stimulatory site derived from the Ty3 retrotransposon of the yeast Saccharomyces cerevisiae (the Ty3 stimulator) comprises a 14 nucleotide sequence with partial complementarity to a Helix 18 of the 18S rRNA, a component of the ribosome's accuracy center. A model for the function of the Ty3 stimulator predicts that it base pairs with Helix 18, reducing the efficiency with which the ribosome rejects erroneous out of frame decoding. We have tested this model by making a saturating set of single-base mutations of the Ty3 stimulator. The phenotypes of these mutations are inconsistent with the Helix 18 base-pairing model. We discuss the phenotypes of these mutations in light of structural data on the path of the mRNA on the ribosome, suggesting that the true target of the Ty3 stimulator may be rRNA and ribosomal protein elements of the ribosomal entry tunnel, as well as unknown constituents of the solvent face of the 40S subunit.