Multiple active centers of multi-subunit RNA polymerases

Abstract

The active center of multi-subunit RNA polymerase consists of two modules, the Mg(2+) module, holding the catalytic Mg(2+) ion, and a module made of a flexible domain, the Trigger Loop. Uniquely, the TL module can be substituted by alternative modules, thus changing the catalytic properties of the active center.

Figure 1. Switching of RNAP active centers. (A) Substitution of the flexible TL in the active center of RNAP by Gfh1 and Gre factors. Left top: the “TL module” is absent from the active center when the TL (dark gray) adopts its open conformation (PDB 2BE5; parts of β and β’ forming “Mg²⁺” module of the active center are in beige and brown spacefill, respectively; RNA is yellow; Mg²⁺I is green sphere). Right top: the “TL active center” is formed when the TL (gray) adopts its closed conformation (PDB 2O5J). Left bottom: the “Gfh1 active center” (PDB 3A0H) is formed when Gfh1 (light gray) substitutes for the TL (dark gray from PDB 2O5J) in the active center. Right bottom: “Gre active center” is formed when the TL (dark gray) is substituted by Gre factor (light gray). The structure of E. coli GreB (PDB 2P4V) was fitted into the structure of Gfh1 bound to the elongation complex (PDB 3A0H). (B) The Gre factor stays bound to the elongation complex after the cleavage reaction is completed (for protein purification, oligonucleotides, elongation complex assembly see ref. 14). Gel at the top: electrophoretic mobility shift assay of elongation complex EC15 assembled with T. aquaticus RNAP, with radioactively labeled non-template strand (black asterisk). Heparin was added to reactions to 100 µg/mL final concentration. Where specified, MgCl₂ was added to 10 mM final concentration. T. aquaticus Gre was added in a two molar excess over the fully assembled elongation complexes for 1 min (concentration of assembled elongation complex was determined by quantification of the amount of the non-template strand that was incorporated in the complex). Gre in the presence of Mg²⁺ converts EC15 to EC13, which is resistant to Gre. Complexes were supplied with 10% glycerol, resolved in a 6% Tris-glycin polyacrylamide gel (19:1), and revealed by PhosphorImaging (GE Healthcare). The mobility of EC13/Gre was slightly different from that of EC13 (not shown). The presence of RNAP and factors in the complexes was confirmed by separation of the cut out complexes by 4–15% gradient SDS PAGE (bottom panel). After electrophoresis, the gel was silver stained. (C) Switching of the active centers of RNAP. In response to some signals, the “TL module” (green) of the active center can be substituted by various “alternative modules” thus converting “TL active center” into corresponding “alternative active centers” (crescents of different colors). “Alternative modules” may stay permanently associated with RNAP, or bind RNAP from solution in response to various signals.