RESUMEN
In this work, we studied how the accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel, ribosomal protein (rp) uS3 and helix (h) 16 of the 18S rRNA, changes upon the translation initiation. In particular, we examined the accessibility of rp uS3 for binding of unstructured RNAs and of riboses in h16 towards attack with benzoyl cyanide (BzCN) in complexes assembled in rabbit reticulocyte lysate utilizing synthetic oligoribonucleotides as well as full-length and truncated up to the initiation AUG codon hepatitis C virus IRES as model mRNAs. With both mRNA types, the rp uS3 peptide recognizing single-stranded RNAs was shown to become shielded only in those 48S preinitiation complexes (PICs) that contained eIF3j bound to 40S subunit in the area between the decoding site and the mRNA entry channel. Chemical probing with BzCN revealed that h16 in the 48S PICs containing eIF3j or scanning factor DHX29 is strongly shielded; the effect was observed with all the mRNAs used, and h16 remained protected as well in 80S post-initiation complexes lacking these factors. Altogether, the obtained results allowed us to suggest that eIF3j bound at the 48S PICs makes the rp uS3 inaccessible for binding of RNAs and this factor subunit is responsible for the decrease of h16 conformational flexibility; the latter is manifested as reduced accessibility of h16 to BzCN. Thus, our findings provide new insights into how eIF3j is implicated in ensuring the proper conformation of the mRNA entry channel, thereby facilitating mRNA loading.
Asunto(s)
Mamíferos/genética , Iniciación de la Cadena Peptídica Traduccional/genética , ARN Mensajero/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Animales , Secuencia de Bases , Codón Iniciador/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Biosíntesis de Proteínas/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 18S/genética , Reticulocitos/metabolismoRESUMEN
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.
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/metabolismoRESUMEN
In this work, intimate contacts of riboses of mRNA stretch from nucleotides in positions +3 to +12 with respect to the first nucleotide of the P site codon were studied using cross-linking of short mRNA analogs with oxidized 3'-terminal riboses bound to human ribosomes in the complexes stabilized by codon-anticodon interactions and in the binary complexes. It was shown that in all types of complexes cross-links of the mRNA analogs to ribosomal protein (rp) uS3 occur and the yield of these cross-links does not depend on the presence of tRNA and on sequences of the mRNA analogs. Site of the mRNA analogs cross-linking in rp uS3 was mapped to the peptide in positions 55-64 that is located away from the mRNA binding site. Additionally, in complexes with P site-bound tRNA, riboses of mRNA nucleotides in positions +4 to +7 cross-linked to the C-terminal tail of rp uS19 displaying a contact specific to the decoding site of the mammalian ribosome, and tRNA bound at the A site completely blocked this cross-linking. Remarkably, rps uS3 and uS19 were also able to cross-link to the fragment of HCV IRES containing unstructured 3'-terminal part restricted by the AUGC tetraplet with oxidized 3'-terminal ribose. However, no cross-linking to rp uS3 was observed in the 48S preinitiation complex assembled in reticulocyte lysate with this HCV IRES derivative. The results obtained show an ability of rp uS3 to interact with single-stranded RNAs. Possible roles of rp uS3 region 55-64 in the functioning of ribosomes are discussed.