RESUMO
Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3' major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion.
Assuntos
Escherichia coli/química , RNA Mensageiro/química , RNA Ribossômico 16S/química , Proteínas Ribossômicas/química , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Divisão Celular/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genótipo , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
A-1 frameshift event is required for expression of the pol gene when ribosomes translate the mRNA of human immunodeficiency virus type-1 (HIV-1). In this study, we inserted the frameshift region of HIV-1 (a slippery heptanucleotide motif followed by a stem-loop) in a reporter gene coding for firefly luciferase. The ability of the corresponding mRNA, generated by in vitro transcription, to be translated in an Escherichia coli cell-free extract is the first demonstration that the HIV-1 frameshift can be reproduced in a bacterial cell-free extract, providing a powerful approach for analysis of the frameshift mechanism. The responses of the frameshift signal to chloramphenicol, an inhibitor of peptide bond formation, and spectinomycin, an inhibitor of translocation, suggest that the frameshift complies with the same rules found in eukaryotic translation systems. Furthermore, when translation was performed in the presence of streptomycin and neamine, two error-inducing antibiotics, or with hyperaccurate ribosomes mutated in S12, the frameshift efficiency was increased or decreased, respectively, but only in the presence of the stem-loop, suggesting that the stem-loop can influence the frameshift through a functional interaction with the ribosomes.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , HIV-1/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Viral/genética , Animais , Antibacterianos/farmacologia , Sequência de Bases , Sistema Livre de Células , Cloranfenicol O-Acetiltransferase/genética , Clonagem Molecular , Besouros , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes Reporter , Genes gag , Genes pol , Humanos , Luciferases/genética , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Viral/química , Proteínas Recombinantes/biossíntese , Deleção de SequênciaRESUMO
Mutagenesis of Escherichia coli K12 cells with ethyl methanesulfonate and selection of streptomycin-resistant mutants after a long delay for phenotypic expression allowed us to isolate new types of streptomycin-resistant ribosomes. Misreading patterns of the ribosomes in the presence of streptomycin revealed that most of the streptomycin-resistant mutants isolated under these conditions differed from the four classical types of streptomycin-resistant mutants studied and characterized by Strigini, P. and Gorini, L. (1970) J. Mol. Biol. 47, 517-530.
Assuntos
Resistência Microbiana a Medicamentos , Escherichia coli/metabolismo , Estreptomicina/farmacologia , Colífagos/metabolismo , Escherichia coli/efeitos dos fármacos , Metanossulfonato de Etila/farmacologia , Mutação , Fenótipo , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/metabolismo , Especificidade da Espécie , Transdução GenéticaRESUMO
A deletion of five nucleotides was introduced at the 5' end of the Escherichia coli 16S rRNA gene cloned in an appropriate vector under control of a T7 promoter. The 16S rRNA generated by in vitro transcription could be assembled into 30S subunits. The deletion did not affect the efficiency of translation of natural messengers and the correct selection of the reading frame. However, it reduced the binding of the messengers, which suggests that the 5' end of 16S rRNA is located on the pathway followed by the messengers on the 30S subunits. The deletion also restricted the stimulation of misreading by streptomycin in a poly(U)-directed system. This is in accord with the proximity of the 5' end of 16S rRNA to proteins S4, S5 and S12, which are known to be involved in the control of translational accuracy.
Assuntos
Deleção Cromossômica , Escherichia coli/genética , Mutagênese Sítio-Dirigida , Biossíntese de Proteínas , RNA Ribossômico 16S/genética , Sequência de Bases , Clonagem Molecular , Genes Bacterianos , Vetores Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Transcrição GênicaRESUMO
The influence of streptomycin and neomycin upon the conformation of the ribosome has been investigated using spin-labeled and fluorescent analogs of the sulfhydryl reagent, N-ethylmaleimide. Changes in the electron paramagnetic resonance spectra or in the polarization of fluorescence of labeled ribosomes reveal that streptomycin alters the mobility of labels bound to the sulfhydryl group of protein S18 while neomycin affects the mobility of labels bound to the sulfhydryl groups of proteins S1, S21 and/or L10. It is also observed that both streptomycin and neomycin interfere with changes in the mobility of labels induced by storage under inactivating conditions. From these results, it is concluded that: 1. streptomycin and neomycin distort the conformation of the ribosome at different sites, streptomycin disturbing preferentially the area around the sulfhydryl group of protein S18 while neomycin affects the environment of the sulfhydryl groups of proteins S1, S21 and/or L10; 2. streptomycin and neomycin interefere with the ability of the ribosome to undergo conformational changes.
Assuntos
Escherichia coli/ultraestrutura , Neomicina/farmacologia , Ribossomos/ultraestrutura , Estreptomicina/farmacologia , Ácido Edético/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Etilmaleimida/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Espectrometria de Fluorescência , Marcadores de SpinRESUMO
Conformational alterations induced by streptomycin in the bacterial ribosome have been investigated using as probes, ethidium bromide, N-[14C]ethylmaleimide and a spin label nitroxide analog of N-ethylmaleimide. 1. The binding of the antibiotic to the ribosome does not affect the reactivity of sulfhydryl groups towards N-ethylmaleimide. 2. The motional freedom of spin labels bound to ribosomal proteins S1 and S18 is increased but it is hardly affected at other labeled sites. This observation suggests that the binding of streptomycin causes a local loosening of the ribosomal structure. 3. Ribosomes are found to bind less ethidium bromide in the presence of streptomycin, which suggests that the binding of streptomycin decreases the degree of organization of ribosomal RNA.
Assuntos
Escherichia coli/ultraestrutura , Ribossomos/ultraestrutura , Estreptomicina/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Etídio , Etilmaleimida , Cinética , Ribossomos/efeitos dos fármacos , Marcadores de SpinRESUMO
In a poly(U)-programmed translation system, neomycin stimulates the misincorporation of tyrosine and of serine which, according to Thompson and Stone (Thompson, R.C. and Stone, P.J. (1977) Proc. Natl. Acad. Sci. USA. 74, 198-202), are normally rejected at an initial discrimination step during the binding of charged tRNAs to the ribosome. In contrast, streptomycin favors the misincorporation of isoleucine which is normally rejected at a subsequent GTP-dependent discrimination step, the so-called proofreading step. The labeling of the ribosome with N-ethylmaleimide mimics the effect of streptomycin in that it stimulates the misincorporation of isoleucine but not of tyrosine or serine. This effect is correlated with the labeling of protein S18 but not with that of protein S1. These observations indicate that the sulfhydryl group of protein S18 is located within a ribosomal domain involved in the proofreading control of tRNA selection. Taking into account our previous results that streptomycin and neomycin perturb ribosomal areas around the sulfhydryl groups of proteins S18 and S1, respectively, we suggest that these antibiotics induce misreading by different mechanisms which are linked to such perturbations.
Assuntos
Neomicina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Estreptomicina/farmacologia , Escherichia coli , Etilmaleimida/farmacologia , Isoleucina/metabolismo , Leucina/metabolismo , Poli U/metabolismo , Ribossomos/efeitos dos fármacos , Serina/metabolismo , Tirosina/metabolismoRESUMO
Streptomycin, an error-inducing aminoglycoside antibiotic, binds to a single site on the small ribosomal subunit of bacteria, but this site has not yet been defined precisely. Here, we demonstrate that streptomycin binds to E. coli 16 S rRNA in the absence of ribosomal proteins, and protects a set of bases in the decoding region against dimethyl sulfate attack. The binding studies were performed in a high ionic strength buffer containing 20 mM Mg2+. The pattern of protection in the decoding region was similar to that observed when streptomycin binds to the 30 S subunit. However, streptomycin also protects the 915 region of 16 S rRNA within the 30 S subunit, whereas it did not protect the 915 region of the naked 16 S rRNA. The interaction of streptomycin with 16 S rRNA was further defined by using two fragments that correspond to the 3' minor domain of 16 S rRNA and to the decoding analog, a portion of this domain encompassing the decoding center. In the presence of streptomycin, the pattern of protection against dimethyl sulfate attack for the two fragments was similar to that seen with the full-length 16 S rRNA. This indicates that the 3' minor domain as well as the decoding analog contain the recognition signals for the binding of streptomycin. However, streptomycin could not bind to the decoding analog in the absence of Mg2+. This contrasts with neomycin, another error-inducing aminoglycoside antibiotic, that binds to the decoding analog in the absence of Mg2+, but not at 20 mM Mg2+. Our results suggest that both neomycin and streptomycin interact with the decoding center, but recognize alternative conformations of this region.
Assuntos
Antibacterianos/metabolismo , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/metabolismo , Estreptomicina/metabolismo , Antibacterianos/farmacologia , Sequência de Bases , Sítios de Ligação , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Magnésio/farmacologia , Dados de Sequência Molecular , Mutagênicos/farmacologia , Neomicina/metabolismo , Neomicina/farmacologia , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/efeitos dos fármacos , RNA Ribossômico 16S/química , RNA Ribossômico 16S/efeitos dos fármacos , Estreptomicina/farmacologia , Ésteres do Ácido Sulfúrico/farmacologiaRESUMO
Ribosomal protein S7 binds to a small RNA fragment of about 100 nucleotides within the lower half of the 3' major domain of E. coli 16 S rRNA. This fragment (D3M) comprises two large internal loops, A and B, connected by helix 29, a six-base-pair helix containing a G.U pair. Two hairpins with non-canonical base-pairs, 42' and 43, protrude from loops A and B, respectively. We used site-directed mutagenesis and molecular probing to further define which parts of D3M are important for S7 binding. Changing the stem of hairpin 42' into a Watson-Crick helix did not affect S7 binding, indicating that the non-canonical pairs of 42' do not provide recognition features for S7. However, deletion of this hairpin decreased S7 binding affinity by about threefold and altered the conformation of loop A. Deletion of the upper part of hairpin 43 (the loop and the adjacent four base-pairs) did not affect S7 binding, whereas the lower part of this hairpin (three base-pairs) was found to be required for proper S7 binding. Moreover, replacing the U.G pair with a C.G pair in this lower part decreased S7 binding affinity by twofold, suggesting that the U.G pair is a recognition signal for S7. S7 binding was also affected by mutations in helix 29. Insertion of one nucleotide 5' to the G or 3' to the U of the G.U pair decreased S7 binding affinity by about threefold and twofold, respectively, whereas replacement of the G.U pair by a G.C pair enhanced the affinity about twofold, and lengthening the helix by inserting a C.G pair upstream from the G.U pair had no effect. Taken together, these results are consistent with a bipartite binding site for S7 on 16 S rRNA, involving two regions of interaction: one centered around helix 29 and extending on the adjacent part of loop A, and the other one centered around the lower part of hairpin 43 and probably extending on the adjoining part of loop B.
Assuntos
Escherichia coli/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Proteínas Ribossômicas/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Análise Mutacional de DNA , Escherichia coli/metabolismo , Sondas Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA/química , RNA/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , Relação Estrutura-AtividadeRESUMO
A novel alternative conformation, which involves an interaction between the 5' terminal and 915 regions (E. coli numbering), is proposed after a screening of compiled sequences of small subunit ribosomal RNAs. This conformation contains a pseudoknot helix between residues 12-16 and 911-915, and its formation requires the partial melting of the 5' terminal helix and the disruption of the 17-19/916-918 pseudoknot helix of the classical 16 S rRNA secondary structure. The alternate pseudoknot helix is proximal to the binding site of streptomycin and various mutations in rRNA which confer resistance to streptomycin have been located in each strand of the proposed helix. It is suggested that the presence of streptomycin favours the shift towards the alternate conformation, thereby stabilizing drug binding. Mutations which destabilize the novel pseudoknot helix would restrict the response to streptomycin.
Assuntos
RNA Ribossômico 16S/ultraestrutura , Ribossomos/ultraestrutura , Sequência de Bases , Escherichia coli/ultraestrutura , Ligação de Hidrogênio , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
The present study shows that a mutation in the 530 loop of 16S rRNA impairs the binding of streptomycin to the bacterial ribosome, thereby restricting the misreading effect of the drug. Previous reports demonstrated that proteins S4, S5 and S12 as well as the 915 region of 16S rRNA are involved in the binding of streptomycin, and indicated that the drug not only interacts with the 30S subunit but also with the 50S subunit. The relationship between the target of streptomycin and its known interference with the proofreading control of translational accuracy is examined in light of these results.
Assuntos
Escherichia coli/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/metabolismo , Estreptomicina/metabolismo , Autorradiografia , Proteínas de Bactérias/biossíntese , Sítios de Ligação , Sulfato de Di-Hidroestreptomicina/metabolismo , Resistência Microbiana a Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Mutação , Conformação de Ácido Nucleico , Plasmídeos , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Estreptomicina/farmacologiaRESUMO
The cDNA for hamster preproenkephalin A (ENK) was cloned from an adrenal gland cDNA library constructed in the lambda ZapII vector. A nearly full-length cDNA was obtained and its 5' end region was completed using the technique of rapid amplification of cDNA ends (RACE). The coding and 3' untranslated regions of the hamster ENK cDNA share a high sequence identity with the rat, human, and bovine cDNAs, whereas the sequence identity is lower for the 5' untranslated region. Southern blot analysis of genomic DNA digests showed that a single copy of the ENK gene is present in the hamster haploid genome. Northern blot analysis of poly(A)+RNA from various hamster tissues indicated the following rank order for ENK messenger RNA abundance: adrenal glands > right atrium > brain > left atrium > right ventricle > ventricular septum > left ventricle, whereas primer extension analysis showed a single, identical transcriptional initiation site for the ENK mRNA in all these tissues. The sequence of the 5' untranslated region of the heart ENK cDNA was found to be identical to that from adrenal glands. This rules out the possibility that structural divergences in the 5' untranslated region of the heart ENK mRNA could decrease its translation efficiency and contribute to the very low level of enkephalin-containing peptides in the heart, compared to the adrenal glands.
Assuntos
DNA Complementar/genética , Encefalinas/genética , Precursores de Proteínas/genética , Glândulas Suprarrenais/química , Animais , Clonagem Molecular , Cricetinae , Encefalinas/química , Dosagem de Genes , Biblioteca Gênica , Humanos , Masculino , Mesocricetus , Dados de Sequência Molecular , Miocárdio/química , Especificidade de Órgãos , Precursores de Proteínas/química , RNA Mensageiro/análise , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Preproenkephalin A mRNA (ppEnk mRNA) and immunoreactive Met-enkephalin (ir-Met-Enk) were measured in the heart of 4, 8 and 16 week-old normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. WKY rats displayed a small decrease in their cardiac concentration of free (1.3 to 1.0 pmol/g) and cryptic (enzyme processed: 5.3 to 3.7 pmol/g) ir-Met-Enk with aging while the abundance of ppEnk mRNA increased by 3.2 fold between 4 and 16 week-old animals. Similar decreases in free (1.5 to 1.0 pmol/g) and cryptic (5.6 to 4.2 pmol/g) ir-Met-Enk levels were observed in SHR with aging but the rise in the level of ppEnk mRNA was much more pronounced reaching at 16 week-old levels of 7.3 times higher than at 4 week-old and 4.3 times higher than in age-matched WKY. The lack of correlation between the concentration of free and cryptic ir-Met-Enk and the abundance of ppEnk mRNA led us to measure the level of peptides in the heart of 16 week-old animals sacrificed at 4 hr intervals over a 24 hr period. SHR rats displayed circadian variations in their heart content of free and cryptic ir-Met-Enk and increased levels (1.6 fold) of cryptic peptide as compared with WKY at the beginnings of light (6 hr) and dark (18 and 22 hr) periods, suggesting the occurrence of cyclic and transitory upregulation of cDNA transcription and/or derepression of mRNA translation.
Assuntos
Ritmo Circadiano , Encefalina Metionina/biossíntese , Encefalinas/biossíntese , Hipertensão/metabolismo , Miocárdio/metabolismo , Precursores de Proteínas/biossíntese , Animais , Northern Blotting , Encefalina Metionina/genética , Encefalinas/genética , Masculino , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
The protein synthesis activity of heart, skeletal muscle and liver polysomes from isoprotenerol-treated and control hamsters has been compared in an in vitro non-initiating translation system. Heart and skeletal muscle polysomes from treated hamsters were less active than controls and required a higher magnesium concentration for optimal protein synthesis. These results suggest that there is a conformational modification in heart and skeletal muscle ribosomes from isoprotenerol-treated hamsters. No such change was observed with ribosomes from the liver of isoproterenol-treated hamsters.
Assuntos
Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Músculos/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Animais , Cricetinae , Técnicas In Vitro , Masculino , Mesocricetus , Músculos/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/biossíntese , Ribossomos/metabolismoRESUMO
Polysomes extracted from cultured fibroblast cells isolated from patients with Duchenne muscular dystrophy (DMD), carriers of the disease, and normal controls were used for in vitro measurement of protein synthesis in a wheat germ extract system. It was observed that polysomes from patients and carriers (seven of each aged 17 years or older) exhibited a 3-fold and a 1.5-fold decrease in the rate of protein synthesis, respectively, as compared with controls. These results are discussed with a view to developing a sensitive and easily available assay for the detection of DMD carriers.
Assuntos
Fibroblastos/metabolismo , Distrofias Musculares/metabolismo , Biossíntese de Proteínas , Pele/metabolismo , Adolescente , Adulto , Triagem de Portadores Genéticos/métodos , Humanos , Distrofias Musculares/genética , Polirribossomos/metabolismoRESUMO
Various approaches have been developed to study how mutations in Escherichia coli ribosomal RNA affect the function of the ribosome. Most of them are in vivo approaches, where mutations are introduced in a specialized plasmid harboring the ribosomal RNA genes. The mutated plasmids are then expressed in an appropriate host, where they can confer resistance to antibiotics whose target is the ribosome. Conditions can be used where the host ribosomal RNA genes or the host ribosomes are selectively inactivated, and the effect of the mutations on ribosome assembly and function can be studied. Another approach, which has been developed mainly with 16S ribosomal RNA, can be used entirely in vitro. In this approach, a plasmid has been constructed which contains the 16S ribosomal RNA gene under control of a T7 promoter. Mutations can be introduced in the 16S ribosomal RNA sequence and the mutated 16S ribosomal RNAs are produced by in vitro transcription. It is then possible to investigate how the mutations affect the assembly of the 16S ribosomal RNA into 30S subunits and the activity of the reconstituted 30S subunits in cell-free protein synthesis assays. Although these approaches are recent, they have already provided a large body of interesting information, relating specific RNA sequences to interactions with ribosomal proteins, to ribosome function, and to its response to antibiotics.
Assuntos
Escherichia coli/genética , RNA Bacteriano/genética , RNA Ribossômico/genética , Sequência de Bases , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ribossomos/metabolismoRESUMO
Polysomes were isolated from the skeletal muscle, the heart, and the liver of dystrophic and normal hamsters and their protein synthesis activity was assessed in a cell-free wheat germ extract as a source factors and tRNAs. Our results show that there is a shift of the optimal magnesium concentration required for protein synthesis with polysomes from the skeletal muscle and the heart of dystrophic hamsters, as compared with control hamsters. As a consequence of this shift, polysomes from the skeletal muscle and the heart of dystrophic hamsters, were less active than normal ones at low magnesium concentrations, but more active at high magnesium concentrations. These changes in activity were age dependent since, with skeletal muscle, they were observed at 30 days and disappeared at 60 days but reappeared at 120 and 200 days. With heart polysomes, on the other hand, the changes in activity were observed at 60 days but not in younger or older animals. No change in activity was observed with liver polysomes. Similar results were obtained when endogenous mRNAs were replaced by an exogenous messenger such as poly(U). This suggests that the differences in protein synthesis activity between polysomes from dystrophic and normal hamsters are not due to changes in the endogenous mRNAs but result from a ribosomal abnormality.
Assuntos
Distrofia Muscular Animal/metabolismo , Polirribossomos/metabolismo , Biossíntese de Proteínas , Animais , Cricetinae , Cinética , Fígado/ultraestrutura , Masculino , Mesocricetus , Músculos/ultraestrutura , Distrofia Muscular Animal/patologia , Miocárdio/ultraestrutura , Poli U/metabolismo , RNA Mensageiro/metabolismoRESUMO
This review surveys the different experimental approaches which describe the binding of tRNA to mRNA-programmed ribosomes and the control of tRNA selection. This selection is best described by the two-step model proposed by Hopfield and demonstrated by Thompson and his collaborators. The model involves a first control at the initial reversible binding of tRNA to the ribosome and a second control, the proofreading control, which promotes rejection of the incorrect tRNA from a high-energy intermediate during the transition from the initial to the final binding state. Streptomycin, neomycin, and ribosomal fidelity mutations appear to affect both control steps. Their effect can be related to the location of the mutated ribosomal proteins and to the conformational changes induced in the ribosome by the misreading agents. An alteration of the first control probably results from a distortion of the codon-anticodon interaction, while an alteration of the second control may be caused by a change in the association between ribosomal subunits.
Assuntos
Proteínas de Bactérias/biossíntese , Escherichia coli/metabolismo , Neomicina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Ribossômicas/genética , Estreptomicina/farmacologia , Anticódon/metabolismo , Proteínas de Bactérias/genética , Códon/metabolismo , Escherichia coli/genética , Cinética , Modelos Biológicos , Mutação , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismoRESUMO
[3H]Dihydrostreptomycin was covalently linked to the 50S subunit of Escherichia coli K12A19 with the bifunctional cross-linking reagent phenyldiglyoxal. The cross-linking was abolished under conditions that prevent the specific interaction of streptomycin with the ribosome. The binding primarily involved the ribosomal RNA and also a limited number of proteins, namely, L2, L6, and L17. This suggests that the binding domain for streptomycin is close to the peptidyl transferase center, in the valley between the central protuberance and the wider lateral protuberance of the 50S subunit. This domain faces the binding domain for streptomycin which we have previously characterized on the 30S subunit [Melançon, P., Boileau, G., & Brakier-Gingras, L. (1984) Biochemistry 23, 6697-6703]. Our results indicate that the 50S subunit is involved in the binding of streptomycin to the bacterial ribosome, in addition to the 30S subunit which is generally considered as the specific target of the antibiotic. They are consistent with the occurrence of a single binding site for streptomycin on the ribosome, comprised of regions of both subunits.
Assuntos
Aldeídos/farmacologia , Reagentes de Ligações Cruzadas , Escherichia coli/metabolismo , Fenilglioxal/farmacologia , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Estreptomicina/metabolismo , Fracionamento Celular , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Fenilglioxal/análogos & derivados , Proteínas Ribossômicas/isolamento & purificação , Ribossomos/efeitos dos fármacos , Ribossomos/ultraestruturaRESUMO
Preproenkephalin A messenger RNA was detected in hamster heart by Northern blot analysis using a human preproenkephalin A cDNA probe. Ventricular levels of this messenger were one order of magnitude lower than atrial levels, which were equivalent to brain levels. Furthermore, in the heart of cardiomyopathic hamsters, an animal model of cardiac hypertrophy and congestive heart failure, the relative abundance of the preproenkephalin A messenger RNA was found to increase three- to four-fold in ventricles while no change was seen in atria. These results support the hypothesis that the heart has the potential for locally synthesizing enkephalins and provide evidence that alterations in preproenkephalin A messenger RNA levels are associated with the development of cardiac hypertrophy and failure.