Kinetic Investigation of Escherichia coli RNA Polymerase Mutants That Influence Nucleotide Discrimination and Transcription Fidelity

Abstract

Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue ␤Asp 675 , which is >10Å away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues ␤Arg 678 and ␤Asp 814 do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the ␤Asp 675 mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.High resolution structures of multisubunit RNA polymerases (RNAPs) 4 have offered insight into many of the regulatory stages and conformational changes associated with the transcription cycle (1-14). Multiple structures of both prokaryotic and eukaryotic RNAPs have revealed a funnel-shaped pore (termed the secondary channel) leading from the surface of the enzyme directly to the active site. This channel has been proposed to be the major, and perhaps only, pathway for entry of nucleoside triphosphates (NTPs) to the active site (1-11, 13, 15).Recent structures of the yeast RNAP II elongation complex revealed that binding of an incorrect NTP for synthesis was at a site (termed the E site) adjacent to the catalytic NTP binding site (termed the A site) with the position of the base in the E site inverted and pointing out into the secondary channel (6). Comparison of structures with the correct and incorrect nucleotides led to the proposal of a two-step model of NTP binding, in which an incoming NTP first binds to the E site and then rotates through a narrow negatively charged pore (13) into the A site where it pairs with the template base (6). An NTP bound in the E site was also suggested to inhibit backtracking (13). This two-step model, however, does not allow for direct base pairing between the incoming NTP and the template base until the incoming NTP is positioned in the active site. Cramer and co-workers (11) observed an NTP analog in a third site in which the incoming NTP was base-paired with the DNA template base but not positioned for catalysis and suggested that this site was a preinsertion (PS) site. They proposed a mecha...

RNA Polymerase Active Center: The Molecular Engine of Transcription

“…In addition to the TL, some secondary channel residues have been implicated in nucleotide discrimination (45), consistent with the hypothesis that NTPs enter through the secondary channel. In contrast, the main-channel model of NTP entry postulates that, prior to the induced-fit step of NTP selection in the active site, NTP substrates are prescreened in the main channel by pairing with the i+2 and i+3 DNA bases, which are separated from the active site by the BH (85,145).…”

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

“…β-R678 binds to the nascent RNA 3′-end and orients it for nucleotide addition, and β-R1106 stabilizes the incoming NTP (22,23). RNAP complexes with alanine substitutions at either residue retained DksA affinity (SI Appendix, Fig.…”

Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

“…1C). This interesting result may be attributable to simultaneous templated binding of multiple NTPs [7, 48–52] and/or to allosteric effects of NTPs [53, 54]. …”