Does the channel for nascent peptide exist inside the ribosome? Immune electron microscopy study

Area of Science:

• Molecular Biology
• Structural Biology
• Ribosome Function

Background:

• Protein synthesis is a fundamental biological process.
• Ribosomes are the molecular machines responsible for translation.
• Understanding ribosome structure and function is crucial for deciphering gene expression.

Purpose of the Study:

• To visualize the N-terminal ends of nascent peptides on Escherichia coli 70S ribosomes.
• To identify the exit site and pathway of nascent peptides during translation.
• To investigate the accessibility of nascent peptides to antibodies.

Main Methods:

• Cell-free protein synthesis system using MS2 phage RNA.
• Initiation of translation with N-dinitrophenyl derivative of methionyl-tRNAFMet.
• Immune electron microscopy to visualize nascent peptide-ribosome complexes.

Main Results:

• Nascent peptides up to 42 amino acid residues were synthesized with a dinitrophenyl hapten at the N-terminus.
• The N-ends of these nascent peptides were accessible to antibodies.
• The nascent peptide exit site was localized to a specific pocket on the 50S ribosomal subunit, likely the peptidyl transferase center.
• A potential pathway for nascent peptide egress along the 50S subunit surface was identified.

Conclusions:

• The N-terminal ends of short nascent peptides are exposed on the ribosome surface.
• The peptidyl transferase center region of the 50S subunit serves as the exit site for nascent peptides.
• Nascent peptides likely follow a specific groove on the 50S subunit for their pathway out of the ribosome.