Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes.
Eukaryotic translation is initiated following binding of ribosomes either to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are both mediated by eukaryotic initiation factor 4F (eIF4F), which consists of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G subunits. Here we present a functional analysis of eIF4F which defines the subunits and subunit domains necessary for its function in initiation mediated by the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified translation components and lacking eIF4A and -4F, IRES-mediated initiation did not require the cap-binding protein eIF4E but was absolutely dependent on eIF4A and the central third of eIF4G. This central domain of eIF4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation codon in an ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES. The central domain of eIF4G was itself UV cross-linked to the IRES and strongly stimulated UV cross-linking of eIF4A to the IRES in conjunction with either eIF4B or with the carboxy-terminal third of eIF4G.
Pestova
T V
TV
Department of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York Health Science Center at Brooklyn, 11203, USA.
Shatsky
I N
IN
Hellen
C U
CU
D004686:Encephalomyocarditis virus / Q000235:genetics*; D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics*; D017421:Sequence Analysis
D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D012367:RNA, Viral