RESUMO
Eukaryotic translation initiation requires unwinding of secondary structures in the 5'-untranslated region of mRNA. The DEAD-box helicase eIF4A is thought to unwind structural elements in the 5'-UTR in conjunction with eIF4G and eIF4B. Both factors jointly stimulate eIF4A activities by modulation of eIF4A conformational cycling between open and closed states. Here we examine how RNA substrates modulate eIF4A activities. The RNAs fall into two classes: Short RNAs only partially stimulate the eIF4A ATPase activity, and closing is rate-limiting for the conformational cycle. By contrast, longer RNAs maximally stimulate ATP hydrolysis and promote closing of eIF4A. Strikingly, the rate constants of unwinding do not correlate with the length of a single-stranded region preceding a duplex, but reach a maximum for RNA with a single-stranded region of six nucleotides. We propose a model in which RNA substrates affect eIF4A activities by modulating the kinetic partitioning of eIF4A between futile, unproductive, and productive cycles.
Assuntos
Regiões 5' não Traduzidas , Adenosina Trifosfatases/química , Fator de Iniciação 4F em Eucariotos/química , RNA Helicases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Clonagem Molecular , Fator de Iniciação 4F em Eucariotos/genética , Hidrólise , Cinética , Nucleotídeos/genética , Domínios Proteicos , RNA/genética , RNA Helicases/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Eukaryotic translation initiation starts with binding of the eIF4F complex to the 5'-m7G cap of the mRNA. Recruitment of the 43S pre-initiation complex (PIC), formed by the 40S ribosomal subunit and other translation initiation factors, leads to formation of the 48S PIC that then scans the 5'-untranslated region (5'-UTR) toward the start codon. The eIF4F complex consists of eIF4E, the cap binding protein, eIF4A, a DEAD-box RNA helicase that is believed to unwind secondary structures in the 5'-UTR during scanning, and eIF4G, a scaffold protein that binds to both eIF4E and eIF4A. The ATPase and helicase activities of eIF4A are jointly stimulated by eIF4G and the translation initiation factor eIF4B. Yeast eIF4B mediates recruitment of the 43S PIC to the cap-bound eIF4F complex by interacting with the 40S subunit and possibly with eIF4A. However, a direct interaction between yeast eIF4A and eIF4B has not been demonstrated yet. Here we show that eIF4B binds to eIF4A in the presence of RNA and ADPNP, independent of the presence of eIF4G. A stretch of seven moderately conserved repeats, the r1-7 region, is responsible for complex formation, for modulation of the conformational energy landscape of eIF4A by eIF4B, and for stimulating the RNA-dependent ATPase- and ATP-dependent RNA unwinding activities of eIF4A. The isolated r1-7 region only slightly stimulates eIF4A conformational changes and activities, suggesting that communication of the repeats with other regions of eIF4B is required for full stimulation of eIF4A activity, for recruitment of the PIC to the mRNA and for translation initiation.
Assuntos
Adenosina Trifosfatases/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Ácido Nucleico , Regiões 5' não Traduzidas , Sítios de Ligação , Ativação Enzimática , Fator de Iniciação 4A em Eucariotos/química , Fatores de Iniciação em Eucariotos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/metabolismo , RNA Mensageiro/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic translation initiation factor eIF4A is a DEAD-box helicase that resolves secondary structure elements in the 5'-UTR of mRNAs during ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. DEAD-box proteins alternate between open and closed conformations during RNA unwinding. The transition to the closed conformation is linked to duplex destabilization. eIF4A is a special DEAD-box protein that can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by the translation initiation factor eIF4G and a closed state in the presence of eIF4G and eIF4B. We show here that eIF4A alone does not measurably sample the closed conformation. The translation initiation factors eIF4B and eIF4G accelerate the eIF4A conformational cycle. eIF4G increases the rate of closing more than the opening rate, and eIF4B selectively increases the closing rate. Strikingly, the rate constants and the effect of eIF4B are different for different RNAs, and are related to the presence of single-stranded regions. Modulating the kinetics of the eIF4A conformational cycle is thus central for the multi-layered regulation of its activity, and for its role as a regulatory hub in translation initiation.