T7 DNA Helicase: A Molecular Motor that Processively and Unidirectionally Translocates Along Single-stranded DNA

Kim, Dong-Eun; Narayan, Murli; Patel, Smita S.

doi:10.1016/s0022-2836(02)00733-7

Cited by 97 publications

(86 citation statements)

References 35 publications

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“…As we derive (see SI Text), the mean stepping velocity of a passive helicase can be written as a function of the rate of helicase translocation (19,20) on ssRNA and the thermodynamic stability of base pairs in front of the helicase (see SI Text):…”

Section: Resultsmentioning

confidence: 99%

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

Cheng¹,

Dumont

Tinoco³

et al. 2007

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

149

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RNA helicases regulate virtually all RNA-dependent cellular processes. Although much is known about helicase structures, very little is known about how they deal with barriers in RNA and the factors that affect their processivity. The hepatitis C virus encodes NS3, an RNA helicase that is essential for viral RNA replication. We have used optical tweezers to determine at the single-molecule level how the local stability of the RNA substrate affects the enzyme rate of strand separation, whether separation occurs by an active or a passive mechanism, and whether processivity is affected. We show that sequence barriers in RNA modulate NS3 activity. NS3 processivity depends on barriers ahead of the opening fork. Our results rule out a model where NS3 passively waits for the thermal fraying of double-stranded RNA. Instead, we find that NS3 destabilizes the duplex before separating the strands. Failure to do so before a strong barrier leads to helicase dissociation and limits the processivity of the enzyme.hairpin ͉ molecular motors ͉ optical tweezers ͉ processivity A wide range of RNA metabolic activities (1) require the breaking of base pairs in double-stranded RNA (dsRNA) by RNA helicases. Despite recent progress in the study of these motor proteins (2-4), the physical mechanisms by which they move and catalyze the strand separation are not well understood. Helicase models ranging from a pure Brownian ratchet to a pure power stroke action have been discussed (5-8), but experimental data to support them for RNA helicases have been lacking. The hepatitis C virus NS3 protein is a superfamily 2, 3Ј to 5Ј RNA helicase (9) known to be essential for virus replication and, thus, an antiviral drug target (10). Recently, NS3 has been the subject of single-molecule manipulation studies (4), which revealed that NS3 makes steps of 11 bp each made up of three substeps (4). RNA molecules contain various kinetic barriers to mechanical unfolding (11). How these barriers influence the activity of motor proteins that work on RNA will depend on the mechanism of the motor. To probe the molecular mechanism of NS3 unwinding activity, we have designed and characterized RNA molecules with various mechanical unfolding barriers and used single-molecule methods to investigate how these barriers affect the velocity, pausing, and processivity of the helicase in real time. ResultsDesign and Characterization of RNA Hairpin Substrates. We first designed two RNA hairpin substrates that terminate both on a tetraloop (Fig. 1A) to monitor the response of a single NS3 helicase to weak and strong sequence barriers. Substrate RNA-AG has 30 A⅐U pairs followed by 30 G⅐C pairs; RNA-GA has the A⅐U and G⅐C sequences interchanged. The sequences within A⅐U and G⅐C regions were chosen to minimize the formation of alternative secondary structures other than the desired 60-bp hairpin. In our experiment, a single RNA hairpin was attached between a microsphere in an optical trap and a microsphere placed on the end of a micropipette through hybrid RNA-DNA handles to se...

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Section: Resultsmentioning

confidence: 99%

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

Cheng¹,

Dumont

Tinoco³

et al. 2007

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

149

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“…This complex that mediates leading strand synthesis is extremely stable with a half-life of more than 10 min (8). The binding between gp4 and ssDNA is stable, with a dissociation rate of 0.002 s Ϫ1 (35). However, the complex of gp5/trx, in the absence of gp4, readily dissociates from a primer/template with a dissociation constant of 0.2 s Ϫ1 (36).…”

Section: Discussionmentioning

confidence: 99%

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

Kulczyk

Akabayov

Lee

et al. 2012

Journal of Biological Chemistry

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Background:Interactions of DNA polymerase and DNA helicase are crucial in DNA synthesis. Results: Two distinct interactions are involved in formation of the DNA polymerase/DNA helicase complex. Conclusion:The multiple interactions between DNA polymerase and DNA helicase account for the high processivity of leading strand synthesis. Significance: Understanding of the replication process in bacteriophage T7 facilitates studies in more complex systems.

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“…Interestingly, a slow mandatory initiation process following dsDNA binding is observed before DNA translocation begins (27,100); it should be noted that it is still unclear if this process was ATP-dependent or represented an ATP-independent conformational change following DNA binding by RSC. A multi-step DNA binding mechanism may be a common theme in all translocases, as it has also been found in the ISW2 chromatin remodeler (103), the human polymerase β (104, 105), and several helicases (24,75,(106)(107)(108).…”

Section: Dna Translocation By Chromatin Remodeling Enzymesmentioning

confidence: 90%

“…The bacteriophage T7 gene product 4 (gp4) is an SF4 hexameric helicase that translocates from 5′ to 3′ along ssDNA (3,74) and is capable of translocating on the order of 75 kb before dissociation (75). During its translocation along ssDNA T7 gp4 moves two to three nucleotides for each molecule of ATP it hydrolyzes (75).…”

Section: Sf3 Through Sf6 Family Helicasesmentioning

confidence: 99%

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All motors have to decide is what to do with the DNA that is given them

Briggs

Fischer

2014

Biomolecular Concepts

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DNA translocases are a diverse group of molecular motors responsible for a wide variety of cellular functions. The goal of this review is to identify common aspects in the mechanisms for how these enzymes couple the binding and hydrolysis of ATP to their movement along DNA. Not surprisingly, the shared structural components contained within the catalytic domains of several of these motors appear to give rise to common aspects of DNA translocation. Perhaps more interesting, however, are the differences between the families of translocases and the potential associated implications both for the functions of the members of these families and for the evolution of these families. However, as there are few translocases for which complete characterizations of the mechanisms of DNA binding, DNA translocation, and DNA-stimulated ATPase have been completed, it is difficult to form many inferences. We therefore hope that this review motivates the necessary further experimentation required for broader comparisons and conclusions.

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T7 DNA Helicase: A Molecular Motor that Processively and Unidirectionally Translocates Along Single-stranded DNA

Cited by 97 publications

References 35 publications

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

All motors have to decide is what to do with the DNA that is given them

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