Mutants of eukaryotic initiation factor eIF-4E with altered mRNA cap binding specificity reprogram mRNA selection by ribosomes in Saccharomyces cerevisiae.
Recognition of the 5'-end of eukaryotic mRNA by the ribosomal 43 S preinitiation complex involves the eukaryotic translation initiation factor eIF-4E (eIF-4alpha). Deletion mutants of the eIF-4E gene of Saccharomyces cerevisiae (CDC33) encoded proteins with reduced affinity for the 5'-cap. One of these mutant proteins lacked any detectable binding to a cap analogue binding column, yet was still able to support cell growth. More than 17% of the total eIF-4E amino acid sequence could be removed without fully inactivating this factor. At least 30 of the N-terminal amino acids are not essential for function. The minimal functional eIF-4E protein segment therefore comprises at most 176 amino acids. The translation and growth defects of the deletion mutants could be at least partially compensated by increases in eIF-4E synthesis, possibly due to a mass-action effect on mRNA binding. Electroporation of yeast spheroplasts with in vitro synthesized mRNA allowed us to characterize the ability of eIF-4E mutant strains to distinguish between capped and uncapped mRNAs in vivo. Our data show that the cap specificity of eIF-4E determines to what extent the translational apparatus differentiates between capped and uncapped mRNAs and indicate the minimum relative mRNA (cap) binding activity of eIF-4E required for yeast cell viability.
Vasilescu
S
S
Department of Gene Expression, National Biotechnology Research Center, Gessellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Federal Republic of Germany.
Ptushkina
M
M
Linz
B
B
Müller
P P
PP
McCarthy
J E
JE
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D039561:Eukaryotic Initiation Factor-4E; D008969:Molecular Sequence Data; D009154:Mutation; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012333:RNA, Messenger