eIF1 Loop 2 interactions with Met-tRNA_i control the accuracy of start codon selection by the scanning preinitiation...

Area of Science:

• Molecular Biology
• Protein Structure and Function
• Gene Expression Regulation

Background:

• The eukaryotic 43S preinitiation complex (PIC) scans mRNA to find the start codon for protein synthesis.
• Initiation involves a ternary complex (TC) of eukaryotic initiation factor 2 (eIF2)-GTP and initiator methionyl transfer RNA (Met-tRNAi).
• eIF1 plays a critical role in start codon selection by modulating PIC conformation and TC binding.

Purpose of the Study:

• To investigate the structural and functional role of eIF1 Loop 2 in the context of the PIC.
• To determine how interactions between eIF1 Loop 2 and Met-tRNAi influence start codon recognition and selection.
• To explore the potential of eIF1 Loop 2 mutations to enhance translation initiation at suboptimal start codons.

Main Methods:

• Site-directed mutagenesis of eIF1 Loop 2 residues (e.g., D71A, M74A).
• Reconstitution of PICs with wild-type and mutant eIF1.
• Biochemical assays to assess TC binding and PIC stability.
• In vivo translation assays to evaluate start codon selection efficiency.

Main Results:

• Alanine substitutions in eIF1 Loop 2 increased translation initiation at both near-cognate UUG and AUG codons in poor contexts.
• Specific double substitutions (D71A-M74A) stabilized TC binding to 48S PICs with UUG start codons.
• Arginine substitutions in Loop 2 showed even stronger effects, stabilizing the closed PIC conformation (P_IN state).
• Loop 2 substitutions did not affect the rate of TC loading onto scanning 40S subunits in vivo.

Conclusions:

• Interactions between eIF1 Loop 2 and the Met-tRNAi D loop specifically impede Met-tRNAi accommodation in the P_IN state.
• Mutations in eIF1 Loop 2 can overcome this impediment, stabilizing the P_IN state.
• These modifications enhance the selection of suboptimal start codons, offering insights into translational control mechanisms.