The eIF1A C-terminal domain promotes initiation complex assembly, scanning and AUG selection in vivo.
Translation initiation factor 1A stimulates 40S-binding of the eukaryotic initiation factor 2 (eIF2)/GTP/Met-tRNA(iMet) ternary complex (TC) and promotes scanning in vitro. eIF1A contains an OB-fold present in bacterial IF1 plus N- and C-terminal extensions. Truncating the C-terminus (deltaC) or mutating OB-fold residues (66-70) of eIF1A reduced general translation in vivo but increased GCN4 translation (Gcd- phenotype) in a manner suppressed by overexpressing TC. Consistent with this, both mutations diminished 40S-bound TC, eIF5 and eIF3 in vivo, and deltaC impaired TC recruitment in vitro. The assembly defects of the OB-fold mutation can be attributed to reduced 40S-binding of eIF1A, whereas deltaC impairs eIF1A function on the ribosome. A substitution in the C-terminal helix (98-101) also reduced 43S assembly in vivo. Rather than producing a Gcd- phenotype, however, 98-101 impairs GCN4 derepression in a manner consistent with defective scanning by reinitiating ribosomes. Indeed, 98-101 allows formation of aberrant 48S complexes in vitro and increases utilization of non-AUG codons in vivo. Thus, the OB-fold is crucial for ribosome-binding and the C-terminal domain of eIF1A has eukaryotic-specific functions in TC recruitment and scanning.
Fekete
Christie A
CA
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA.
Applefield
Drew J
DJ
Blakely
Stephen A
SA
Shirokikh
Nikolay
N
Pestova
Tatyana
T
Lorsch
Jon R
JR
Hinnebusch
Alan G
AG
D018387:Codon, Initiator / Q000378:metabolism*; D015977:Eukaryotic Initiation Factor-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D017434:Protein Structure, Tertiary / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1; D029701:Saccharomyces cerevisiae Proteins; C026022:eukaryotic peptide initiation factor-1A