Millisecond phase kinetic analysis of elongation catalyzed by human, yeast, and Escherichia coli RNA polymerase

Kireeva, Maria L.; Nedialkov, Yuri A.; Gong, Xue; Zhang, Chunfen; Xiong, Yalin; Moon, Woo; Burton, Zachary F.; Kashlev, Mikhail

doi:10.1016/j.ymeth.2009.04.008

Cited by 30 publications

(40 citation statements)

References 46 publications

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“…In contrast, EDTA removes Mg 2þ complexed with free NTPs in solution, which renders the NTPs inactive for catalysis; therefore, quenching with EDTA measures the amount of product formation at the time of quench plus any RNAP∶NTP complexes that are able to proceed to product formation without dissociation of the NTP (ES Ã þ EP þ P) (16,30,31). For a single nucleotide addition, quenching with EDTA has been demonstrated to be equivalent to pulse-chase experiments in which unlabeled RNAP elongation complexes are incubated with a radioactively labeled NTP and then an excess of unlabeled NTP is added (32). Comparison of nucleotide incorporation kinetics between these two quenching agents can be used to reveal if there is an accumulation of the enzyme-substrate complex (i.e., RNAP:NTP) prior to phosphodiester bond formation and/or pyrophosphate release (16,30,31,33).…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

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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“…Alternatively, the TECs purified by membrane filtration were preincubated with 5 μM Nun or a comparable volume of Nun buffer at room temperature for 10 min and directly used in the transcription assays. The reactions were stopped manually by addition of an equal volume of gelloading buffer [10 M urea, 50 mM EDTA (pH 7.9), 0.05% bromophenol blue, and xylene cyanol] or, for incubation times less than 5 s, by addition of HCl using an RQF-3 rapid quench flow instrument (Kintek) as described (31). Transcription products were resolved in 20% or 23% denaturing polyacrylamide gels (acrylamide/methylene-bis-acrylamide ratio was 19:1) containing 1× Tris-Borate-EDTA (TBE) and 7 M urea.…”

Section: Methodsmentioning

confidence: 99%

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Vitiello

Kireeva

Lubkowska

et al. 2014

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

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The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β′ subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5′ edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.T ranscription elongation is highly processive, yet the rate of nucleotide addition varies significantly for different ternary elongation complexes (TECs). In the normal elongation pathway, each nucleotide addition is followed by translocation of RNA polymerase (RNAP) along DNA in single-nucleotide increments. This process transfers the RNA 3′ end from the i + 1 site to the i site of the active center, thus allowing binding of the next NTP and subsequent phosphodiester bond formation. Translocation is thought to be a stochastic, rapid, and fully reversible process and not rate-limiting for elongation (1, 2). Instead, NTP sequestration, phosphodiester bond formation, or pyrophosphate release has been suggested to be rate-limiting for transcription elongation (3, 4). However, several reports strongly suggest that translocation may be at least partially rate-limiting for elongation (5-9).Immediately following bond formation, a catalytically inactive and highly pyrophosphorolytic (pretranslocated) state is formed. In this state, the 3′ RNA end remains in the i + 1 site of the active center. The 3′ RNA thus prevents NTP binding and generates a substrate for pyrophosphorolysis. To bind the next NTP, RNAP must translocate 1-bp forward along the DNA register to form a catalytically active and pyrophosphate-resistant (posttranslocated) state. Stationary RNAP has been suggested to "ratchet" between the two states via Brownian motion (5). NTP bound to the transient posttranslocated state acts as a pawl that interferes with backward translocation, thereby favoring the formation of a phosphodiester bond. Retention of this NTP in the posttranslocated TEC is facilitated by isomerization of the active site that blocks substrate exit, and aligns it for catalysis (10, 11)...

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“…The rpoB mutants Q513P, S522Y, and P564L were "up" mutants, and D516N, I572N, and I572T, were "down" mutants. These strains, as indicated above, also carried a functional hexahistidine-tagged ␤Ј subunit, which allowed the rapid pulldown and purification of RNAP from the crude lysate by immobilization on Ni 2ϩ -NTA-agarose beads (49,55). Each of the purified enzymes was assembled into a TEC with a synthetic RNA-DNA scaffold, and the bulk elongation rate for each mutant RNAP was tested in a single-round "runoff" assay with 10 M NTP (Fig.…”

Section: Rifmentioning

confidence: 99%

Isolation and Characterization of RNA Polymerase rpoB Mutations That Alter Transcription Slippage during Elongation in Escherichia coli

Zhou¹,

Lubkowska²,

Hui³

et al. 2013

Journal of Biological Chemistry

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Background:The domains in RNA polymerase involved in elongation slippage are unknown. Results: We isolated E. coli RNA polymerase rpoB mutants with altered transcriptional slippage. Conclusion:The fork domain of RNA polymerase controls slippage. Biochemical analysis of the mutants validates the genetic schemes. Significance: Our work sheds light on the mechanism for maintenance of RNA-DNA register during transcription.

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Millisecond phase kinetic analysis of elongation catalyzed by human, yeast, and Escherichia coli RNA polymerase

Cited by 30 publications

References 46 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

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Isolation and Characterization of RNA Polymerase rpoB Mutations That Alter Transcription Slippage during Elongation in Escherichia coli

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