Multisubunit RNA Polymerase Cleavage Factors Modulate the Kinetics and Energetics of Nucleotide Incorporation: An RNA Polymerase I Case Study

3 years ago

Multisubunit RNA Polymerase Cleavage Factors Modulate the Kinetics and Energetics of Nucleotide Incorporation: An RNA Polymerase I Case Study

Francis D. Appling, David A. Schneider, Aaron L. Lucius

All cellular RNA polymerases are influenced by protein factors that stimulate RNA polymerase-catalyzed cleavage of the nascent RNA. Despite divergence in amino acid sequence, these so-called “cleavage factors” appear to share a common mechanism of action. Cleavage factors associate with the polymerase through a conserved structural element of the polymerase known as the secondary channel or pore. This mode of association enables the cleavage factor to reach through the secondary channel into the polymerase active site to reorient the active site divalent metal ions. This reorientation converts the polymerase active site into a nuclease active site. Interestingly, eukaryotic RNA polymerases I and III (Pols I and III, respectively) have incorporated their cleavage factors as bona fide subunits known as A12.2 and C11, respectively. Although it is clear that A12.2 and C11 dramatically stimulate the polymerase’s cleavage activity, it is not known if or how these subunits affect the polymerization mechanism. In this work we have used transient-state kinetic techniques to characterize a Pol I isoform lacking A12.2. Our data clearly demonstrate that the A12.2 subunit profoundly affects the kinetics and energetics of the elementary steps of Pol I-catalyzed nucleotide incorporation. Given the high degree of conservation between polymerase–cleavage factor interactions, these data indicate that cleavage factor-modulated nucleotide incorporation mechanisms may be common to all cellular RNA polymerases.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00370

DOI: 10.1021/acs.biochem.7b00370