RESUMO
The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2'-O-methyl oligoribonucleotides (2'-OMe RNAs). Further, the inhibition efficiencies of the designed ribosome-interfering 2'-OMe RNAs were tested using a ß-galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC50 values below 1 µM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2'-OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double-filter retention assay. Further, we applied heat-shock chemical transformation to introduce 2'-OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC50 in the cell-free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter-subunit bridges can help design efficient antisense oligomers to probe the ribosome function.
Assuntos
Oligonucleotídeos/metabolismo , RNA Ribossômico/metabolismo , Sequência de Bases , Sítios de Ligação , Desenho Assistido por Computador , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Pactamicina/química , Pactamicina/metabolismo , Pactamicina/farmacologia , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Estrutura Terciária de Proteína , RNA Ribossômico/antagonistas & inibidores , RNA Ribossômico/química , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismoRESUMO
Biosynthetically and chemically derived analogs of the antibiotic pactamycin and de-6-methylsalicylyl (MSA)-pactamycin have attracted recent interest as potential antiprotozoal and antitumor drugs. Here, we report a 3.1-Å crystal structure of de-6-MSA-pactamycin bound to its target site on the Thermus thermophilus 30S ribosomal subunit. Although de-6-MSA-pactamycin lacks the MSA moiety, it shares the same binding site as pactamycin and induces a displacement of nucleic acid template bound at the E-site of the 30S. The structure highlights unique interactions between this pactamycin analog and the ribosome, which paves the way for therapeutic development of related compounds.
Assuntos
Pactamicina/química , Pactamicina/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Pactamicina/análogos & derivados , Ligação Proteica , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , Thermus thermophilus/metabolismoRESUMO
Mutants of an archaeon Halobacterium halobium, resistant to the universal inhibitor of translation, pactamycin, were isolated. Pactamycin resistance correlated with the presence of mutations in the 16 S rRNA gene of H. halobium single rRNA operon. Three types of mutations were found in pactamycin resistant cells, A694G, C795U and C796U (Escherichia coli 16 S rRNA numeration) located distantly in rRNA primary structure but probably neighboring each other in the three-dimensional structure. Pactamycin resistance mutations either overlapped (C795U) or were located in the immediate vicinity of nucleotides protected by the drug in E. coli and H. halobium 16 S rRNA indicating that corresponding rRNA sites might be directly involved in pactamycin binding. Ribosomal functions were not affected significantly either by mutation of C795 (one of the positions protected by the P-site-bound tRNA), or by mutations of A694 and C796 (which neighbor nucleotides protected by tRNA) suggesting that tRNA-dependent protections of C795 and G693 are explained by a conformational change in the ribosome induced by the P-site-bound tRNA. A novel mode of pactamycin action is proposed suggesting that pactamycin restricts structural transitions in 16 S rRNA preventing the ribosome from adopting a functional conformation induced by tRNA binding.
Assuntos
Halobacterium salinarum/genética , Mutagênese Sítio-Dirigida , Pactamicina/metabolismo , RNA Ribossômico 16S/genética , Sequência de Bases , Sítios de Ligação/genética , Resistência Microbiana a Medicamentos , Halobacterium salinarum/efeitos dos fármacos , Dados de Sequência Molecular , Pactamicina/farmacologia , RNA Ribossômico 16S/efeitos dos fármacosRESUMO
The binding sites of the antibiotics pactamycin and celesticetin on the rRNAs of Escherichia coli ribosomes were investigated by a chemical footprinting procedure. Pactamycin protected residues G-693 and C-795 in 16S RNA which are located in an important functional region of the 30S subunit participating in initiation complex formation and ribosomal subunit interaction. Celesticetin altered the reactivities of 5 residues A-2058, A-2059, A-2062, A-2451 and G-2505 within the central loop of domain V of 23S RNA which has been implicated in peptidyltransferase activity. Inferences are drawn concerning the mode of action of the antibiotics.
Assuntos
Pactamicina/metabolismo , RNA Ribossômico/metabolismo , Antibacterianos/metabolismo , Sequência de Bases , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Lincomicina/análogos & derivados , Lincomicina/metabolismo , Lincosamidas , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismoRESUMO
The antitumoral and antibacterial drug pactamycin can be radioactively labeled by iodination without loss of biological activity. Using the labeled pactamycin, the ribosomal binding site of the drug on rat liver ribosomes has been studied by affinity labeling techniques taking advantage of the photoreactive acetophenone group present in the molecule. When 40 S ribosomal subunits are labeled, one major spot of radioactivity is found associated to protein S25. In addition, weaker spots related to proteins S14/15, S10, S17 and S7 can also be detected in the autoradiogram of the two-dimensional gel slab. Since pactamycin inhibits protein synthesis initiation, the proteins forming its binding site must be related to some step of this process. By comparison with results from pactamycin affinity labeling of Escherichia coli ribosomes (Tejedor, F., Amils, R. and Ballesta, J.P.G. (1985) Biochemistry 24, 3667-3672) these proteins could lie in the mRNA and initiation factors binding region of the rat liver ribosome.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Radioisótopos do Iodo/metabolismo , Fígado/metabolismo , Pactamicina/metabolismo , Ribossomos/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Cinética , Masculino , Pactamicina/farmacologia , Fotoquímica , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Reticulócitos/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [125I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis.