Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription

Abstract

The regulation of RNA synthesis by RNA polymerase (RNAP) is essential for proper gene expression. Crystal structures of RNAP reveal two channels: the main channel that contains the downstream DNA and a secondary channel that leads directly to the catalytic site. Although nucleoside triphosphates (NTPs) have been seen only in the catalytic site and the secondary channel in these structures, several models of transcription elongation, based on biochemical studies, propose that template-dependent binding of NTPs in the main channel regulates RNA synthesis. These models, however, remain controversial. We used transient state kinetics and a mutant of RNAP to investigate the role of the main channel in regulating nucleotide incorporation. Our data indicate that a NTP specific for the i þ 2 template position can bind to a noncatalytic site and increase the rate of RNA synthesis and that the NTP bound to this site can be shuttled directly into the catalytic site. We also identify fork loop 2, which lies across from the downstream DNA, as a functional component of this site. Taken together, our data support the existence of a noncatalytic template-specific NTP binding site in the main channel that is involved in the regulation of nucleotide incorporation. NTP binding to this site could promote high-fidelity processive synthesis under a variety of environmental conditions and allow DNA sequence-mediated regulatory signals to be communicated to the active site.T he central role of RNA polymerase (RNAP) in transcription is to catalyze the processive synthesis of the growing RNA transcript with high-fidelity and at reasonable rates. This process is regulated both intrinsically, by RNA and DNA sequence elements, and extrinsically, by nucleotide availability and proteins that interact with the elongating RNAP (1-10). These interactions modulate the conformations of the RNAP ternary elongation complexes (RNAP, DNA, and RNA), which, in turn, affect the rate and fidelity of nucleotide addition and the response of RNAP to regulatory signals. The rate of nucleotide incorporation and the recognition of pause and termination signals by RNAP can be greatly influenced by the sequence of downstream DNA, as well as by the RNA transcript. Subtle changes in the sequence of the downstream DNA can result in dramatic changes in the rates of nucleotide incorporation and the efficiencies of pausing and termination (3, 10-15). The mechanism(s) by which the downstream DNA regulates these processes remains a mystery; however, it has been suggested that nucleoside triphosphates (NTPs) may interact with the downstream DNA to modulate nucleotide incorporation (10,16,17).Crystal structures of prokaryotic and eukaryotic RNAPs reveal two channels: the main channel, which is filled with the downstream DNA, and the secondary channel, which is a negatively charged, funnel-shaped pore that leads from the surface of the enzyme to the active site. NTPs have been observed bound in the catalytic site and secondary channel, but not in the main channel, and there...

Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase

“…There is a strong correlation between the stable sequestration of the cognate NTP in the RNAP II active site and the magnitude of the incoming NTP effect on the pre- to post-translocated border transition in the exonuclease III assay [20,36], showing that the post-translocated state is stabilized by binding of the cognate NTP in the active site [8]. Alternatively, the apparent stimulation of translocation might reflect an allosteric effect of the incoming NTP previously proposed for mammalian RNAP II [4,37] and Ec RNAP [6,7,38]. …”

“…Two models have been put forward for translocation. In one model, incoming NTP substrates act as allosteric effectors driving forward translocation [3-7]. In another model, thermally driven oscillations of RNAP elements stimulate nucleic acids to slide forward [8-11].…”

Basic mechanisms of RNA polymerase II activity and alteration of gene expression in Saccharomyces cerevisiae

“…Second, the location of a templated, non-hydrolysable NTP analog present in a conformation not thought to efficiently support catalysis has been interpreted as a pre-insertion (PI) site [25]. Third biochemical data from kinetic analyses on RNAP and Pol II examining next-nucleotide-dependent substrate effects on transcription have been interpreted to support pre-loading of substrates base-pairing with the DNA template in positions downstream of the active site [26-28]. Binding of substrates in downstream templated positions may allosterically affect msRNAP activity without necessarily transferring those exact substrates to the active site.…”

Structural basis of transcription elongation

“…In addition, a non-templated NTP site was observed [47], but its functional significance remains unclear. Further, a DNA-templated NTP site at position + 2, downstream of the active site, was proposed based on kinetic studies [65,66], but was detected neither biochemically nor structurally thus far.…”