Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex.
We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S.eIF3. Met-tRNAf.eIF2.GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf.eIF2.GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.
Chaudhuri
J
J
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Jack and Pearl Resnick Campus, Bronx, New York 10461, USA.
Chowdhury
D
D
Maitra
U
U
D000818:Animals; D001156:Artemia; D002499:Centrifugation, Density Gradient; D015977:Eukaryotic Initiation Factor-1; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3; D014176:Protein Biosynthesis; D012358:RNA, Transfer, Met / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes; D012270:Ribosomes / Q000378:metabolism*
D015977:Eukaryotic Initiation Factor-1; D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D012358:RNA, Transfer, Met; C026022:eukaryotic peptide initiation factor-1A; C089238:fMet-tRNA(fMet)