The +2 NTP Binding Drives Open Complex Formation in T7 RNA Polymerase

Abstract

The efficiency of transcription is regulated to produce the required amount of RNA in the cell, and this can occur in numerous ways. Transcriptional efficiency is intrinsically regulated by the promoter DNA sequence during initiation through complex protein-DNA interactions. In addition, several extrinsic factors such as accessory proteins or ligands that bind to the RNAP 1 -DNA complex regulate transcription by allosteric mechanisms. The efficiency of transcription is also regulated by the concentration of substrate NTPs when the cellular NTP concentration is close to the K d of NTPs. This is found in Escherichia coli RNAP-rRNA promoter complexes, such as the rrnB P1 promoters, that have relatively weak affinity for initiating NTP, and thus rRNA synthesis is controlled by the initiating NTP concentration that varies with the cellular growth rate (1). The bacteriophage T7 RNAP is similar in that it also has a relatively weak affinity for initiating NTPs; hence, transcription is regulated by the initiating NTP concentration (2-4). It is interesting that RNAP-promoter complexes with the same initiation start sequence but different promoter core sequences respond differently to initiating NTP concentrations, because they show different affinities for initiating NTPs (3, 5). In these promoters, the template sequence at the initiation site is the same, and thus the interactions of the incoming NTP with the template and RNAP are expected to be the same; thus, it is not obvious how the core sequence regulates the affinity of initiating NTP.Both rrn P1 and T7 promoters show a common feature, and that is an unfavorable equilibrium constant for closed to open complex formation in the absence of initiating NTP. The observed K d of the initiating NTP is dependent on the efficiency of steps preceding NTP binding, such as promoter binding and opening; hence, these steps can potentially modulate the K d of the initiating NTP. Kinetic studies of T7 RNAP have shown that the observed rate of promoter opening is fast (Ͼ100 s Ϫ1 ) in the absence of initiating NTP (6 -8). However, both kinetic and structural studies suggest that the equilibrium constant of the promoter opening step is unfavorable. Fluorescence studies indicate that open complex formation is incomplete even at RNAP and DNA concentrations much above the K d of the T7 RNAP-DNA complex (8). Similarly, much of the Ϫ4 to Ϫ1 TATA region of the T7 dsDNA promoter that melts in the open complex cannot be modified by KMnO 4 , unless initiating nucleotides are present (9 -11).Incomplete conversion of closed to open complex is also observed in the rrn P1 promoters of E. coli RNAP. A closed complex between E. coli RNAP with rrn P1 promoter is formed readily, but it is not stoichiometrically converted to the open complex in the absence of initiating nucleotides (1,12,13). The presence of ATP and CTP, the initiating nucleotides, is required to both make the complex heparin-resistant and open the Ϫ10 dsDNA region (1,12,13). The instability of the open complex may be the reason for t...

Functional Architecture of T7 RNA Polymerase Transcription Complexes

“…An enzyme mutated in the downstream positive patch behaved like the WT RNAP in the 100 and 800 mM NaCl conditions (lanes 9-12), but reproducibly exhibited less transcript release in 0 mM NaCl (lanes 7-8). Enzymes mutated in the upstream positively charged region showed increased transcript release, with a quadruply substituted enzyme releasing most transcripts under all 3 NaCl conditions (lanes 13-18) and a doubly substituted enzyme releasing most of the transcripts in the 100 and 800 mM NaCl reactions (lanes [19][20][21][22][23][24] …”

“…Effects of mutations in up-and downstream positively charged regions on EC stability. A: Transcripts (13mers) retained ("R") or freed ("F") from halted ECs formed with WT (lanes 1-6), K711C/K713E/K714E (lanes 7-12), K407E/K494E/K332E/K412C (lanes 13-18), or K407E/K412E (lanes 19-24) mutant T7RNAPs in the presence of 0 mM (lanes 1,2,7,8,13,14,19,20), 100 mM (lanes 3,4,9,10,15,16,21,22), or 800 mM (lanes 5,6,11,12,17,18,23,24) NaCl. B: Ratio of Free/Retained transcripts from the experiment in A plotted vs. enzyme and NaCl concentration (error bars give ranges from n=2).…”

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase

“…All of these species are poised at position +3 of the template. Alternatively, addition of 3′dGTP to the above complexes allows binding of the first two substrates into positions +1 and +2 of the active site (without phosphodiester bond formation) and has been shown to drive binding and melting to completion (20). Crystal structures with and without a three-base transcript show no significant change in the complex structure near the active site and the fluorescence measurements following addition of 3′dGTP, shown in Figure 2C, confirm this prediction (similar results are obtained for the addition of GTP, not shown).…”

Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase

“…S8). In the transcription model, we assumed that promoter binding and opening are fast steps relative to the 2-nt synthesis step (25,27,32,34,35). The fittings provided the elementary rate constants of RNA synthesis and RNA dissociation (Fig.…”