Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by Escherichia coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex
Abstract:FRET (fluorescence
resonance energy transfer) between far-upstream
(−100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed
extensive bending and wrapping of λPR promoter DNA
on Escherichia coli RNA polymerase (RNAP) in closed
and open complexes (CC and OC, respectively). Here we determine the
kinetics and mechanism of DNA bending and wrapping by FRET and of
formation of RNAP contacts with −100 and +14 DNA by single-dye
protein-induced fluorescence enhancement (PIFE). FRET and PIFE kinetics
exhibit two phase… Show more
“…In the later stage of the promoter melting, the loaded dsDNA will be separated into single-stranded DNA, and this step may require clamp opening. Furthermore, we also note that the structure of the promoter dsDNA may be more complicated than the DNA model used here, where the upstream promoter DNA (position −100) was found to wrap around the E. coli RNAP during promoter melting (71,72). In this situation, opening of the clamp was required even at the beginning of promoter melting.…”
Section: Elucidation Of the Clamp Domain Dynamics Reveals The Recognitionmentioning
To initiate transcription, the holoenzyme (RNA polymerase [RNAP] in complex with σ factor) loads the promoter DNA via the flexible loading gate created by the clamp and β-lobe, yet their roles in DNA loading have not been characterized. We used a quasi-Markov State Model (qMSM) built from extensive molecular dynamics simulations to elucidate the dynamics of Thermus aquaticus holoenzyme’s gate opening. We showed that during gate opening, β-lobe oscillates four orders of magnitude faster than the clamp, whose opening depends on the Switch 2’s structure. Myxopyronin, an antibiotic that binds to Switch 2, was shown to undergo a conformational selection mechanism to inhibit clamp opening. Importantly, we reveal a critical but undiscovered role of β-lobe, whose opening is sufficient for DNA loading even when the clamp is partially closed. These findings open the opportunity for the development of antibiotics targeting β-lobe of RNAP. Finally, we have shown that our qMSMs, which encode non-Markovian dynamics based on the generalized master equation formalism, hold great potential to be widely applied to study biomolecular dynamics.
“…In the later stage of the promoter melting, the loaded dsDNA will be separated into single-stranded DNA, and this step may require clamp opening. Furthermore, we also note that the structure of the promoter dsDNA may be more complicated than the DNA model used here, where the upstream promoter DNA (position −100) was found to wrap around the E. coli RNAP during promoter melting (71,72). In this situation, opening of the clamp was required even at the beginning of promoter melting.…”
Section: Elucidation Of the Clamp Domain Dynamics Reveals The Recognitionmentioning
To initiate transcription, the holoenzyme (RNA polymerase [RNAP] in complex with σ factor) loads the promoter DNA via the flexible loading gate created by the clamp and β-lobe, yet their roles in DNA loading have not been characterized. We used a quasi-Markov State Model (qMSM) built from extensive molecular dynamics simulations to elucidate the dynamics of Thermus aquaticus holoenzyme’s gate opening. We showed that during gate opening, β-lobe oscillates four orders of magnitude faster than the clamp, whose opening depends on the Switch 2’s structure. Myxopyronin, an antibiotic that binds to Switch 2, was shown to undergo a conformational selection mechanism to inhibit clamp opening. Importantly, we reveal a critical but undiscovered role of β-lobe, whose opening is sufficient for DNA loading even when the clamp is partially closed. These findings open the opportunity for the development of antibiotics targeting β-lobe of RNAP. Finally, we have shown that our qMSMs, which encode non-Markovian dynamics based on the generalized master equation formalism, hold great potential to be widely applied to study biomolecular dynamics.
“…Mechanism of rRNA-specific transcription inhibition by DksA/ppGpp. Structural and biochemical studies of bacterial RNAP transcription suggest that the order of DNA loading around the TSS and DNA opening may be interchangeable during promoter recognition (i.e., DNA melts first outside RNAP (melt-load) or DNA melts after loading inside the RNAP cleft (load-melt)) depending on σ factors, promoters, transcription factors and conditions 15 , 33 , 41 , 42 . By combining structural and biochemical data from this and previous studies, we propose two pathways of RPo formation (Fig.…”
Section: Discussionmentioning
confidence: 99%
“…However, the rpsTP2 promoter for expressing ribosomal protein S20 is distinct from the rrnBP1 promoter that it contains G + C-rich DNA upstream of the −35 element and the TSS 7 bases downstream from the −10 element; therefore, it does not reveal the pathway for rRNA promoter complex formation and the mechanism of rRNA transcription regulation. In addition, the presence of TraR does not allow to infer the unperturbed pathway of the open complex formation by RNAP [15][16][17] . Here, we used cryo-EM to visualize the RNAP and rrnBP1 complexes and two additional complexes with DksA/ ppGpp on the way to RPo formation.…”
Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β’ lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.
“…The holo docks on the promoter via concerted interactions with specific promoter regions known as UP (from around -60 to -40 region of the promoter relative to transcription start site at +1 position), -35, spacer, and -10 elements (8,(18)(19)(20)(21)(22). This initial unstable RNAP-promoter complex (RPC, where no DNA melting has occurred) isomerizes to more stable forms when the upstream and downstream regions of the promoter bend along the RNAP surface and into the DNA binding cleft, respectively (23)(24)(25)(26). The formation of catalytically active holo-promoter open complex (RPO) is completed when the -11/+2 region of the promoter DNA duplex unwinds and the template DNA strand enters the active site cleft of the RNAP (27)(28)(29)(30).…”
Section: Introductionmentioning
confidence: 99%
“…Most studies report the existence of two (32)(33)(34) or more (35) open complex structures (RPO, intermediates), but also several closed complex intermediates have recently been identified (26). In most cases, less than half of apparent the RPO's appear capable of productive promoter escape followed by full-length RNA synthesis (32,(36)(37)(38)(39).…”
Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor σ to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high – throughput single-molecule magnetic tweezers. We probed the key protein – DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RPO is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence – independent interactions between the DNA and the holoenzyme, driven by a non – Coulombic effect consistent with the non-template DNA strand interacting with σ and the RNAP β subunit. The temperature dependence provides the energy scale of open complex formation and further supports the existence of additional intermediates.
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