Affinity labeling of the ribosomal decoding site with an AUG-substrate analog

Area of Science:

• Molecular Biology
• Ribosome Function
• Protein Synthesis

Background:

• The initiation codon AUG is crucial for starting protein synthesis.
• Understanding the precise binding sites of initiation factors and mRNA on ribosomes is essential for deciphering translation regulation.

Purpose of the Study:

• To synthesize and characterize an AUG-affinity label to identify components of the mRNA binding site on Escherichia coli 70S ribosomes.
• To investigate the specific ribosomal proteins that interact with the AUG initiation codon.

Main Methods:

• Chemical synthesis of a bromoacetylated AUG analog.
• Irreversible cross-linking of the AUG analog to 70S ribosomes.
• Isolation of labeled 30S ribosomal subunits.
• Affinity labeling followed by polyacrylamide gel electrophoresis (SDS-PAGE, Sarkosyl, and urea PAGE).

Main Results:

• The AUG-affinity label reacted irreversibly with the mRNA binding site of E. coli 70S ribosomes.
• Labeled 30S subunits were isolated and shown to be programmed for initiation-factor-dependent fMet-tRNAfMet binding.
• The AUG-affinity label was found to be covalently bound to ribosomal proteins S4 (ram gene product) and S18.

Conclusions:

• The AUG-affinity label successfully identified the decoding site for fMet-tRNAfMet on the ribosome.
• Ribosomal proteins S4 and S18 are located at or near the mRNA binding site and are involved in translation initiation.