Thermosensitive mutations affecting ribonucleic acid polymerases in Saccharomyces cerevisiae

Area of Science:

• Molecular Biology
• Yeast Genetics
• Biochemistry

Background:

• Ribonucleic acid (RNA) synthesis is crucial for cellular function.
• Temperature-sensitive mutants are valuable tools for studying essential genes.
• Saccharomyces cerevisiae is a model organism for eukaryotic gene research.

Purpose of the Study:

• To investigate the genetic basis of RNA synthesis in Saccharomyces cerevisiae.
• To identify and characterize temperature-sensitive mutants affecting RNA synthesis.
• To explore the potential for common subunits among RNA polymerases.

Main Methods:

• Isolation and characterization of 150 temperature-sensitive Saccharomyces cerevisiae mutants.
• Detailed analysis of four mutants exhibiting drastic defects in RNA synthesis.
• Genetic analysis including complementation tests and segregation studies.
• Biochemical assays including in vivo and in vitro RNA synthesis, thermodenaturation, and polyacrylamide gel electrophoresis.

Main Results:

• Fifteen mutants were specifically affected in RNA synthesis, with four showing severe defects.
• In these four mutants, RNA synthesis was immediately blocked at the nonpermissive temperature (37°C).
• Deoxyribonucleic acid synthesis increased by approximately 40% in the mutants.
• Mutant RNA polymerases showed significantly reduced activity in vitro and in vivo, with shorter half-lives at elevated temperatures.
• All species of RNA synthesis were found to be thermosensitive.
• The four mutants belonged to three complementation groups, indicating three distinct genes.

Conclusions:

• The study identified three distinct genes essential for the activity of Saccharomyces cerevisiae RNA polymerases.
• The results strongly support the hypothesis that the three RNA polymerases share common subunits.
• These findings contribute to understanding the regulation and structure of RNA synthesis machinery in eukaryotes.