Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active center

Batada, Nizar N.; Westover, Kenneth D.; Bushnell, David; Levitt, Michael; Kornberg, Roger D.

doi:10.1073/pnas.0408168101

Cited by 69 publications

(101 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our previous simulation study has suggested that the PPi release is rapid [at ∼1 μs (9)]. NTP loading has been suggested to occur in a few milliseconds (45), whereas the existence of a prebinding site may further accelerate this process. In the current study, we show that translocation can occur at timescales of tens of microseconds, which is relatively fast compared with the elongation rate (∼100 ms per base pair in vivo).…”

Section: Discussionmentioning

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.

Self Cite

144

199

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

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.

Self Cite

144

199

View full text Add to dashboard Cite

show abstract

“…NTPs have been observed bound in the catalytic site and secondary channel, but not in the main channel, and there is no obvious path for NTPs to move from the main channel to the active site (18)(19)(20)(21)(22)(23). These observations have led to the proposal that the secondary channel is the major, and perhaps only, pathway for NTPs to enter the active site (24)(25)(26)(27). Biochemical studies, however, indicate that there may be NTP binding site (s) located in the main channel of RNAP (10,16,17).…”

mentioning

confidence: 95%

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

Kennedy

Erie²

2011

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

View full text Add to dashboard Cite

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...

show abstract

“…5B), thereby changing the surrounding structure and potentially creating a looser configuration in the tunnel around the small pore that separates the E and PS sites. In addition, removal of the charged ␤Asp 675 residue will reduce the negative electrostatic potential of this pore (13).…”

Section: Table 1 Rates Of Misincorporation For Variant and Wild-type mentioning

confidence: 99%

“…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)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(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.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Holmes¹,

Santangelo

Cunningham³

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active center

Cited by 69 publications

References 15 publications

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

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

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

Contact Info

Product

Resources

About