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

Abstract

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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Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

“…S1). This behavior may reflect sequence-dependent unwinding and/or pause kinetics, as has been observed for other superfamily (SF) 1 and 2 helicases including hepatitis virus NS3 helicase (18), XPD helicase (19), and RecBCD helicase (20). Additional experiments will be required to characterize the sequence dependence of RecQ unwinding and pausing, and the role of the HRDC domain in these processes.…”

The Protease Domain Increases the Translocation Stepping Efficiency of the Hepatitis C Virus NS3-4A Helicase

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