Translation arrest requires two-way communication between a nascent polypeptide and the ribosome

Area of Science:

• Molecular Biology
• Protein Synthesis
• Biochemistry

Background:

• Bacterial protein export is crucial for cellular function.
• Translation arrest is a regulatory mechanism in protein synthesis.
• The Escherichia coli SecM protein is involved in protein export and exhibits regulated translation arrest.

Purpose of the Study:

• To elucidate the mechanism of translation arrest induced by the E. coli SecM protein.
• To investigate the role of the C-terminal motif in SecM-mediated translation arrest.
• To understand the conformational changes and ribosome interactions during arrest.

Main Methods:

• Utilized fluorescence resonance energy transfer (FRET) to monitor protein conformation.
• Synthesized and analyzed the SecM protein with its C-terminal arrest motif.
• Performed puromycin sensitivity assays on arrested peptidyl-tRNA.
• Characterized changes in the ribosome-nascent SecM complex.

Main Results:

• Synthesis of the SecM arrest motif induced a compact C-terminal conformation.
• This compaction was ribosome-dependent, not spontaneous.
• Translation arrest required both the compact conformation and specific residues in the motif.
• Arrested peptidyl-tRNA showed resistance to puromycin.
• Observed additional alterations in the ribosome-nascent SecM complex.

Conclusions:

• SecM translation arrest results from reciprocal interactions between the ribosome and the nascent polypeptide C terminus.
• The ribosome actively induces conformational changes in SecM that lead to arrest.
• Specific residues within the arrest motif are critical for the arrest mechanism.