RESUMO
In eukaryotic ribosomes, the conserved protein uS19, formerly known as S15, extends with its C-terminal tail to the decoding site. The cross-linking of uS19 to the A site codon has been detected using synthetic mRNAs bearing 4-thiouridine (s4U) residues. Here, we showed that the A-site tRNA prevents this cross-linking and that the P site codon does not contact uS19. Next, we focused on determining uS19-mRNA interactions in vivo by applying the photoactivatable-ribonucleoside enhancing cross-linking and immunoprecipitation method to a stable HEK293 cell line producing FLAG-tagged uS19 and grown in a medium containing s4U. We found that when translation was stopped by cycloheximide, uS19 was efficiently cross-linked to mRNA regions with a high frequency of Glu, Lys and, more rarely, Arg codons. The results indicate that the complexes, in which the A site codon is not involved in the formation of the mRNA-tRNA duplex, are present among the cycloheximide-arrested 80S complexes, which implies pausing of elongating ribosomes at the above mRNA regions. Thus, our findings demonstrate that the human ribosomal protein uS19 interacts with mRNAs during translation elongation and highlight the regions of mRNAs where ribosome pausing occurs, bringing new structural and functional insights into eukaryotic translation in vivo.
Assuntos
RNA Mensageiro/química , Proteínas Ribossômicas/química , Ribossomos/química , Códon , Eucariotos/genética , Células HEK293 , Humanos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA de Transferência/química , RNA de Transferência/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Tiouridina/químicaRESUMO
Translation termination in eukaryotes is mediated by release factors: eRF1, which is responsible for stop codon recognition and peptidyl-tRNA hydrolysis, and GTPase eRF3, which stimulates peptide release. Here, we have utilized ribose-specific probes to investigate accessibility of rRNA backbone in complexes formed by association of mRNA- and tRNA-bound human ribosomes with eRF1â¢eRF3â¢GMPPNP, eRF1â¢eRF3â¢GTP, or eRF1 alone as compared with complexes where the A site is vacant or occupied by tRNA. Our data show which rRNA ribose moieties are protected from attack by the probes in the complexes with release factors and reveal the rRNA regions increasing their accessibility to the probes after the factors bind. These regions in 28S rRNA are helices 43 and 44 in the GTPase associated center, the apical loop of helix 71, and helices 89, 92, and 94 as well as 18S rRNA helices 18 and 34. Additionally, the obtained data suggest that eRF3 neither interacts with the rRNA ribose-phosphate backbone nor dissociates from the complex after GTP hydrolysis. Taken together, our findings provide new information on architecture of the eRF1 binding site on mammalian ribosome at various translation termination steps and on conformational rearrangements induced by binding of the release factors.
Assuntos
Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , RNA Mensageiro/química , RNA Ribossômico 18S/química , RNA Ribossômico 28S/química , Aminoacil-RNA de Transferência/química , Sítios de Ligação , Códon de Terminação , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Conformação de Ácido Nucleico , Fatores de Terminação de Peptídeos/genética , Placenta/química , Gravidez , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Ribosomal protein eL42 (formerly known as L36A), a small protein of the large (60S) subunit of the eukaryotic ribosome, is a component of its exit (E) site. The residue K53 of this protein resides within the motif QSGYGGQTK mainly conserved in eukaryotes, and it is located in the immediate vicinity of the CCA-terminus of the ribosome-bound tRNA in the hybrid P/E state. To examine the role of this eL42 motif in translation, we obtained HEK293T cells producing the wild-type FLAG-tagged protein or its mutant forms with either single substitutions of conserved amino acid residues in the above motif, or simultaneous replacements in positions 45 and 51 or 45 and 53. Examination of the level of exogenous eL42 in fractions of polysome profiles from the target protein-producing cells by the Western blotting revealed that neither single substitution affects the assembly of 60S ribosomal subunits and 80S ribosomes or critically decreases the level of polysomes, but the latter was observed with the double replacements. Analysis of tRNAs bound to 80S ribosomes containing eL42 with double substitutions and examination their peptidyl transferase activity enabled estimation the stage of the elongation cycle, in which amino acid residues of the conserved eL42 motif are involved. We clearly show that cooperative interactions implicating the eL42 residues Q45, Q51, and K53 play a critical role in the ability of the human ribosome to perform properly elongation cycle at the step of deacylated tRNA dissociation from the E site in the human cell.
Assuntos
Proteínas Ribossômicas , Ribossomos , Humanos , Proteínas Ribossômicas/metabolismo , Células HEK293 , Ribossomos/metabolismo , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Aminoácidos/metabolismoRESUMO
Positioning of release factor eRF1 toward adenines and the ribose-phosphate backbone of the UAAA stop signal in the ribosomal decoding site was studied using messenger RNA (mRNA) analogs containing stop signal UAA/UAAA and a photoactivatable cross-linker at definite locations. The human eRF1 peptides cross-linked to these analogs were identified. Cross-linkers on the adenines at the 2nd, 3rd or 4th position modified eRF1 near the conserved YxCxxxF loop (positions 125-131 in the N domain), but cross-linker at the 4th position mainly modified the tripeptide 26-AAR-28. This tripeptide cross-linked also with derivatized 3'-phosphate of UAA, while the same cross-linker at the 3'-phosphate of UAAA modified both the 26-28 and 67-73 fragments. A comparison of the results with those obtained earlier with mRNA analogs bearing a similar cross-linker at the guanines indicates that positioning of eRF1 toward adenines and guanines of stop signals in the 80S termination complex is different. Molecular modeling of eRF1 in the 80S termination complex showed that eRF1 fragments neighboring guanines and adenines of stop signals are compatible with different N domain conformations of eRF1. These conformations vary by positioning of stop signal purines toward the universally conserved dipeptide 31-GT-32, which neighbors guanines but is oriented more distantly from adenines.
Assuntos
Adenina/química , Códon de Terminação/química , Guanina/química , Fatores de Terminação de Peptídeos/química , Humanos , Modelos Moleculares , Terminação Traducional da Cadeia Peptídica , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/química , Proteínas Ribossômicas/químicaRESUMO
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
The protein eS26 is a structural component of the eukaryotic small ribosomal subunit involved in the formation of the mRNA binding channel in the region of the exit site. By applying site-directed cross-linking to mammalian 80S ribosomes, it has been shown that the same mRNA nucleotide residues are implicated in the interaction with both eS26 and translation initiation factor 3 (eIF3) and that contacts of the protein with mRNAs are mediated by its eukaryote-specific motif YxxPKxYxK. To examine the role of eS26 in translation, we transfected HEK293T cells with plasmid constructs encoding the wild-type FLAG-labeled protein (wt-eS26FLAG) or its forms with either a single substitution of any conserved amino acid residue in the above motif, or a simultaneous replacement of all the five ones (5A). The western blot analysis of fractions of polysome profiles from the transfected cells revealed no effects of the single mutations in eS26, but showed that the replacement of the five conserved residues led to the increased share of the light polysome fraction compared to that detected with control, wt-eS26FLAG-producing cells. In addition, the above fraction exhibited the enhanced content of the eIF3e subunit that is known to promote selective translation. These findings, together with real-time PCR data on the relative contents of specific mRNAs in light and heavy polysomes from cells producing the mutant 5A compared to those from control cells, suggest a possible involvement of the YxxPKxYxK motif of eS26 in the fine regulation of translation to maintain the required balance of synthesized proteins.
Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Animais , Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/genética , Células HEK293 , Humanos , Mamíferos/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/química , Ribossomos/genéticaRESUMO
The conformation of mRNA in the region of the human 80S ribosome decoding site was monitored using 11-mer mRNA analogues that bore nitroxide spin labels attached to the terminal nucleotide bases. Intramolecular spin-spin distances were measured by DEER/PELDOR spectroscopy in model complexes mimicking different states of the 80S ribosome during elongation and termination of translation. The measurements revealed that in all studied complexes, mRNA exists in two alternative conformations, whose ratios are different in post-translocation, pre-translocation and termination complexes. We found that the presence of a tRNA molecule at the ribosomal A site decreases the relative share of the more extended mRNA conformation, whereas the binding of eRF1 (alone or in a complex with eRF3) results in the opposite effect. In the termination complexes, the ratios of mRNA conformations are practically the same, indicating that a part of mRNA bound in the ribosome channel does not undergo significant structural alterations in the course of completion of the translation. Our results contribute to the understanding of mRNA molecular dynamics in the mammalian ribosome channel during translation.
RESUMO
Here we employed site-directed cross-linking with the application of tRNA and mRNA analogues bearing an oxidized ribose at the 3'-terminus to investigate mutual arrangement of the main components of translation termination complexes formed on the human 80S ribosome bound with P site deacylated tRNA using eRF1â¢eRF3â¢GTP or eRF1 alone. In addition, we applied a model complex obtained in the same way with eRF1â¢eRF3â¢GMPPNP. We found that eRF3 content in the complexes with GTP and GMPPNP is similar, proving that eRF3 does not leave the ribosome after GTP hydrolysis. Our cross-linking data allowed determining locations of the 3'-terminus of the P site tRNA relatively the eRF1 M domain and of the mRNA stop signal toward the N domain and the ribosomal decoding site at the nucleotide-peptide resolution level. Our results indicate that locations of these components do not change after peptide release up to post-termination pre-recycling state, and the positioning of the mRNA stop signal remains similar to that when eRF1 recognizes it. Besides, we found that in all the complexes studied eRF1 shielded the N-terminal part of ribosomal protein eS30 from the interaction with the nucleotide adjacent to stop codon observed with pre-termination ribosome free of eRFs. Altogether, our findings brought important information on contacts of the key structural elements of eRF1, tRNA and mRNA in the ribosomal complexes including those mimicking different translation termination steps, thereby providing a deeper understanding of molecular mechanisms underlying events occurring in the course of protein synthesis termination in mammals.
Assuntos
Códon de Terminação/genética , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Humanos , Ligação Proteica/genética , Proteínas Ribossômicas/genéticaRESUMO
Ribosomal proteins neighboring the mRNA downstream of the codon bound at the decoding site of human 80S ribosomes were identified using three sets of mRNA analogues that contained a UUU triplet at the 5' terminus and a perfluorophenylazide cross-linker at guanosine, adenosine or uridine residues placed at various locations 3' of this triplet. The positions of modified mRNA nucleotides on the ribosome were governed by tRNA(Phe) cognate to the UUU triplet targeted to the P site. Upon mild UV-irradiation, the mRNA analogues cross-linked preferentially to the 40S subunit, to the proteins and to a lesser extent to the 18S rRNA. Cross-linked nucleotides of 18S rRNA were identified previously. In the present study, it is shown that among the proteins the main target for cross-linking with all the mRNA analogues tested was protein S3 (homologous to prokaryotic S3, S3p); minor cross-linking to protein S2 (S5p) was also detected. Both proteins cross-linked to mRNA analogues in the ternary complexes as well as in the binary complexes (without tRNA). In the ternary complexes protein S15 (S19p) also cross-linked, the yield of the cross-link decreased significantly when the modified nucleotide moved from position +5 to position +12 with respect to the first nucleotide of the P site bound codon. In several ternary complexes minor cross-linking to protein S30 was likewise detected. The results of this study indicate that S3 is a key protein at the mRNA binding site neighboring mRNA downstream of the codon at the decoding site in the human ribosome.
Assuntos
RNA/genéticaRESUMO
Protein uL2 is essential for the catalytic activity of the ribosome and has a conserved shape in ribosomes from all domains of life. However, the sequence of its unstructured C-terminal loop apex that contacts the conserved 23S/28S rRNA helix (H) 93 near the ribosomal peptidyl transferase center differs in bacteria, archaea and eukaryotes. Eukaryote-specific residue His216 located in this loop in mammalian uL2 is hydroxylated in ribosomes. We used a set of chemical probes to explore the structure of an RNA that mimicked a segment of 28S rRNA domain V containing part of the uL2 binding site including H93, complexed with either natural (hydroxylated) or recombinant (unmodified) human uL2. It was found that both protein forms engage H93 during binding, but only natural uL2 (uL2n) protects it from hydroxyl radicals. The association of uL2n with RNA leads to changes in its structure at U4532 adjacent to the universally conserved U4531 (U2585, Escherichia coli numbering) involved in peptidyl transferase center formation, and at the universally conserved C4447 (2501) located in the ribosome near A4397 (2451) and C3909 (2063) belonging to the peptidyl transferase center. As a result, both nucleotides become strongly exposed to hydroxyl radicals. Our data argue that the hydroxyl group at His216 in the C-terminal loop apex of mammalian uL2 contributes to stabilization of a protein conformation that is favorable for binding to H93 of 28S rRNA and that this binding induces structural rearrangement in the regions close to the peptidyl transferase center in the mature ribosome.
Assuntos
Histidina/química , Peptidil Transferases/metabolismo , Placenta/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Feminino , Células HeLa , Humanos , Hidroxilação , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Gravidez , RNA Ribossômico 28S , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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
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.