A conformational change in the eukaryotic translation preinitiation complex and release of eIF1 signal recognition of the start codon.
During eukaryotic translation initiation, ribosomal 43S complexes scan mRNAs for the correct AUG codon at which to begin translation. Start codon recognition triggers GTP hydrolysis, committing the complex to engagement at that point on the mRNA. While fidelity at this step is essential, the nature of the codon recognition event and the mechanism by which it activates GTP hydrolysis are poorly understood. Here we report the changes that occur within the 43S.mRNA complex in response to AUG codon recognition. eIF1 and eIF1A are key players in assembly of 43S.mRNA complexes capable of locating initiation codons. We observed FRET between these two factors when bound to the 40S subunit. Using steady-state FRET, anisotropy, and kinetic analyses, we demonstrate that start codon recognition results in a conformational change and release of eIF1 from the ribosome. These rearrangements probably play a role in triggering GTP hydrolysis and committing the complex to downstream events.
Maag
David
D
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Fekete
Christie A
CA
Gryczynski
Zygmunt
Z
Lorsch
Jon R
JR
D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1 / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D008968:Molecular Conformation; D010442:Peptide Chain Initiation, Translational; D011485:Protein Binding; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D021122:Protein Subunits; D012333:RNA, Messenger; D012235:Rhodamines; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate