A Ratchet Mechanism of Transcription Elongation and Its Control

Bar-Nahum, Gil; Epshtein, Vitaly; Ruckenstein, Andrei E.; Rafikov, Ruslan; Mustaev, Arkady; Nudler, Evgeny

doi:10.1016/j.cell.2004.11.045

Cited by 338 publications

(466 citation statements)

References 45 publications

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“…The incoming nucleotide substrate acts like a pawl of a ratchet that traps the complex in its post-translocational state, which prevents the reverse motion. A related, more complex version of this model has also been proposed for multisubunit RNA polymerases (34,35). Our previous site-specific footprinting experiments with stalled HIV-1 RT complexes revealed that the presence of the templated nucleotide can stabilize and trap the post-translocated complex (27).…”

Section: Discussionmentioning

confidence: 99%

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

Marchand

Tchesnokov

Götte

2007

Journal of Biological Chemistry

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The pyrophosphate (PPi) analogue phosphonoformic acid (PFA or foscarnet) inhibits the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1); however, the mechanisms of drug action and resistance remain elusive. Here we studied the effects of the translocational status of HIV-1 RT on drug binding and inhibition of DNA synthesis. We identified "hot spots" for inhibition during active elongation. Site-specific footprinting analyses revealed that the corresponding complexes exist predominantly in the pre-translocational state. The sensitivity to PFA is significantly reduced with sequences that show a bias toward the post-translocational state. Binding studies showed that PFA stabilizes selectively the complex in the pre-translocated configuration. These findings are consistent with a Brownian ratchet model of polymerase translocation. The enzyme can rapidly shuttle between pre-and post-translocated states. The bound inhibitor acts like a pawl of a ratchet and prevents the forward motion of HIV-1 RT, whereas the bound nucleotide binds to the post-translocated complex and prevents the reverse motion. The proposed mechanisms of RT translocation and drug action are consistent with the PFA-resistant phenotypes. We show that certain sequences and the PFA-resistant E89K mutant diminishes the stability of the pre-translocated complex. In these cases, the enzyme is seen at multiple positions around the 3 end of the primer, which provides a novel mechanism for resistance. These findings validate the pre-translocated complex as a target for the development of novel, perhaps less toxic and more potent inhibitors that block HIV-1 RT translocation.Different classes of inhibitors that target the reverse transcriptase (RT) 4 of the human immunodeficiency virus type 1 (HIV-1) have been developed (1). Nucleoside analogue reverse transcriptase inhibitors (NRTIs) are major components in drug regimens that are currently used in the clinic. Two different NRTIs are usually combined with a non-nucleoside analogue RT inhibitor (NNRTI) or a protease inhibitor. The triphosphate forms of NRTIs compete with natural nucleotide pools for incorporation, and, once incorporated, the monophosphate acts as a chain terminator. NNRTIs bind to a hydrophobic pocket in the vicinity but not at the active site of HIV-1 RT. Previous studies have suggested that these compounds interfere with the chemical step, whereas nucleotide binding does not appear to be largely affected (2, 3). Here we studied the mechanism of action of the pyrophosphate (PP i ) analogue phosphonoformic acid (PFA or foscarnet), which represents a third class of RT inhibitors.PFA shows a broad spectrum of antiviral activities against various members of the Herpesviridae and Retroviridae (4). The inhibitor is used in the clinic to treat infection with herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), the human cytomegalovirus, and other related herpesviruses when firstline agents have failed (5). Toxic side effects and its poor bioavailability limit its clini...

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Section: Discussionmentioning

confidence: 99%

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

Marchand

Tchesnokov

Götte

2007

Journal of Biological Chemistry

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“…Two mutations in the trigger loop of E. coli RNAP affect misincorporation [52]. Mutation of Rpb1 residue Glu1103 in the RNAP II trigger loop also promotes incorporation of incorrect substrates .…”

Section: Rnap Fidelity Mechanismsmentioning

confidence: 99%

RNA polymerase fidelity and transcriptional proofreading

Sydow

Cramer

2009

Current Opinion in Structural Biology

148

133

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Whereas mechanisms underlying the fidelity of DNA polymerases (DNAPs) have been investigated in detail, RNA polymerase (RNAP) fidelity mechanisms remained poorly understood. New functional and structural studies now suggest how RNAPs select the correct nucleoside triphosphate (NTP) substrate to prevent transcription errors, and how the enzymes detect and remove a misincorporated nucleotide during proofreading. Proofreading begins with fraying of the misincorporated nucleotide away from the DNA template, which pauses transcription. Subsequent backtracking of RNAP by one position enables nucleolytic cleavage of an RNA dinucleotide that contains the misincorporated nucleotide. Since cleavage occurs at the same active site that is used for polymerization, the RNAP proofreading mechanism differs from that used by DNAPs, which contain a distinct nuclease specific active site.

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“…In the "Brownian ratchet" model, random thermal fluctuations between the pre-and post-translocated states of the enzyme are mechanically rectified by NTP binding and hydrolysis, leading to unidirectional motion. Experimental (80,102) and theoretical (103,104) evidence has often been interpreted in terms of a ratchet-like mechanism, although other interpretations certainly cannot be ruled out. The power-stroke model was inspired by crystal structures of T7 RNAP obtained in nominally pre-and posttranslocated states.…”

Section: On-pathway Elongationmentioning

confidence: 99%

Single-Molecule Studies of RNA Polymerase: Motoring Along

Herbert

Greenleaf

Block

2008

Annu. Rev. Biochem.

189

141

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Single-molecule techniques have advanced our understanding of transcription by RNA polymerase. A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNA polymerase identifies a promoter; initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNA polymerase, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates.

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A Ratchet Mechanism of Transcription Elongation and Its Control

Cited by 338 publications

References 45 publications

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

RNA polymerase fidelity and transcriptional proofreading

Single-Molecule Studies of RNA Polymerase: Motoring Along

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