View the Current Issue

Article

  • The EMBO Journal (2005) 24, 3588 - 3601
  • doi:10.1038/sj.emboj.7600821

Published online: 29 September 2005

The eIF1A C-terminal domain promotes initiation complex assembly, scanning and AUG selection in vivo

Christie A Fekete1, Drew J Applefield2, Stephen A Blakely1, Nikolay Shirokikh3, Tatyana Pestova3, Jon R Lorsch2 and Alan G Hinnebusch1

  1. Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA
  2. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA
  3. Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, Brooklyn, NY, USA

Correspondence to:

Alan G Hinnebusch, Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Building 6A/Room B1A-13, Bethesda, MD 20892, USA. Tel.: +1 301 496 4480; Fax: +1 301 496 6828; E-mail: ahinnebusch@nih.gov

Received 2 June 2005; Accepted 26 August 2005

Translation initiation factor 1A stimulates 40S-binding of the eukaryotic initiation factor 2 (eIF2)/GTP/Met-tRNAiMet 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.

  • Keywords:

    • eIF1A,
    • eIF2,
    • GCN4,
    • scanning,
    • translation