RESUMO
A mouse model has often been used in studies of p53 gene expression. Detailed interpretation of functional studies is, however, hampered by insufficient knowledge of the impact of mouse p53 mRNA's structure and its interactions with proteins in the translation process. In particular, the 5'-terminal region of mouse p53 mRNA is an important region which takes part in the regulation of the synthesis of p53 protein and its N-truncated isoform Δ41p53. In this work, the spatial folding of the 5'-terminal region of mouse p53 mRNA and its selected sub-fragments was proposed based on the results of the SAXS method and the RNAComposer program. Subsequently, RNA-assisted affinity chromatography was used to identify proteins present in mouse fibroblast cell lysates that are able to bind the RNA oligomer, which corresponds to the 5'-terminal region of mouse p53 mRNA. Possible sites to which the selected, identified proteins can bind were proposed. Interestingly, most of these binding sites coincide with the sites determined as accessible to hybridization of complementary oligonucleotides. Finally, the high binding affinity of hnRNP K and PCBP2 to the 5'-terminal region of mouse p53 mRNA was confirmed and their possible binding sites were proposed.
Assuntos
RNA Mensageiro/química , Proteína Supressora de Tumor p53/genética , Animais , Camundongos , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Espalhamento a Baixo Ângulo , Proteína Supressora de Tumor p53/metabolismo , Difração de Raios XRESUMO
The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5' terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3' end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5' terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression.
Assuntos
Regiões 5' não Traduzidas , Biossíntese de Proteínas , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Baixo , Células HCT116 , Humanos , Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The p53 protein is one of the transcription factors responsible for cell cycle regulation and prevention of cancer development. Its expression is regulated at the transcriptional, translational and post-translational levels. Recent years of research have shown that the 5' terminus of p53 mRNA plays an important role in this regulation. This region seems to be a docking platform for proteins involved in p53 expression, particularly under stress conditions. Here, we applied RNA-centric affinity chromatography to search for proteins that bind to the 5' terminus of p53 mRNA and thus may be able to regulate the p53 expression profile. We found heterogeneous nuclear ribonucleoprotein K, hnRNP K, to be one of the top candidates. Binding of hnRNP K to the 5'-terminal region of p53 mRNA was confirmed in vitro. We demonstrated that changes in the hnRNP K level in the cell strongly affected the p53 expression profile under various stress conditions. Downregulation or overexpression of hnRNP K caused a decrease or an increase in the p53 mRNA amount, respectively, pointing to the transcriptional mode of expression regulation. However, when hnRNP K was overexpressed under endoplasmic reticulum stress and the p53 amount has elevated no changes in the p53 mRNA level were detected suggesting translational regulation of p53 expression. Our findings have shown that hnRNP K is not only a mutual partner of p53 in the transcriptional activation of target genes under stress conditions but it also acts as a regulator of p53 expression at the transcriptional and potentially translational levels.
Assuntos
Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Estresse Fisiológico , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Cromatografia de Afinidade , Humanos , Espectrometria de Massas , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The recently discovered group of noncoding RNAs, which are fragments of tRNA molecules (tRFs), has not been fully characterized and its potential functions still require investigation. Porcine tRFs were characterized and compared to mouse and human tRFs. Two tRFs, 5' 32-33â¯nt and 3' 41-42â¯nt that are derived from the mature tRNAVal(CAC) and tRNAGly(GCC) were detected with the use of bioinformatics and the Northern blot method. The abundance of these tRFs in the case of Sus scrofa is restricted to the ovary and the kidney. The same tRFs were found in human cancer cells and in mouse sperm, circulating blood and its serum. The binding of selected sncRNAs (piRNA, 5'tRFVal(CAC) and miRNA) to the overexpressed PAZ domain of the PIWIL4 protein was also studied. It is noteworthy that porcine 5'tRFVal(CAC) and human 5'tRFVal(CAC)as well as 5'tRFGly(GCC) are bound to the PIWIL4 protein. The potential role of the analyzed tRFs in the development of mammals is also discussed.
Assuntos
Mamíferos/crescimento & desenvolvimento , Mamíferos/genética , RNA de Transferência/genética , Sus scrofa/crescimento & desenvolvimento , Sus scrofa/genética , Animais , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sequência Conservada , Evolução Molecular , Feminino , Humanos , Masculino , Mamíferos/metabolismo , Camundongos , MicroRNAs/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA , Especificidade da Espécie , Sus scrofa/metabolismoRESUMO
Transcription initiation sites of Trp53 gene in mice were determined using the 5'RACE method. Based on sequence alignment of the 5'-terminal regions of p53 mRNA in mammals, the site for the most abundant transcript turned out to be essentially identical with that determined for human TP53 gene and slightly differed for the longest transcripts, in mice and humans. Secondary structures of the 5' -terminal regions of the shorter, most abundant and the longest mouse transcripts were determined in vitro and the shorter transcript was also mapped in transfected mouse cells. For the first time, secondary structure models of the 5' terminus of two mouse p53 mRNAs were proposed. Comparing these models with the conservativeness of the nucleotide sequence of the 5'-terminal region of mRNA in mouse and other mammals, the possible function of the selected structural domains of this region was discussed. To elucidate the translation mechanisms, the two studied mRNAs were translated in the presence of an increasing concentration of the cap analog. For the longest transcript, the data suggested that IRES element(s) was/were involved in translation initiation. Additionally, changes in p53 synthesis under genotoxic and endoplasmic reticulum stress conditions in mouse cells were analyzed.
Assuntos
Regiões 5' não Traduzidas , Regulação da Expressão Gênica , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/genética , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada , Sítios Internos de Entrada Ribossomal , Camundongos , Iniciação Traducional da Cadeia Peptídica , Dobramento de RNA , Elementos de Resposta , Sítio de Iniciação de TranscriçãoRESUMO
In this review, the latest research concerning the structure and function of the 5'-terminal region of p53 mRNA was discussed. Special attention was focused on defined structural motifs which are present in this region, as well as their conservation and plausible functional role in translation. It is known that the length of the 5'-terminal region and the structural environment of initiation codons can strongly modulate translation initiation. The ability of this region of p53 mRNA to bind protein factors was also described with special emphasis on general principles that govern, such RNA-protein interactions. The structural alterations within the 5'-terminal region of p53 mRNA and proteins that bind to this region have a strong impact on the rate of mRNA scanning and on translation efficiency in in vitro assays, in selected cell lines, and under stress conditions. Thus, the structural features of the 5'-terminal region of p53 mRNA seem to be very important for translation and for translation regulation mechanisms. Finally, we suggested topics that, in our opinion, should be further explored for better understanding of the mechanisms of the p53 gene expression regulation at the translational level.
Assuntos
Regiões 5' não Traduzidas/fisiologia , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regiões 5' não Traduzidas/genética , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas/fisiologiaRESUMO
BACKGROUND: Bacitracin is a polypeptide antibiotic active against Gram-positive bacterial strains. Its mechanism of action postulates disturbing the cell wall synthesis by inhibiting dephosphorylation of the lipid carrier. We have discovered that bacitracin induces degradation of nucleic acids, being particularly active against RNA. METHODS: In the examination of the nucleolytic activity of bacitracin several model RNA and DNA oligomers were used. The oligomers were labeled at their 5' ends with (32)P radioisotope and following treatment with bacitracin the cleavage sites and efficiency were determined. RESULTS AND CONCLUSIONS: Bacitracin induces degradation of RNA at guanosine residues, preferentially in single-stranded RNA regions. Bacitracin is also able to degrade DNA to some extent but comparable effects to those observed with RNA require its 10-fold higher concentration. The sites of degradation in DNA are very infrequent and preferentially occur near cytidine residues. Free radicals are not involved in the reaction, and which probably proceeds via a hydrolytic mechanism. The phosphate groups at the cleavage sites are present at the 3' ends of RNA products and at the 5' ends of DNA fragments. Importantly, the presence of EDTA does not influence RNA degradation but completely inhibits the degradation of DNA. For DNA degradation divalent metal ions like Mg(2+), Mn(2+) or Zn(2+) are absolutely necessary. GENERAL SIGNIFICANCE: The ability of bacitracin to degrade nucleic acids via a hydrolytic mechanism was a surprising observation, and it is of interest whether these properties can contribute to its mechanisms of action during antibiotic treatment.
Assuntos
Antibacterianos/farmacologia , Bacitracina/farmacologia , DNA/química , RNA/química , HidróliseRESUMO
Recently, we have determined the secondary structure of the 5'-terminal region of p53 mRNA that starts from the P1 transcription initiation site and includes two major translation initiation codons responsible for the synthesis of p53 and ΔNp53 isoform. Here, we showed that when this region was extended into 5' direction to the P0 transcription start site, the two characteristic hairpin motifs found in this region were preserved. Moreover, the presence of alternatively spliced intron 2 did not interfere with the formation of the larger hairpin in which the initiation codon for p53 was embedded. The impact of the different variants of p53 5'-terminal region, which start at P0 or P1 site and end with the initiation codon for p53 or ΔNp53, on the translation of luciferase reporter protein was compared. Strikingly, the efficiency of translation performed in rabbit reticulocyte lysate differed by two orders of magnitude. The toe-printing analysis was also applied to investigate the formation of the ribosomal complex on the model mRNA constructs. The relative translation efficiencies in HeLa and MCF-7 cells were similar to those observed in the cell lysate, although some differences were noted in comparison with cell-free conditions. The results were discussed in terms of the role of secondary structure folding of the 5'-terminal region of p53 mRNA in translation and possible modes of p53 and ΔNp53 translation initiation.
Assuntos
Regiões 5' não Traduzidas/fisiologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , Isoformas de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Processamento Alternativo , Animais , Códon de Iniciação , Células HeLa , Humanos , Íntrons , Células MCF-7 , Modelos Genéticos , Conformação Molecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Dobramento de RNA , RNA Mensageiro/genética , CoelhosRESUMO
The p53 protein is one of the major factors involved in cell cycle control, DNA repair, and induction of apoptosis. We determined the secondary structure of the 5'-terminal region of p53 mRNA that includes two major translation initiation codons AUG1 and AUG2, responsible for the synthesis of p53 and its N-truncated isoform ΔN-p53. It turned out that a part of the coding sequence was involved in the folding of the 5' untranslated region for p53. The most characteristic structural elements in the 5'-terminal region of p53 mRNA were two hairpin motifs. In one of them, the initiation codon AUG1 was embedded while the other hairpin has been earlier shown to bind the Mdm2 protein. Alternative mechanisms of p53 mRNA translation initiation were investigated in vitro using model mRNA templates. The results confirmed that initiation from AUG1 was mostly cap-dependent. The process was stimulated by a cap structure and strongly inhibited by a stable hairpin at the template 5' end. Upon inhibition, the remaining protein fraction was synthesized in a cap-independent process, which was strongly stimulated by the addition of a cap analogue. The translation initiation from AUG2 showed a largely cap-independent character. The 5' cap structure actually decreased initiation from this site which argues against a leaky scanning mechanism but might suggest the presence of an IRES. Moreover, blocking cap-dependent translation from AUG1 by the stable hairpin did not change the level of initiation from AUG2. Upon addition of the cap analogue, translation from this site was even increased.
Assuntos
Regiões 5' não Traduzidas/genética , Códon de Iniciação/genética , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , RibossomosRESUMO
The p53 protein is expressed as at least twelve protein isoforms. Within intron 4 of the human TP53 gene, a P2 transcription initiation site is located and this transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. Here, the secondary structure of the 5'-terminal region of P2-initiated mRNA was characterized by means of the SHAPE and Pb2+-induced cleavage methods and for the first time, a secondary structure model of this region was proposed. Surprisingly, only Δ133p53 isoform was synthetized in vitro from the P2-initiated p53 mRNA while translation from both initiation codons occurred after the transfection of vector-encoded model mRNA to HCT116 cells. Interestingly, translation performed in the presence of the cap analogue suggested that the cap-independent process contributes to the translation of P2-initiated p53 mRNA. Subsequently, several antisense oligonucleotides targeting the 5'-terminal region of P2-initiated p53 mRNA were designed. The selected oligomers were applied in in vitro translation assays as well as in cell lines and their impact on the Δ133p53 synthesis and on cell viability was investigated. The results show that these oligomers are attractive tools in the modulation of the translation of P2-initiated p53 mRNA through attacking the 5' terminus of the transcript. Since cell proliferation is also reduced by antisense oligomers that lower the level of Δ133p53, this demonstrates an involvement of this isoform in tumorigenesis.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/genética , Proteína Supressora de Tumor p53/genética , Sobrevivência Celular/efeitos dos fármacos , Códon de Iniciação/antagonistas & inibidores , Células HCT116 , Humanos , Íntrons/genética , Isoformas de Proteínas/antagonistas & inibidores , RNA Mensageiro/antagonistas & inibidores , Sítio de Iniciação de Transcrição/efeitos dos fármacos , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
Two early nodulin 40 (enod40) genes, ENOD40-1, the shortest legume ENOD40 gene, and ENOD40-2, were isolated from Lupinus luteus, a legume with indeterminate nodules. Both genes were expressed at similar levels during symbiosis with nitrogen-fixing bacteria. ENOD40 phylogeny clustered the L. luteus genes with legumes forming determinate nodules and revealed peptide similarities. The ENOD40-1 small ORF A fused to a reporter gene was efficiently expressed in plant cells, indicating that the start codon is recognized for translation. The ENOD40-1 RNA structure predicted based on Pb(II)-induced cleavage and modeling revealed four structurally conserved domains, an absence of domain 4 characteristic for legumes of indeterminate nodules, and interactions between the conserved region I and a region located upstream of domain 6. Domain 2 contains Mg(II) ion binding sites essential for organizing RNA secondary structure. The differences between L. luteus and Glycine max ENOD40 RNA models suggest the possibility of a switch between two structural states of ENOD40 transcript.
Assuntos
Lupinus/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Biossíntese de Proteínas , RNA de Plantas/química , Southern Blotting , Genes Reporter , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The hepatitis delta virus (HDV) ribozyme is an RNA enzyme that catalyzes the site-specific trans-esterification reaction. Using high hydrostatic pressure (HHP) technique we showed that HDV ribozyme catalyzes the reaction of RNA cleavage in the absence of magnesium ions according to mechanism of acidic hydrolysis of esters. HHP induces changes of water structure, lowering pH and effect ribozyme catalytic site structure formation without magnesium. HHP, similarly to magnesium ion at ambient pressure stabilizes the higher order RNA structure of HDV, but Mg(2+) is not involved in the catalysis. Our results clearly support the new mechanism of HDV hydrolysis and show advantages of using HHP in analysis of macromolecules interaction.
Assuntos
Ácidos/metabolismo , Ésteres/metabolismo , Vírus Delta da Hepatite/enzimologia , RNA Catalítico/metabolismo , Sequência de Bases , Biocatálise , Genoma Viral/genética , Hidrólise , Pressão Hidrostática , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/genéticaRESUMO
Here we present the results of a structural analysis of the 3'-terminal region of the replicative strand of hepatitis C virus (HCV), IRES(-), by the Pb (2+)-induced cleavage approach and partial digestion with T1 ribonuclease. Oligoribonucleotides that represent selected domains of the earlier proposed in the literature secondary structure models of this region were also synthesized, their structures were analyzed in solution, and the results were compared to those obtained with the full-length molecule. Such "structural fingerprinting" gave better insight into the structure of the IRES(-) region. We showed that in the case of the IRES(-) fragment, which consists of 374 nucleotides, its three domains, D3 (nucleotides 1-104), DM (nucleotides 105-222), and D5 (nucleotides 223-374), independently fold on one another. However, when the IRES(-) molecule is extended by 25 nucleotides of the upstream viral sequence, domains D3 and DM fold autonomously, but a part of domain D5 interacts with that additional RNA stretch. Analysis in silico suggests that similar interactions involving the IRES(-) region and upstream sequences are also possible in other fragments of viral RNA, several hundreds of nucleotides in length. The results of experimental probing are supported by secondary structure predictions in silico and phylogenetic analysis.
Assuntos
Regiões 3' não Traduzidas/química , Hepacivirus/fisiologia , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/química , Replicação Viral/fisiologia , Regiões 3' não Traduzidas/metabolismo , Chumbo/química , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , Filogenia , RNA Viral/metabolismoRESUMO
We have used the in vitro selection method to search for catalytically active variants of the antigenomic delta ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2. In the initial combinatorial library 16 nt positions were randomized and the library contained a full representation of all possible sequences. Following ten cycles of selection-amplification several catalytically active ribozyme variants were identified. It turned out that one-third of the variants contained only single mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next one-third of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position to the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial 'structural plasticity' and the capacity of functional adaptation. Four selected ribozyme variants were subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards Mg2+, Ca2+ and Mn2+ ions. Thus, the functional properties of the variants were dependent on both the structure of their catalytic sites and divalent metal ions performing catalysis.
Assuntos
Vírus Delta da Hepatite/enzimologia , RNA Catalítico/química , Sequência de Bases , Domínio Catalítico , Técnicas de Química Combinatória , Evolução Molecular Direcionada , Variação Genética , Genoma Viral , Vírus Delta da Hepatite/genética , Cinética , Metais/química , Dados de Sequência Molecular , Mutação , RNA Catalítico/genética , RNA Catalítico/metabolismoRESUMO
At the 3' end of genomic hepatitis C virus (HCV) RNA there is a highly conserved untranslated region, the 3'X-tail, which forms part of the 3'UTR. This region plays key functions in regulation of critical processes of the viral life cycle. The 3'X region is essential for viral replication and infectivity. It is also responsible for regulation of switching between translation and transcription of the viral RNA. There is some evidence indicating the contribution of the 3'X region to the translation efficiency of the viral polyprotein and to the encapsidation process. Several different secondary structure models of the 3'X region, based on computer predictions and experimental structure probing, have been proposed. It is likely that the 3'X region adopts more than one structural form in infected cells and that a specific equilibrium between the various forms regulates several aspects of the viral life cycle. The most intriguing explanations of the structural heterogeneity problem of the 3'X region came with the discovery of its involvement in long-range RNA-RNA interactions and the potential for homodimer formation. This article summarizes current knowledge on the structure and function of the 3'X region of hepatitis C genomic RNA, reviews previous opinions, presents new hypotheses and summarizes the questions that still remain unanswered.
Assuntos
Regiões 3' não Traduzidas/genética , Hepacivirus/genética , Hepatite C/terapia , Conformação de Ácido Nucleico , RNA Viral/química , Antivirais/farmacologia , Antivirais/uso terapêutico , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/imunologia , Hepacivirus/patogenicidade , Hepatite C/epidemiologia , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Modelos Moleculares , Interferência de RNA , RNA Viral/antagonistas & inibidores , RNA Viral/genética , Relação Estrutura-Atividade , Resultado do Tratamento , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia , Vacinas contra Hepatite Viral/uso terapêutico , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
The p53 protein is one of the major cell cycle regulators. The protein is expressed as at least twelve protein isoforms resulting from the use of alternative promoters, alternative splicing or downstream initiation codons. Importantly, there is growing evidence that translation initiation of p53 mRNA may be regulated by the structure and length of the naturally occurring variants of the 5'-terminal region of p53 mRNA transcripts. Here, several mRNA constructs were synthesized with variable length of the p53 5'-terminal regions and encoding luciferase reporter protein, and their translation was monitored continuously in situ in a rabbit reticulocyte lysate system. Moreover, four additional mRNA constructs were prepared. In two constructs, the structural context of AUG1 initiation codon was altered while in the other two constructs, characteristic hairpin motifs present in the p53 5'-terminal region were changed. Translation of the last two constructs was also performed in the presence of the cap analogue to test the function of the 5'-terminal region in cap-independent translation initiation. Superposition of several structural factors connected with the length of the 5'-terminal region, stable elements of the secondary structure, structural environment of the initiation codon and IRES elements greatly influenced the ribosomal scanning and translation efficiency.
Assuntos
Regiões 5' não Traduzidas/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Fusão Gênica Artificial , Sistema Livre de Células , Genes Reporter , Luciferases/análise , Luciferases/genética , CoelhosRESUMO
Oligoribonucleotides that corresponded to the X regions of the (+) and (-) polarity strands of HCV RNA, as well as several shorter oligomers comprising defined stem-loop motifs of their predicted secondary structure models, were analyzed by Pb2+-induced cleavage, partial digestion with specific nucleases and chemical modification. Patterns characteristic of the motifs were compared with those obtained for the full-length molecules and on the basis of such 'structural fingerprinting' conclusions concerning folding of regions X were formulated. It turned out that the secondary structure model of X(+) RNA proposed earlier, the three-stem-loop model composed of hairpins SL1, SL2 and SL3, was only partially consistent with our experimental data. We confirmed the presence of SL1 and SL3 motifs and showed that the single-stranded stretch adjacent to the earlier proposed hairpin SL2 contributed to the folding of that region. It seemed to be arranged into two hairpins, which might form a hypothetical pseudoknot by changing their base-pairing systems. These data were discussed in terms of their possible biological significance. On the other hand, analysis of the X(-) RNA and its sub-fragments supported a three-stem-loop secondary structure model for this RNA.
Assuntos
Hepacivirus/genética , RNA Viral/química , Sequência de Bases , Sequência Conservada , Genoma Viral , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , RNA Viral/metabolismo , Replicação ViralRESUMO
All eukaryotic mRNA molecules have a cap structure at the 5' ends which plays a crucial role in the scanning model of their translation initiation. In an alternative way of translation, the active ribosome is formed in a cap-independent mode due to the presence of IRES, internal ribosome entry site, in the 5' untranslated region of certain mRNAs. This region folds into a distinct secondary and tertiary structure, which binds the 40S ribosomal subunit and some protein factors, and subsequently forms the initiation complex and the translationally active 80S ribosome. This enables the synthesis of specific proteins under the conditions when cap-dependent translation is inhibited or strongly reduced. The cap-independent mode of translation initiation concerns proteins that play very important roles during cell cycle, apoptosis, response to stress stimuli and cancer development.
Assuntos
Células Eucarióticas/metabolismo , Modelos Biológicos , Biossíntese de Proteínas/fisiologia , Proteínas de Ligação ao Cap de RNA/metabolismo , Animais , Apoptose/genética , Ciclo Celular/genética , Humanos , Neoplasias/genética , Dobramento de Proteína , Estresse Fisiológico/genéticaRESUMO
Here, we describe the characterization of new RNA-cleaving DNAzymes that showed the highest catalytic efficiency at pHâ 4.0 to 4.5, and were completely inactive at pH values higher than 5.0. Importantly, these DNAzymes did not require any divalent metal ion cofactors for catalysis. This clearly suggests that protonated nucleic bases are involved in the folding of the DNAzymes into catalytically active structures and/or in the cleavage mechanism. The trans-acting DNAzyme variants were also catalytically active. Mutational analysis revealed a conservative character of the DNAzyme catalytic core that underpins the high structural requirements of the cleavage mechanism. A significant advantage of the described DNAzymes is that they are inactive at pH values close to physiological pH and under a wide range of conditions in the presence of monovalent and divalent metal ions. These pH-dependent DNAzymes could be used as molecular cassettes in biotechnology or nanotechnology, in molecular processes that consist of several steps. The results expand the repertoire of DNAzymes that are active under nonphysiological conditions and shed new light on the possible mechanisms of catalysis.
RESUMO
In most in vitro run-off transcription reactions with T7 RNA polymerase, transcripts with heterogeneous ends are commonly obtained. Towards the goal of finding a simple and effective procedure for correct processing of their 3' ends we propose the use of trans-acting antigenomic delta ribozyme. We demonstrate that the extension of nascent transcripts with only seven nucleotides complementary to the ribozyme's recognition site, and subsequently, the removal of those nucleotides with the ribozyme acting in trans, is an efficient procedure for generating transcripts with homogenous 3' ends. This approach was tested on two model RNA molecules: an in vitro transcript of yeast tRNA(Phe) and a delta ribozyme, which processed itself during transcription. The proposed procedure is a simple alternative to the use of ribozymes as cis-cleaving autocatalytic cassettes attached to transcript 3' ends. As there is little possibility that the required additional stretch, only seven nucleotides long, enters into stable interactions with other parts of the transcripts, it can be cleaved off with high efficacy.