DNA-dependent RNA polymerase III from cauliflower. Characterization and template specificity

Area of Science:

• Molecular Biology
• Plant Biochemistry
• Enzymology

Background:

• Class III DNA-dependent RNA polymerase (EC 2.7.7.6) plays a crucial role in transcription.
• Understanding plant RNA polymerases is essential for deciphering gene regulation in crops like cauliflower (Brassica oleracea).

Purpose of the Study:

• To highly purify Class III RNA polymerase from cauliflower.
• To characterize its enzymatic activity, template specificity, and binding affinities.
• To compare cauliflower enzyme properties with those of mammalian counterparts.

Main Methods:

• Enzyme purification using polyethyleneimine precipitation.
• Glycerol gradient sedimentation for determining sedimentation coefficient.
• Inhibition assays with alpha-amanitin.
• Kinetic analysis (Km value determination).
• Template specificity assays using synthetic polynucleotides (homopolymers and their pairs).

Main Results:

• Highly purified cauliflower RNA polymerase III exhibited specific activity comparable to mammalian enzymes.
• Sedimentation analysis revealed a sedimentation coefficient of 23 S.
• The enzyme showed preference for pyrimidine homopolymers, particularly poly(dT), and transcribed both strands of synthetic homopolymer pairs.
• Alpha-amanitin inhibited the enzyme at high concentrations (200 μg/ml).
• Binding and template specificities differed for synthetic polymers, suggesting sequence discrimination.

Conclusions:

• Cauliflower RNA polymerase III is a distinct enzyme with properties similar to mammalian Class III enzymes.
• The enzyme possesses specific binding and template recognition mechanisms, enabling discrimination among different DNA sequences.
• Findings contribute to understanding eukaryotic transcription and gene regulation in plants.