The Elongation-Termination Decision in Transcription

Transcription 3' movement of the protein along the nascent RNA to elongation complexes paused at specific Rho-dependent termination sites on the DNA template. Rho then engages its ATPase-dependent RNA-DNA helicase activity to release the RNA from the transcription complex (5). The sequence specificity of Rho-dependent termination sites appears to reflect the extended dwell-time of the elongation complex at these positions (14-16). This extensive pausing allows Rho proteins that are translocating along the nascent RNA to "catch up" with the transcription complex at these sites, with the resultant termination efficiency depending on the "kinetic coupling" ofthe rate ofmovement ofRho along the nascent transcript and of RNA polymerase along the DNA template (1,17,18).The development of this view of Rho function has been paralleled by progress in elucidating its structural and enzymatic properties. It has been shown that Rho exists under physiological conditions as a hexamer of identical subunits (19-23), organized as a trimer of asymmetric dimers with overall D3 symmetry (24). This symmetry is reflected functionally in the presence of three strong and three weak ATP (substrate) and three strong and three weak RNA (cofactor) binding sites per Rho hexamer (25)(26)(27)(28) (37,38), and subunit interaction sites have been proposed in the C-terminal region (35). Rho monomers associate to form a hexamer with D3 symmetry under physiological conditions.

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confidence: 99%

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confidence: 99%

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confidence: 99%

See 1 more Smart Citation

A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho.

Geiselmann

Wang

Seifried

et al. 1993

“…In classic rho-independent or ''intrinsic'' termination, dissociation is thought to occur as the polymerase slows in response to sequence, and structure begins to form concurrently in the nascent transcript (6)(7)(8). A run of encoded U's has been proposed both to slow transcription and to weaken the RNA-DNA hybrid.…”

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confidence: 99%

Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward-translocation mechanism

Zhou¹,

Navaroli²,

Enuameh

et al. 2007

hypertranslocation ͉ hybrid ͉ stability ͉ topological lock T ranscription lies at the heart of cellular gene expression. Unlike replication, transcription is not distributive; that is, any dissociation of an elongation complex is a terminal event. Consequently, elongation complexes must be highly stable while transcribing at up to several hundred bases per second. Recent studies demonstrate, however, that elongation is not a uniform process, with reasonably long-lived, sequence-dependent pauses occurring stochastically (1-4). Indeed, the elongation phase is well known to be subject to regulation through attenuation: sequence-dependent signals leading to pause, arrest, slippage, or termination (5). Elongating RNA polymerases must hold the DNA template and the RNA product tightly enough to be highly stable through nonterminating pauses, yet they must be able to dissociate the complex in response to specific sequences in the DNA. Understanding this interplay requires understanding mechanisms of dissociation.In classic rho-independent or ''intrinsic'' termination, dissociation is thought to occur as the polymerase slows in response to sequence, and structure begins to form concurrently in the nascent transcript (6-8). A run of encoded U's has been proposed both to slow transcription and to weaken the RNA-DNA hybrid. Recently, it has been proposed that, rather than direct dissociation of the nascent transcript from a complex halted at the primary termination site (with or without allosteric assistance), dissociation occurs primarily from forward-translocated states (9-11). In forwardtranslocated (hypertranslocated) states, the hybrid is shortened, weakening the binding of the RNA to the complex. Additionally, shortening of the hybrid may also serve to remove a topological locking of the RNA onto the template strand. As illustrated in Fig. 1, dissociation of the RNA transcript would be substantially faster from forward translocated states than from the initially paused state. Although forward-translocated states are expected to be energetically disfavored relative to the initial on-pathway state and so would be expected to exist at lower populations, the difference in the kinetics of dissociation could compensate such that the dominant dissociative pathway proceeds via forward translocated states.The model shown in Fig. 1 predicts certain behaviors. In general, forward translocation requires melting of the DNA duplex downstream and dissociation of the most upstream base(s) in the hybrid. These costs are partially offset by the energy gained by collapse of a base pair in the DNA at the upstream edge of the bubble. Thus, one would expect that altering the balance between these effects could shift the distribution toward more forward-translocated states and therefore toward or away from dissociation. As demonstrated previously, a direct crosslink between the DNA strands downstream of the halt site should prevent forward translocation and is in fact seen to reduce termination from an intrinsic terminator (9).We propose t...

“…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)(2)(3)(4)(5)(6)(7)(8)(9)(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.…”

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confidence: 99%

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

Kennedy

Erie²

2011

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...