RESUMEN

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.

Asunto(s)

Proteínas Ribosómicas , Ribosomas , Humanos , Proteínas Ribosómicas/metabolismo , Células HEK293 , Ribosomas/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia/metabolismo , Aminoácidos/metabolismo

RESUMEN

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.

Asunto(s)

Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo , Animales , Eucariontes/genética , Factor 3 de Iniciación Eucariótica/genética , Células HEK293 , Humanos , Mamíferos/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/química , Ribosomas/genética

RESUMEN

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.

RESUMEN

The eukaryotic ribosomal protein uS19 has a C-terminal tail that is absent in its bacterial homologue. This tail has been shown to be involved in the formation of the decoding site of human ribosomes. We studied here the previously unexplored functional significance of the 15 C-terminal amino acid residues of human uS19 for the assembly of ribosomes and translation using HEK293-based cell cultures capable of producing FLAG-labeled uS19 (uS19FLAG) or its mutant form deprived of the mentioned amino acid ones. The examination of polysome profiles of cytoplasmic extracts from the respective cells revealed that the deletion of the above uS19 amino acid residues barely affected the assembly and maturation of 40S subunits and the initiation of translation, but completely prevented the formation of polysomes. This implied the crucial importance of the uS19 tail in the elongation process. Analysis of tRNAs associated with 40S subunits and 80S ribosomes containing wild type uS19FLAG or its truncated form showed that the deletion of the C-terminal pentadecapeptide fragment of uS19 did not interfere with the binding of aminoacyl-tRNA (aa-tRNA) at the ribosomal A site. The results led to the conclusion that the transpeptidation, which occurs on the large ribosomal subunit after decoding the A site codon by the incoming aa-tRNA, is the most likely elongation stage, where this uS19 fragment can play a critical role. Our findings suggest that the uS19 tail is a keystone player in the accommodation of aa-tRNA at the A site, which is a pre-requisite for the peptide transfer.

Asunto(s)

Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Secuencia de Aminoácidos , Células HEK293 , Humanos , Polirribosomas/metabolismo , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Eliminación de Secuencia

RESUMEN

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.

Asunto(s)

ARN Mensajero/química , Proteínas Ribosómicas/química , Ribosomas/química , Codón , Eucariontes/genética , Células HEK293 , Humanos , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Tiouridina/química

RESUMEN

The GGQ minidomain of the ribosomal protein eL42 was previously shown to contact the CCA-arm of P-site bound tRNA in human ribosome, indicating a possible involvement of the protein in the catalytic activity. Here, using Schizosaccharomyces pombe (S. pombe) cells, we demonstrate that the GGQ minidomain and neighboring region of eL42 is critical for the ribosomal function. Mutant eL42 proteins containing amino acid substitutions within or adjacent to the GGQ minidomain failed to complement the function of wild-type eL42, and expression of the mutant eL42 proteins led to severe growth defects. These results suggest that the mutations in eL42 interfere with the ribosomal function in vivo. Furthermore, we show that some of the mutations associated with the conserved GGQ region lead to reduced activities in the poly(Phe) synthesis and/or in the peptidyl transferase reaction with respect to puromycin, as compared with those of the wild-type ribosomes. A pK value of 6.95 was measured for the side chain of Lys-55/Arg-55, which is considerably less than that of a Lys or Arg residue. Altogether, our findings suggest that eL42 contributes to the 80S ribosome's peptidyl transferase activity by promoting the course of the elongation cycle.

Asunto(s)

Mutación Missense , Extensión de la Cadena Peptídica de Translación/fisiología , Proteínas Ribosómicas , Ribosomas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Sustitución de Aminoácidos , Catálisis , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Ribosomas/genética , Ribosomas/metabolismo , Schizosaccharomyces/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo

RESUMEN

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 eRF1•eRF3•GTP or eRF1 alone. In addition, we applied a model complex obtained in the same way with eRF1•eRF3•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.

Asunto(s)

Codón de Terminación/genética , Terminación de la Cadena Péptídica Traduccional/genética , Factores de Terminación de Péptidos/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Ribosomas/genética , Humanos , Unión Proteica/genética , Proteínas Ribosómicas/genética

RESUMEN

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 eRF1•eRF3•GMPPNP, eRF1•eRF3•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.

Asunto(s)

Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos/metabolismo , ARN Mensajero/química , ARN Ribosómico 18S/química , ARN Ribosómico 28S/química , Aminoacil-ARN de Transferencia/química , Sitios de Unión , Codón de Terminación , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Conformación de Ácido Nucleico , Factores de Terminación de Péptidos/genética , Placenta/química , Embarazo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Ribosómico 28S/genética , ARN Ribosómico 28S/metabolismo , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo

RESUMEN

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.

Asunto(s)

Histidina/química , Peptidil Transferasas/metabolismo , Placenta/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Femenino , Células HeLa , Humanos , Hidroxilación , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Embarazo , ARN Ribosómico 28S , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

We have demonstrated previously that the E-site specific protein RPL36AL present in human ribosomes can be crosslinked with the CCA-end of a P-tRNA in situ. Here we report the following: (i) We modeled RPL36AL into the structure of the archaeal ortholog RPL44E extracted from the known X-ray structure of the 50S subunit of Haloarcula marismortui. Superimposing the obtained RPL36AL structure with that of P/E tRNA observed in eukaryotic 80S ribosomes suggested that RPL36AL might in addition to its CCA neighbourhood interact with the inner site of the tRNA elbow similar to an interaction pattern known from tRNA•synthetase pairs. (ii) Accordingly, we detected that the isolated recombinant protein RPL36AL can form a tight binary complex with deacylated tRNA, and even tRNA fragments truncated at their CCA end showed a high affinity in the nanomolar range supporting a strong interaction outside the CCA end. (iii) We constructed programmed 80S complexes containing the termination factor eRF1 (stop codon UAA at the A-site) and a 2',3'-dialdehyde tRNA (tRNAox) analog at the P-site. Surprisingly, we observed a crosslinked ternary complex containing the tRNA, eRF1 and RPL36AL crosslinked both to the aldehyde groups of tRNAox at the 2'- and 3'-positions of the ultimate A. We also demonstrated that, upon binding to the ribosomal A-site, eRF1 induces an alternative conformation of the ribosome and/or the tRNA, leading to a novel crosslink of tRNAox to another large-subunit ribosomal protein (namely L37) rather than to RPL36AL, both ribosomal proteins being labeled in a mutually exclusive fashion. Since the human 80S ribosome in complex with P-site bound tRNAox and A-site bound eRF1 corresponds to the post-termination state of the ribosome, the results represent the first biochemical evidence for the positioning of the CCA-arm of the P-tRNA in close proximity to both RPL36AL and eRF1 at the end of the translation process.

RESUMEN

Nucleotides of 28S rRNA involved in binding of the human 80S ribosome with acceptor ends of the A site and the P site tRNAs were determined using two complementary approaches, namely, cross-linking with application of tRNA(Asp) analogues substituted with 4-thiouridine in position 75 or 76 and hydroxyl radical footprinting with the use of the full sized tRNA and the tRNA deprived of the 3'-terminal trinucleotide CCA. In general, these 28S rRNA nucleotides are located in ribosomal regions homologous to the A, P and E sites of the prokaryotic 50S subunit. However, none of the approaches used discovered interactions of the apex of the large rRNA helix 80 with the acceptor end of the P site tRNA typical with prokaryotic ribosomes. Application of the results obtained to available atomic models of 50S and 60S subunits led us to a conclusion that the A site tRNA is actually present in both A/A and A/P states and the P site tRNA in the P/P and P/E states. Thus, the present study gives a biochemical confirmation of the data on the structure and dynamics of the mammalian ribosomal pretranslocation complex obtained with application of cryo-electron microscopy and single-molecule FRET [Budkevich et al., 2011]. Moreover, in our study, particular sets of 28S rRNA nucleotides involved in oscillations of tRNAs CCA-termini between their alternative locations in the mammalian 80S ribosome are revealed.

Asunto(s)

Biosíntesis de Proteínas , ARN de Transferencia de Aspártico/química , Ribosomas/química , Tiouridina/química , Animales , Emparejamiento Base , Sitios de Unión , Reactivos de Enlaces Cruzados , Microscopía por Crioelectrón , Transferencia Resonante de Energía de Fluorescencia , Humanos , Secuencias Invertidas Repetidas/genética , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , ARN Ribosómico 28S , ARN de Transferencia de Aspártico/genética , ARN de Transferencia de Aspártico/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Tiouridina/metabolismo

RESUMEN

Previously we have shown that the CCA end of a P-tRNA can be crosslinked with the RPL36AL protein of the large subunit of mammalian ribosomes; it belongs to the L44e protein family present in all eukaryotic and archaeal ribosomes. Here we confirm and extend this finding and demonstrate that: 1) this crosslink is specific for a tRNA at the P/E hybrid site, as a tRNA in all other tRNA positions of pre-translocational ribosomes could not be crosslinked with a ribosomal protein, 2) the crosslink was formed most efficiently with C74 and C75 of P/E-tRNA, but could also connect the ultimate A of this tRNA with Lys53 of protein RPL36AL, 3) this protein contains seven monomethylated residues (three lysyl and three arginyl residues, as well as glutaminyl residue 51), 4) Q51 is part of a conserved GGQ motif in the L44e proteins in eukaryotic 80S ribosomes that is identical to the universally conserved motif of release factors implicated in promoting peptidyl-tRNA hydrolysis, and 5) the large number of modifications, in which some of the residues were methylated to about 50 %, might indicate that protein RPL36AL is a preferential target for regulation.

Asunto(s)

Lisina/química , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/química , Proteínas Ribosómicas/química , Ribosomas/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Bovinos , Reactivos de Enlaces Cruzados , Humanos , Lisina/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Aminoacil-ARN de Transferencia/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

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.

Asunto(s)

Adenina/química , Codón de Terminación/química , Guanina/química , Factores de Terminación de Péptidos/química , Humanos , Modelos Moleculares , Terminación de la Cadena Péptídica Traduccional , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/química , Proteínas Ribosómicas/química

RESUMEN

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.

Asunto(s)

Codón de Terminación/genética , Codón de Terminación/metabolismo , Factores de Terminación de Péptidos/metabolismo , Secuencia de Bases , Reactivos de Enlaces Cruzados , Humanos , Técnicas In Vitro , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Terminación de la Cadena Péptídica Traduccional , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Conformación Proteica , Estructura Terciaria de Proteína , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribosomas/genética , Ribosomas/metabolismo

RESUMEN

The eukaryotic ribosomal protein S15 is a key component of the decoding site in contrast to its prokaryotic counterpart, S19p, which is located away from the mRNA binding track on the ribosome. Here, we determined the oligopeptide of S15 neighboring the A site mRNA codon on the human 80S ribosome with the use of mRNA analogues bearing perfluorophenyl azide-modified nucleotides in the sense or stop codon targeted to the 80S ribosomal A site. The protein was cross-linked to mRNA analogues in specific ribosomal complexes that were obtained in the presence of eRF1 in the experiments with mRNAs bearing stop codon. Digestion of modified S15 with various specific proteolytic agents followed by identification of the resulting modified oligopeptides showed that cross-link was in C-terminal fragment in positions 131-145, most probably, in decapeptide 131-PGIGATHSSR-140. The position of cross-linking site on the S15 protein did not depend on the nature of the A site-bound codon (sense or stop codon) and on the presence of polypeptide chain release factor eRF1 in the ribosomal complexes with mRNA analogues bearing a stop codon. The results indicate an involvement of the mentioned decapeptide in the formation of the ribosomal decoding site during elongation and termination of translation. Alignment of amino acid sequences of eukaryotic S15 and its prokaryotic counterpart, S19p from eubacteria and archaea, revealed that decapeptide PGIGATHSSR in positions 131-140 is strongly conserved in eukaryotes and has minor variations in archaea but has no homology with any sequence in C-terminal part of eubacterial S19p, which suggests involvement of the decapeptide in the translation process in a eukaryote-specific manner.

Asunto(s)

Codón/metabolismo , Eucariontes , Biosíntesis de Proteínas , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Archaea , Codón/genética , Bromuro de Cianógeno/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie

RESUMEN

Periodate-oxidized tRNA (tRNAox), the 2',3'-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the binding site for the 3'-end of tRNA on human 80S ribosomes. When human 80S ribosomes were reacted with tRNA(Asp)ox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins (rPs) was observed. The majorily labeled protein was identified as the large subunit rP L36a-like (RPL36AL) by means of mass spectrometry. Intact tRNA(Asp) competed with tRNA(Asp)ox for the binding to the P-site, by preventing tRNA-protein cross-linking with RPL36AL. Altogether, the data presented in this report are consistent with the presence of RPL36AL at or near the binding site for the CCA end of the tRNA substrate positioned at the P-site of human 80S ribosomes. It is the first time that a ribosomal protein is found in an intimate contact (i.e. at a zero-distance) with a nucleotide of the conserved CCA terminus of P-site tRNA which is the substrate of peptidyl transferase reaction. RPL36AL which is strongly conserved in eukaryotes belongs to the L44e family of rPs, a representative of which is Haloarcula marismortui RPL44e.

Asunto(s)

Sitios de Unión , ARN de Transferencia/química , Proteínas Ribosómicas/genética , Emparejamiento Base , Escherichia coli/genética , Humanos , Modelos Moleculares , Proteínas Ribosómicas/metabolismo

RESUMEN

Long synthetic mRNAs were used to study the positioning of the E site codon, the 2nd and 3rd nucleotides of the A site bound codon and a nucleotide 3' of this codon with respect to the 18S rRNA in the human 80S ribosome. The mRNAs contained a GAC triplet coding for Asp and a single 4-thiouridine residue (s(4)U) upstream or downstream of the GAC codon. In the presence of tRNA(Asp), the GAC codon of the mRNAs was targeted to the ribosomal P site thus placing s(4)U in one of the following positions -3, -2, -1, +5, +6 or +7 with respect to the first nucleotide of the P site bound codon. It was found that mRNAs that bore s(4)U in positions +5 to +7 cross-linked to the 18S rRNA nucleotides C1696, C1698 and 1820-1825, the distribution of cross-links among these targets depending on the position of s(4)U. Cross-links of mRNAs containing s(4)U in positions -3 to -1 were found in the region 1699-1704 of the 18S rRNA. In the absence of tRNA, all mRNAs cross-linked only to C1696 and C1698. Absence of the cross-linked nucleotides C1696 and C1698 in the case of mRNAs containing s(4)U in positions -3 to -1 confirmed that tRNA(Asp) actually phased the mRNA on the ribosome.

Asunto(s)

Codón/genética , ARN Mensajero/química , ARN Ribosómico 18S/química , Ribosomas/química , Tiouridina/química , Secuencia de Bases , Sitios de Unión/genética , Codón/química , Reactivos de Enlaces Cruzados/química , Humanos , ARN Ribosómico 18S/genética , ARN de Transferencia de Aspártico/química , ARN de Transferencia de Aspártico/genética , Ribosomas/genética

RESUMEN

This study is directed towards an important problem concerning the organization of the peptidyl transferase center (PTC) on the mammalian ribosome that cannot be studied by X-ray analysis since crystals of 80S ribosomes are still unavailable. Here, we investigated the arrangement of the 3'-end of tRNA in the 80S ribosomal A and P sites using a tRNA(Asp) analogue that bears a 4-thiouridine (s(4)U) attached to the 3'-terminal adenosine. It was shown that an additional nucleotide s(4)U77 on the 3'-end does not impede codon-dependent binding of the tRNA to the A and P sites of 80S ribosome. Mild UV-irradiation of the ribosomal complexes containing a short appropriately designed mRNA and the tRNA analogue resulted in cross-linking of the analogue exclusively to 28S rRNA. The cross-linking site was detected in the 4302-4540 fragment of the 28S rRNA which belongs to the highly conserved domain V that in prokaryotic ribosomes is involved in the formation of the PTC. Nucleotides cross-linked to the tRNA analogue were determined by means of reverse transcription. A comparison of the results obtained with a dynamic model of mutual arrangement of s(4)U77 of the A site tRNA and nucleotides of 23S rRNA built on the basis of an atomic model for the prokaryotic PTC led to the conclusion that environments of the tRNA 3'-terminus in prokaryotic and eukaryotic ribosomes share a significant extent of similarity, although pronounced differences are also detectable.

Asunto(s)

Conformación de Ácido Nucleico , ARN de Transferencia de Aspártico/química , ARN de Transferencia de Fenilalanina/química , Ribosomas/metabolismo , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , ARN Mensajero/química , ARN Ribosómico 23S/química , ARN Ribosómico 28S/química

RESUMEN

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.

Asunto(s)

ARN/genética

RESUMEN

Messenger RNA analogues (42-mers) containing a GAC codon (aspartic acid) in the middle of their sequence followed by a s(4)UGA stop codon were used to identify the components of the human ribosomal A site in direct contact with the photoactivatable 4-thiouridine (s(4)U) residue. We compared the behavior of the nonphased ribosome-mRNA complex, (-)tRNA(Asp), to the one of the phased complex, (+)tRNA(Asp), in the absence and in the presence of eRF1, the eukaryotic class 1 translation termination factor of human origin. The patterns of cross-links obtained for the three complexes were similar to those previously reported for rabbit ribosomes [Chavatte, L., et al. (2001) Eur. J. Biochem. 268, 2896-2904]. Cross-links involving proteins S2, S3, S3a, and S30 were poorly dependent on the presence of tRNA(Asp) and eRF1. Cross-linking to nucleotide C1696 of 18S rRNA occurred in all complexes, but its yield was at least two times higher in the phased complex with an empty A site than in the nonphased complex or when the A site was occupied by eRF1. In contrast, protein S15 cross-linked only in the phased complex in the absence of eRF1. The data obtained point to notable differences in organization of the decoding site between mammalian and prokaryotic ribosomes and to large internal mobility of the components of the tRNA (eRF1)-free A site.

Asunto(s)

Codón/genética , Citosina/química , ARN Ribosómico 18S/química , Proteínas Ribosómicas/genética , Ribosomas/química , Secuencia de Bases , Sitios de Unión/genética , Codón/química , Reactivos de Enlaces Cruzados/química , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , ARN Mensajero/química , ARN Ribosómico 18S/genética , ARN de Transferencia de Aspártico/química , ARN de Transferencia de Aspártico/genética , Proteínas Ribosómicas/química , Ribosomas/genética , Moldes Genéticos , Tiouridina/química