A shared subunit belongs to the eukaryotic core RNA polymerase

Area of Science:

• Molecular Biology
• Biochemistry
• Yeast Genetics

Background:

• Yeast RNA polymerase I (Pol I) is a complex enzyme with 14 subunits.
• The function of shared subunits, like ABC23, in Pol I structure and activity remains unclear.

Purpose of the Study:

• To investigate the specific role of the ABC23 subunit in yeast RNA polymerase I function.
• To determine if ABC23 is essential for Pol I activity and structural integrity.

Main Methods:

• In vitro reconstitution of an inactive RNA Pol I complex (Pol I delta) lacking ABC23, A43, and A14 subunits.
• Biochemical assays to measure enzyme activity and subunit reassociation.
• Gel retardation and surface plasmon resonance to assess DNA binding.
• Electron microscopy and electrophoretic analysis for structural changes.

Main Results:

• Recombinant ABC23 subunit alone reactivated the inactive Pol I delta enzyme to 50% of wild-type activity.
• ABC23 stably reassociated with the complex and was essential for the first phosphodiester bond formation.
• ABC23 binding induced a significant conformational change, rendering the enzyme transcriptionally competent.
• ABC23 was not involved in DNA binding and did not recycle during transcription.

Conclusions:

• The ABC23 subunit is indispensable for the structural and functional integrity of yeast RNA polymerase I.
• ABC23 should be considered a core component of the RNA Pol I enzyme, essential for transcription initiation.