NTP-driven translocation and regulation of downstream template opening by multi-subunit RNA polymerases

Burton, Zachary F.; Feig, Michael; Gong, Xue; Zhang, Chunfen; Nedialkov, Yuri A.; Xiong, Yalin

doi:10.1139/o05-059

Cited by 33 publications

(42 citation statements)

References 42 publications

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“…3A, Fig. S2F) support models of nucleotide addition that suggest that NTPs can bind into a site in main chan- nel and subsequently be loaded into the catalytic site (10,16,29,40). To further test this idea, we performed experiments in which unlabeled SECs were preincubated with 10 μM [α-32 P]-ATP prior to initiating the reaction with unlabeled ATP and CTP, and then measured the extent of [ 32 P]-AMP incorporation into the transcript as a function of the concentration of unlabeled ATP (Fig.…”

Section: Resultssupporting

confidence: 57%

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

Kennedy

Erie²

2011

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultssupporting

confidence: 57%

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

Kennedy

Erie²

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Structures were modified (iϩ2 and iϩ3 NTPs placed by modeling, DNA strands extended, non-template DNA placed), as described previously (10). Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Mg 2ϩ atoms are green; the DNA template strand is green; the non-template DNA strand is white; RNA is red; phosphorous atoms are indicated as tan spheres; basic amino acids (Rpb1 is purple; phenylalanine residues found at the N-terminal end of the bridge ␣-helix (Rpb1 813 to 815) are white. DNA missing from available structures was placed by modeling (10). Multiple crystal structures were used to generate this figure (6, 19).…”

Section: Isomerization Reversal and The Trigger Loop Hypothesis-inmentioning

confidence: 99%

“…In this model, the secondary pore is the route of pyrophosphate release and may be a route for expulsion of NTP substrates rejected at the active site. Furthermore, NTP-driven translocation can be utilized as a means to dislodge an NTP from the active site in response to a translocation block, indicating a role for downstream NTPs in dynamic error avoidance and error correction (10,12). Normally, a translocation block signals a transcription error.…”

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confidence: 99%

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A Tunable Ratchet Driving Human RNA Polymerase II Translocation Adjusted by Accurately Templated Nucleoside Triphosphates Loaded at Downstream Sites and by Elongation Factors

Xiong

Burton

2007

Journal of Biological Chemistry

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When nucleoside triphosphate (NTP) substrates and ␣-amanitin are added to a human RNA polymerase II elongation complex simultaneously, the reaction becomes stalled in the core of the bond synthesis mechanism. The mode of stalling is influenced by NTP substrates at the active site and at downstream sites and by transcription factor IIF (TFIIF) and TFIIS. NTP substrates templated at i؉2, i؉3, and i؉4 downstream DNA sites can reverse the previously stable binding of an NTP loaded at the i؉1 substrate site. Deoxy-(d)NTPs and NDPs (nucleoside diphosphates) do not substitute for NTPs at the i؉2 and i؉3 positions (considered together) or the i؉4, i؉5, and i؉6 positions (considered together). The mode of stalling is altered by changing the number of downstream template sites that are accurately occupied by NTPs and by changing NTP concentration. In the presence of the translocation blocker ␣-amanitin, a steady state condition is established in which RNA polymerase II stably loads an NTP substrate at i؉1 and forms a phosphodiester bond but cannot rapidly complete bond synthesis by releasing pyrophosphate. These observations support a role for incoming NTP substrates in stimulating translocation; results appear inconsistent with the secondary pore being the sole route of NTP entry for human RNA polymerase II, and results indicate mechanisms of dynamic error avoidance and error correction during rapid RNA synthesis.A goal of transient state kinetic analyses is to reveal the internal mechanism of an enzyme reaction by observing synchronized millisecond events on a millisecond time scale. In this work, the mushroom toxin ␣-amanitin is utilized as a transient state inhibitor, locking human RNA polymerase II in the core of the elongation mechanism. By altering the conditions of stalling and by employing two distinct reaction quenching methods, details of the core mechanism of human RNA polymerase II become apparent. Recent x-ray crystal structures of Thermus thermophilus RNA polymerase elongation complexes indicate a simple thermal ratchet mechanism for elongation (1, 2), with nucleoside triphosphate (NTP) 2 substrates loading through the secondary pore, a solvent accessible channel, to the active site (designated iϩ1). Very little space is available in these structures to load NTPs through the main enzyme channel, which holds the DNA duplex and RNA-DNA hybrid. The downstream transcription bubble is closed at the iϩ2 position by base pairing, so it is difficult to imagine how NTP substrates could interact at iϩ2 or iϩ3 template sites. T. thermophilus ␤ Arg 422 makes a specific contact to the iϩ1 DNA template phosphate, and appears to provide a specific mechanism for closing the downstream bubble at the iϩ2 position. This mechanism for closing the downstream bubble is not conserved in human or yeast RNA polymerase II, in which the corresponding residue to ␤ Arg 422 is Rpb2 Gly 493 (human) and Rpb2 Gly 506 (yeast). Furthermore, even if the downstream bubble were open in the T. thermophilus structure, little space is available to...

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“…Transcription is a multistep process consisting of initiation, elongation, and termination, where elongation is composed of consecutive nucleotide addition cycles (NACs). In each NAC, the NTP substrate first diffuses into Pol II active site through the secondary channel (3)(4)(5) or alternatively the main channel (6). Upon correct NTP binding to the Pol II active site, the trigger loop (TL) conformation switches from an inactive open state to an active closed state (7).…”

mentioning

confidence: 99%

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Silva

Weiss

Avila

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

144

199

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Transcription is a central step in gene expression, in which the DNA template is processively read by RNA polymerase II (Pol II), synthesizing a complementary messenger RNA transcript. At each cycle, Pol II moves exactly one register along the DNA, a process known as translocation. Although X-ray crystal structures have greatly enhanced our understanding of the transcription process, the underlying molecular mechanisms of translocation remain unclear. Here we use sophisticated simulation techniques to observe Pol II translocation on a millisecond timescale and at atomistic resolution. We observe multiple cycles of forward and backward translocation and identify two previously unidentified intermediate states. We show that the bridge helix (BH) plays a key role accelerating the translocation of both the RNA:DNA hybrid and transition nucleotide by directly interacting with them. The conserved BH residues, Thr831 and Tyr836, mediate these interactions. To date, this study delivers the most detailed picture of the mechanism of Pol II translocation at atomic level.Markov state model | molecular dynamics | trigger loop T he RNA polymerase is the central component of gene expression in all living organisms, transferring genetic information from DNA to RNA. In eukaryotes, the RNA polymerase II (Pol II) enzyme is responsible for transcribing DNA into messenger RNA. In the past decade, a number of X-ray crystallographic structures of Pol II have been obtained at different stages of the transcription process, providing a static picture of how this complex machine performs its function (1, 2). Transcription is a multistep process consisting of initiation, elongation, and termination, where elongation is composed of consecutive nucleotide addition cycles (NACs). In each NAC, the NTP substrate first diffuses into Pol II active site through the secondary channel (3-5) or alternatively the main channel (6). Upon correct NTP binding to the Pol II active site, the trigger loop (TL) conformation switches from an inactive open state to an active closed state (7). The closure of the active site subsequently facilitates the catalysis of the nucleotide addition reaction (7), followed by release of the pyrophosphate ion (PPi). To proceed to the next NAC, Pol II must translocate from a pretranslocation state, in which the active site is still occupied by the newly added nucleotide at 3′-RNA, to a posttranslocation state. During translocation, the template DNA and RNA must move by exactly one register, once again creating a free insertion site (i site) (1,2,5,(8)(9)(10)(11)(12)(13).Although static snapshots of X-ray structures of Pol II pretranslocation and posttranslocation states are valuable, the dynamics underlying the fundamental RNA polymerase translocation mechanism remain poorly understood (14). Two models of translocation have been proposed based on structural, biochemical, and genetic approaches. On one hand, for single subunit T7 RNAP, PPi release is suggested to be mechanically coupled to the opening motion of the O-helix (co...

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NTP-driven translocation and regulation of downstream template opening by multi-subunit RNA polymerases

Cited by 33 publications

References 42 publications

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

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

A Tunable Ratchet Driving Human RNA Polymerase II Translocation Adjusted by Accurately Templated Nucleoside Triphosphates Loaded at Downstream Sites and by Elongation Factors

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

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