A temperature-sensitive mutation affecting the mammalian 60 S ribosome

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Protein synthesis is essential for cell function.
• Temperature-sensitive mutants provide valuable tools for studying cellular processes.
• Ribosomes are complex molecular machines responsible for protein biosynthesis.

Purpose of the Study:

• To characterize the protein biosynthetic machinery of a temperature-sensitive Chinese hamster cell mutant, ts14.
• To identify the specific component responsible for the temperature-sensitive defect in protein synthesis.
• To elucidate structure-function relationships within the eukaryotic ribosome using a genetic approach.

Main Methods:

• Cell-free extracts from ts14 and wild-type cells were used for protein synthesis assays.
• Fractionation and complementation experiments were performed on mutant extracts.
• Sucrose gradient sedimentation analysis was employed to study ribosome stability.
• High ionic strength buffer treatments were used to assess ribosomal subunit integrity.

Main Results:

• ts14 cell extracts ceased protein synthesis at 40°C, unlike wild-type extracts.
• A thermolabile component associated with polyribosomes was identified in ts14.
• Complementation experiments revealed that the defect resides in the 60S ribosomal subunit.
• Mutant 60S ribosomal subunits showed instability, preferentially disrupting in high ionic strength buffers.

Conclusions:

• The ts14 mutation specifically affects the stability of the 60S ribosomal subunit.
• This genetic defect provides insights into the structural integrity and function of eukaryotic ribosomes.
• A genetic approach is effective for dissecting ribosome structure-function relationships.