The interaction between eukaryotic initiation factor 1A and eIF5 retains eIF1 within scanning preinitiation complexes

Biochemistry. 2013 Dec 31;52(52):9510-8. doi: 10.1021/bi4009775. Epub 2013 Dec 19.

Abstract

Scanning of the mRNA transcript by the preinitiation complex (PIC) requires a panel of eukaryotic initiation factors, which includes eIF1 and eIF1A, the main transducers of stringent AUG selection. eIF1A plays an important role in start codon recognition; however, its molecular contacts with eIF5 are unknown. Using nuclear magnetic resonance, we unveil eIF1A's binding surface on the carboxyl-terminal domain of eIF5 (eIF5-CTD). We validated this interaction by observing that eIF1A does not bind to an eIF5-CTD mutant, altering the revealed eIF1A interaction site. We also found that the interaction between eIF1A and eIF5-CTD is conserved between humans and yeast. Using glutathione S-transferase pull-down assays of purified proteins, we showed that the N-terminal tail (NTT) of eIF1A mediates the interaction with eIF5-CTD and eIF1. Genetic evidence indicates that overexpressing eIF1 or eIF5 suppresses the slow growth phenotype of eIF1A-NTT mutants. These results suggest that the eIF1A-eIF5-CTD interaction during scanning PICs contributes to the maintenance of eIF1 within the open PIC.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Eukaryotic Initiation Factor-1 / chemistry
  • Eukaryotic Initiation Factor-1 / genetics
  • Eukaryotic Initiation Factor-1 / metabolism*
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Initiation Factors / chemistry
  • Peptide Initiation Factors / genetics
  • Peptide Initiation Factors / metabolism*
  • Protein Binding
  • Protein Biosynthesis
  • Protein Multimerization
  • Protein Structure, Tertiary
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Alignment

Substances

  • Eukaryotic Initiation Factor-1
  • Peptide Initiation Factors
  • RNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins
  • eukaryotic peptide initiation factor-1A
  • eukaryotic translation initiation factor 5A