A multicomponent mitochondrial RNA polymerase from Saccharomyces cerevisiae

Area of Science:

• Molecular Biology
• Yeast Genetics
• Enzymology

Background:

• Mitochondrial gene expression relies on specific RNA polymerases.
• Previous studies identified a mitochondrial RNA polymerase in purified yeast mitochondria.
• The precise nature and components of this polymerase were not fully elucidated.

Purpose of the Study:

• To detect and characterize selective RNA polymerase activity in whole cell extracts of Saccharomyces cerevisiae.
• To determine if this activity recognizes mitochondrial promoter sequences.
• To separate and analyze the components responsible for selective transcription.

Main Methods:

• In vitro transcription assays using whole cell yeast extracts.
• Chromatographic separation techniques to isolate RNA polymerase activities.
• Characterization of enzyme properties using different DNA templates (poly[d(AT)] and cloned mitochondrial promoters).

Main Results:

• A selective RNA polymerase activity recognizing mitochondrial promoters was detected in yeast whole cell extracts.
• This activity was chromatographically separated from nuclear RNA polymerases (I, II, and III).
• The selective RNA polymerase activity was resolved into two nonfunctional components, which restored activity upon recombination, suggesting a multi-subunit enzyme or complex.

Conclusions:

• Yeast whole cell extracts contain a distinct RNA polymerase activity capable of selective transcription of mitochondrial promoters.
• This activity is separable from nuclear RNA polymerases and appears to be composed of at least two distinct components.
• Further investigation is needed to determine if the poly[d(AT)] polymerase activity represents a nonspecific component or a separate enzyme within the selective transcription apparatus.