Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo.
RNA
; 6(10): 1380-92, 2000 Oct.
Article
em En
| MEDLINE
| ID: mdl-11073214
ABSTRACT
The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein-RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1-2, 3, and 5 of the IRES did not contribute to this interaction. In agreement with this, binding to domain 4 alone was as efficient as to the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient to interact with the IRES or to its domain 4 alone. Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 3' region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Iniciação Traducional da Cadeia Peptídica
/
Ribossomos
/
RNA Viral
/
Fatores de Iniciação de Peptídeos
/
Sequências Reguladoras de Ácido Nucleico
/
Proteínas de Ligação a RNA
/
Aphthovirus
/
Fatores de Iniciação em Eucariotos
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2000
Tipo de documento:
Article