RESUMO
To study positioning of the polypeptide release factor eRF1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives of oligoribonucleotides. The human eRF1 peptides cross-linked to these short mRNAs were identified. Cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31-33, and to lesser extent amino acids within region 121-131 (the "YxCxxxF loop") in the N domain. Hence, both regions are involved in the recognition of the purines. A cross-linker at the first uridine of the stop codon modifies Val66 near the NIKS loop (positions 61-64), and this region is important for recognition of the first uridine of stop codons. Since the N domain distinct regions of eRF1 are involved in a stop-codon decoding, the eRF1 decoding site is discontinuous and is not of "protein anticodon" type. By molecular modeling, the eRF1 molecule can be fitted to the A site proximal to the P-site-bound tRNA and to a stop codon in mRNA via a large conformational change to one of its three domains. In the simulated eRF1 conformation, the YxCxxxF motif and positions 31-33 are very close to a stop codon, which becomes also proximal to several parts of the C domain. Thus, in the A-site-bound state, the eRF1 conformation significantly differs from those in crystals and solution. The model suggested for eRF1 conformation in the ribosomal A site and cross-linking data are compatible.
Assuntos
Códon de Terminação/genética , Códon de Terminação/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Sequência de Bases , Reagentes de Ligações Cruzadas , Humanos , Técnicas In Vitro , Modelos Moleculares , Mutagênese Sítio-Dirigida , Terminação Traducional da Cadeia Peptídica , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Conformação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Inhibition of primer extension by ribosome-mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture.
Assuntos
Eletroforese Capilar , Biossíntese de Proteínas , RNA Mensageiro/análise , Ribossomos/metabolismo , Primers do DNA , Corantes Fluorescentes , RNA Mensageiro/metabolismo , Transcrição ReversaRESUMO
BACKGROUND: Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas. METHODOLOGY/PRINCIPAL FINDINGS: We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1-2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3-5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines. CONCLUSIONS/SIGNIFICANCE: This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread.
Assuntos
Mutação/genética , Neoplasias/genética , Proteínas Supressoras de Tumor/genética , Desaminase APOBEC-1 , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , Biologia Computacional , Citidina Desaminase/metabolismo , DNA Bacteriano/genética , DNA Complementar/genética , Proteínas de Escherichia coli/genética , Etiquetas de Sequências Expressas , Efeito Fundador , Genoma/genética , Hematopoese/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Camundongos , Camundongos SCID , Reação em Cadeia da PolimeraseRESUMO
To analyze RNA interactions with RNA binding molecules an RNA microchip containing immobilized oligoribonucleotides with protective groups [t-butyldimethylsilyl (tBDMS)] at 2'-O- positions was developed. The oligonucleotides were immobilized within three-dimensional (3-D) hydrogel pads fixed on a glass support. The protective groups preserved the oligoribonucleodes from degradation and were suitable to be removed directly on the microchip when needed, right before its use. These immobilized, deprotected oligoribonucleotides were tested for their interaction with afluorescently labeled oligodeoxyribonucleotide and analyzed for their availability to be cleaved enzymatically by the RNase binase. Stability of tBDMS-protected immobilized oligoribonucleotides after 2.5 years of storage as well as after direct RNase action was also tested. Melting curves of short RNA/DNA hybrids that had formed into gel pads of the microchip were found to exhibit clearly defined S-like shapes, with the melting temperatures in full accordance with those theoretically predicted for the same ionic strength. This approach, based on keeping the protective groups attached to oligoribonucleotides, can be applied for manufacturing any RNA microchips containing immobilized oligoribonucleotides, including microchips with two-dimensional (2-D) features. These RNA microchips can be used to measure thermodynamic parameters of RNA/RNA or RNA/DNA duplexes as well as to study ligand- or protein-RNA interactions.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligorribonucleotídeos/metabolismo , RNA/metabolismo , Sequência de Bases , DNA/metabolismo , Endorribonucleases/metabolismo , Corantes Fluorescentes/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato , Cinética , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes , Oligorribonucleotídeos/genética , TermodinâmicaRESUMO
Eukaryotic translational termination is triggered by polypeptide release factors eRF1, eRF3, and one of the three stop codons at the ribosomal A-site. Isothermal titration calorimetry shows that (i) the separated MC, M, and C domains of human eRF1 bind to eRF3; (ii) GTP binding to eRF3 requires complex formation with either the MC or M + C domains; (iii) the M domain interacts with the N and C domains; (iv) the MC domain and Mg2+ induce GTPase activity of eRF3 in the ribosome. We suggest that GDP binding site of eRF3 acquires an ability to bind gamma-phosphate of GTP if altered by cooperative action of the M and C domains of eRF1. Thus, the stop-codon decoding is associated with the N domain of eRF1 while the GTPase activity of eRF3 is controlled by the MC domain of eRF1 demonstrating a substantial structural uncoupling of these two activities though functionally they are interrelated.
Assuntos
Guanosina Trifosfato/metabolismo , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Calorimetria , Códon de Terminação , GTP Fosfo-Hidrolases/metabolismo , Humanos , TermodinâmicaRESUMO
The eukaryotic class 1 polypeptide chain release factor is a three-domain protein involved in the termination of translation, the final stage of polypeptide biosynthesis. In attempts to understand the roles of the middle domain of the eukaryotic class 1 polypeptide chain release factor in the transduction of the termination signal from the small to the large ribosomal subunit and in peptidyl-tRNA hydrolysis, its high-resolution NMR structure has been obtained. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal. However, the orientation of the functionally critical GGQ loop and neighboring alpha-helices has genuine and noticeable differences in solution and in the crystal. Backbone amide protons of most of the residues in the GGQ loop undergo fast exchange with water. However, in the AGQ mutant, where functional activity is abolished, a significant reduction in the exchange rate of the amide protons has been observed without a noticeable change in the loop conformation, providing evidence for the GGQ loop interaction with water molecule(s) that may serve as a substrate for the hydrolytic cleavage of the peptidyl-tRNA in the ribosome. The protein backbone dynamics, studied using 15N relaxation experiments, showed that the GGQ loop is the most flexible part of the middle domain. The conformational flexibility of the GGQ and 215-223 loops, which are situated at opposite ends of the longest alpha-helix, could be a determinant of the functional activity of the eukaryotic class 1 polypeptide chain release factor, with that helix acting as the trigger to transmit the signals from one loop to the other.
Assuntos
Fatores de Terminação de Peptídeos/química , Aminoacil-RNA de Transferência/química , Ribossomos/química , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Células Eucarióticas/metabolismo , Hidrólise , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We report NMR assignments of the protein backbone of the C-terminal domain (163 a.a.) of human class 1 translation termination factor eRF1. It was found that several protein loop residues exist in two slowly interconverting conformational states.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Fatores de Terminação de Peptídeos/química , Sequência de Aminoácidos , Humanos , Estrutura Terciária de ProteínaRESUMO
GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg2+. We show that (i) eRF3 binds GDP (K(d) = 1.9 microM) and this interaction depends only minimally on the Mg(2+) concentration; (ii) GTP binds to eRF3 (K(d) = 0.5 microM) only in the presence of eRF1 and this interaction depends on the Mg2+ concentration; (iii) GTP displaces GDP from the eRF1*eRF3*GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1*eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1*eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1*eRF3*GTP*Mg2+, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3*eRF1 complex profoundly differ from those of prokaryotic RF3.
Assuntos
Guanosina Trifosfato/metabolismo , Magnésio/metabolismo , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/fisiologia , Proteínas de Bactérias/fisiologia , Calorimetria , Guanosina Difosfato/metabolismo , Humanos , Modelos BiológicosRESUMO
Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes all three termination codons and induces hydrolysis of peptidyl tRNA, eRF3's function remains obscure. Here, we reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits; initiation, elongation, and termination factors; and aminoacyl tRNAs. This allowed us to investigate termination using pretermination complexes assembled on mRNA encoding a tetrapeptide and to propose a model for translation termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. In this model, binding of eRF1, eRF3, and GTP to pretermination complexes first induces a structural rearrangement that is manifested as a 2 nucleotide forward shift of the toeprint attributed to pretermination complexes that leads to GTP hydrolysis followed by rapid hydrolysis of peptidyl tRNA. Cooperativity between eRF1 and eRF3 required the eRF3 binding C-terminal domain of eRF1.
Assuntos
Fatores de Terminação de Peptídeos/química , Biossíntese de Proteínas , Animais , Códon de Terminação , Guanosina Trifosfato/fisiologia , Hidrólise , Modelos Biológicos , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/fisiologia , Ligação Proteica , Subunidades Proteicas/química , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/fisiologia , Coelhos , Ribossomos/química , Ribossomos/fisiologiaRESUMO
Termination of protein synthesis is promoted in ribosomes by proper stop codon discrimination by class 1 polypeptide release factors (RFs). A large set of prokaryotic RFs differing in stop codon specificity, RF1 for UAG and UAA, and RF2 for UGA and UAA, was analyzed by means of a recently developed computational method allowing identification of the specificity-determining positions (SDPs) in families composed of proteins with similar but not identical function. Fifteen SDPs were identified within the RF1/2 superdomain II/IV known to be implicated in stop codon decoding. Three of these SDPs had particularly high scores. Five residues invariant for RF1 and RF2 [invariant amino acid residues (IRs)] were spatially clustered with the highest-scoring SDPs that in turn were located in two zones within the SDP/IR area. Zone 1 (domain II) included PxT and SPF motifs identified earlier by others as 'discriminator tripeptides'. We suggest that IRs in this zone take part in the recognition of U, the first base of all stop codons. Zone 2 (domain IV) possessed two SDPs with the highest scores not identified earlier. Presumably, they also take part in stop codon binding and discrimination. Elucidation of potential functional role(s) of the newly identified SDP/IR zones requires further experiments.
Assuntos
Fatores de Terminação de Peptídeos/química , Algoritmos , Sequência de Aminoácidos , Aminoácidos/química , Códon de Terminação , Biologia Computacional , Modelos Moleculares , Dados de Sequência Molecular , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/classificação , Fatores de Terminação de Peptídeos/metabolismo , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Polypeptide release factors from eubacteria and eukaryotes, although similar in function, belong to different protein families. They share one sequence motif, a GGQ tripeptide that is vital to release factor (RF) activity in both kingdoms. In bacteria, the Gln residue of the motif in RF1 and RF2 is modified to N(5)-methyl-Gln by the S-adenosyl l-methionine-dependent methyltransferase PrmC and the absence of Gln methylation decreases the release activity of Escherichia coli RF2 in vitro severalfold. We show here that the same modification is made to the GGQ motif of Saccharomyces cerevisiae release factor eRF1, the first time that N(5)-methyl-Gln has been found outside the bacterial kingdom. The product of the YDR140w gene is required for the methylation of eRF1 in vivo and for optimal yeast cell growth. YDR140w protein has significant homology to PrmC but lacks the N-terminal domain thought to be involved in the recognition of the bacterial release factors. Overproduced in S. cerevisiae, YDR140w can methylate eRF1 from yeast or man in vitro using S-adenosyl l-methionine as methyl donor provided that eRF3 and GTP are also present, suggesting that the natural substrate of the methyltransferase YDR140w is the ternary complex eRF1.eRF3.GTP.
Assuntos
Glutamina/química , Metiltransferases/fisiologia , Fatores de Terminação de Peptídeos/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Histidina/química , Espectrometria de Massas , Metilação , Metiltransferases/química , Modelos Moleculares , Dados de Sequência Molecular , Fatores de Terminação de Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We searched for chromosome 3p homo- and hemizygous losses in 23 lung cancer cell lines, 53 renal cell and 22 breast carcinoma biopsies using 31 microsatellite markers located in frequently deleted 3p regions. In addition, two sequence-tagged site markers (NLJ-003 and NL3-001) located in the Alu-PCR clone 20 region (AP20) and lung cancer (LUCA) regions, respectively, were used for quantitative real-time PCR (QPCR). We found frequent (10-18%) homozygous deletions (HDs) in both 3p21.3 regions in the biopsies and lung cancer cell lines. In addition, we discovered that amplification of 3p is a very common (15-42.5%) event in these cancers and probably in other epithelial malignancies. QPCR showed that aberrations of either NLJ-003 or NL3-001 were detected in more than 90% of all studied cases. HDs were frequently detected simultaneously both in NLJ-003 or NL3-001 loci in the same tumour (P<3-10(-7)). This observation suggests that tumour suppressor genes (TSG) in these regions could have a synergistic effect. The exceptionally high frequency of chromosome aberrations in NLJ-003 and NL3-001 loci suggests that multiple TSG(s) involved in different malignancies are located very near to these markers. Precise mapping of 15 independent HDs in the LUCA region allowed us to establish the smallest HD region in 3p21.3C located between D3S1568 (CACNA2D2 gene) and D3S4604 (SEMA3F gene). This region contains 17 genes. Mapping of 19 HDs in the AP20 region resulted in the localization of the minimal region to the interval flanked by D3S1298 and D3S3623 markers. Only four genes were discovered in this interval, namely, APRG1, ITGA9, HYA22 and VILL.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Neoplasias Renais/genética , Neoplasias Pulmonares/genética , Deleção de Sequência , Canais de Cálcio/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Pequenas/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 3 , Feminino , Rearranjo Gênico , Marcadores Genéticos , Homozigoto , Humanos , Perda de Heterozigosidade , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase/métodos , Semaforinas , Células Tumorais CultivadasRESUMO
Chromosome 3p21.3 region is frequently (>90%) deleted in lung and other major human carcinomas. We subdivided 3p21.3 into LUCA and AP20 subregions and discovered frequent homozygous deletions (10-18%) in both subregions. This finding strongly implies that they harbor multiple tumor suppressor genes involved in the origin and/or development of major epithelial cancers. In this study, we performed an initial analysis of RBSP3/HYA22, a candidate tumor suppressor genes located in the AP20 region. Two sequence splice variants of RBSP3/HYA22 (A and B) were identified, and we provide evidence for their tumor suppressor function. By sequence analysis RBSP3/HYA22 belongs to a gene family of small C-terminal domain phosphatases that may control the RNA polymerase II transcription machinery. Expression of the gene was drastically (>20-fold) decreased in 11 of 12 analyzed carcinoma cell lines and in three of eight tumor biopsies. We report missense and nonsense mutations in tumors where RBSP3/HYA22 was expressed, growth suppression with regulated transgenes in culture, suppression of tumor formation in severe combined immunodeficient mice, and dephosphorylation of ppRB by RBSP3/HYA22, presumably leading to a block of the cell cycle at the G1/S boundary.
Assuntos
Genes Supressores de Tumor , Proteínas Supressoras de Tumor/genética , Sequência de Aminoácidos , Sequência de Bases , Divisão Celular/genética , Linhagem Celular Tumoral , Metilação de DNA , Primers do DNA , Sondas de DNA , Deleção de Genes , Humanos , Repetições de Microssatélites , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase , Splicing de RNA , Homologia de Sequência de Aminoácidos , Sitios de Sequências Rotuladas , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Positioning of the mRNA codon towards the 18S ribosomal RNA in the A site of human 80S ribosomes has been studied applying short mRNA analogs containing either the stop codon UAA or the sense codon UCA with a perfluoroaryl azide group at the uridine residue. Bound to the ribosomal A site, a modified codon crosslinks exclusively to the 40S subunits under mild UV irradiation. This result is inconsistent with the hypothesis [Ivanov et al. (2001) RNA 7, 1683-1692] which requires direct contact between the large rRNA and the stop codon of the mRNA as recognition step at translation termination. Both sense and stop codons crosslink to the same A1823/A1824 invariant dinucleotide in helix 44 of 18S rRNA. The data point to the resemblance between the ternary complexes formed at elongation (sense codon.aminoacyl-tRNA.AA dinucleotide of 18S rRNA) and termination (stop codon.eRF1.AA dinucleotide of 18S rRNA) steps of protein synthesis and support the view that eRF1 may be considered as a functional mimic of aminoacyl-tRNA.
Assuntos
Códon de Terminação , Códon , RNA Ribossômico 18S/metabolismo , Azidas , Sequência de Bases , Sítios de Ligação , Reagentes de Ligações Cruzadas , Fosfatos de Dinucleosídeos , Oligorribonucleotídeos/metabolismo , Biossíntese de Proteínas/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/química , Aminoacil-RNA de Transferência , RNA de Transferência de FenilalaninaRESUMO
Release factors RF1 and RF2 are required in bacteria for the cleavage of peptidyl-tRNA. A single sequence motif, GGQ, is conserved in all eubacterial, archaebacterial and eukaryotic release factors and may mimic the CCA end of tRNA, although the position of the motif in the crystal structures of human eRF1 and Escherichia coli RF2 is strikingly different. Mutations have been introduced at each of the three conserved positions. Changing the Gln residue to Ala or Glu allowed the factors to retain about 22% of tetrapeptide release activity in vitro, but these mutants could not complement thermosensitive RF mutants in vivo. None of several mutants with altered Gly residues retained activity in vivo or in vitro. Many GGQ mutants were poorly expressed and are presumably unstable; many were also toxic to the cell. The toxic mutant factors or their degradation products may bind to ribosomes inhibiting the action of the normal factor. These data are consistent with a common role for the GGQ motif in bacterial and eukaryotic release factors, despite strong divergence in primary, secondary and tertiary structure, but are difficult to reconcile with the hypothesis that the amide nitrogen of the Gln plays a vital role in peptidyl-tRNA hydrolysis.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Fatores de Terminação de Peptídeos/metabolismo , Proteínas de Bactérias/genética , Western Blotting , Sequência Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/antagonistas & inibidores , Fatores de Terminação de Peptídeos/genética , Transativadores/metabolismoRESUMO
A set of 22 551 unique human NotI flanking sequences (16.2 Mb) was generated. More than 40% of the set had regions with significant similarity to known proteins and expressed sequences. The data demonstrate that regions flanking NotI sites are less likely to form nucleosomes efficiently and resemble promoter regions. The draft human genome sequence contained 55.7% of the NotI flanking sequences, Celera's database contained matches to 57.2% of the clones and all public databases (including non-human and previously sequenced NotI flanks) matched 89.2% of the NotI flanking sequences (identity > or =90% over at least 50 bp, data from December 2001). The data suggest that the shotgun sequencing approach used to generate the draft human genome sequence resulted in a bias against cloning and sequencing of NotI flanks. A rough estimation (based primarily on chromosomes 21 and 22) is that the human genome contains 15 000-20 000 NotI sites, of which 6000-9000 are unmethylated in any particular cell. The results of the study suggest that the existing tools for computational determination of CpG islands fail to identify a significant fraction of functional CpG islands, and unmethylated DNA stretches with a high frequency of CpG dinucleotides can be found even in regions with low CG content.
Assuntos
DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Análise de Sequência de DNA/métodos , Linhagem Celular Transformada , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 22/genética , Ilhas de CpG/genética , DNA/química , DNA/genética , Bases de Dados de Ácidos Nucleicos , Genes/genética , Genoma Humano , Humanos , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3'-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA(Phe), targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3'-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.
Assuntos
Códon de Terminação/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Fator de Iniciação 1 em Eucariotos/química , Fator de Iniciação 1 em Eucariotos/metabolismo , Humanos , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
We have identified a novel human gene UNC93B1 encoding a protein related to unc-93 of Caenorhabditis elegans. The combined sequence derived from several cDNA clones is 2282 bp and comparison with genomic sequence shows that the gene contains 11 exons. The longest open reading frame encodes a deduced sequence of 597 amino acids. Homology analysis shows that the hUNC93B1 gene is highly conserved and related to sequences in Arabidopsis thaliana, C. elegans, Drosophila melanogaster, chicken and mouse. Structural analysis of the deduced amino acid sequence of hUNC93B1 points to possible existence of multiple membrane-spanning domains. hUNC93B1 protein also displays some similarities to the bacterial ABC-2 type transporter signature and to ion transporters of Deinococcus radiodurans and Helicobacter pylori. As revealed by Northern analysis, the level of expression varies significantly between tissues, with the highest level detected in the heart. The gene was mapped to chromosomal band 11q13 by fluorescence in situ hybridization. We suggest that this gene is a member of a novel hUNC93B-related gene family.