RNA helicases are implicated in most cellular RNA-dependent events. In eukaryotes however, only few have been functionally characterized. Upf1 is a RNA helicase essential for nonsense-mediated mRNA decay (NMD). Here, using magnetic tweezers and bulk assays, we observe that human Upf1 is able to translocate slowly over long single-stranded nucleic acids with a processivity >10 kb. Upf1 efficiently translocates through double-stranded structures and protein-bound sequences, demonstrating that Upf1 is an efficient ribonucleoprotein complex remodeler. Our observation of processive unwinding by an eukaryotic RNA helicase reveals that Upf1, once recruited onto NMD mRNA targets, can scan the entire transcript to irreversibly remodel the mRNP, facilitating its degradation by the NMD machinery.

No abstract

Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA that forms due to thermal fraying. Here we show that Dda translocates unidirectionally on single-stranded DNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (Vun) by the velocity of translocation on single-stranded DNA in nucleotides per second (Vtrans). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the “active” range. In the case of Dda, the average DNA unwinding velocity was 257 ± 42 bp/s and the average translocation velocity was 267 ± 15 nucleotides/s. The Vun/Vtrans value of 0.96 places Dda in a unique category of being an essentially “perfectly” active helicase.

Small angle X-ray scattering (SAXS) studies in poly[2-methoxy-5-(2 ' -ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV) with varying conjugation, and polyethylene dioxythiophene complexed with polystyrene sulfonate (PEDOT-PSS) in different solvents have shown the important role of -electron conjugation and solvent-chain interactions in controlling the chain conformation and assembly. In MEH-PPV, by increasing the extent of conjugation from 30 to 100 %, the persistence length (l p ) increases from 20 to 66 Å. Moreover, a pronounced second peak in the pair distribution function has been observed in fully conjugated chain, at larger length scales. This feature indicates that the chain segments tend to self-assemble as the conjugation along the chain increases. In case of PEDOT-PSS, the chains undergo solvent induced expansion and enhanced chain organization. The clusters formed by chains are better correlated in dimethyl sulfoxide (DMSO) solution than water, as observed in the scattered intensity profiles. The values of radius of gyration and the exponent (water: 2.6, DMSO: 2.31) of * Electronic address: s_paramita@physics.iisc.ernet.in Fax: +91-80-2360 2602 Tel: +91-80-2293 2859power-law decay, obtained from the unified scattering function (Beaucage) analysis, give evidence for chain expansion from compact (in water) to extended coil in DMSO solutions, which is consistent with the Kratky plot analysis. The mechanism of this transition and the increase in dc conductivity of PEDOT-PSS in DMSO solution are discussed. The onset frequency for the increase in ac conduction as well as its temperature dependence probes the extent of connectivity in PEDOT-PSS system. The enhanced charge transport in PEDOT-PSS in DMSO is attributed to the extended chain conformation as observed in SAXS results.

Most RecQ DNA helicases share a conserved domain arrangement that mediates their activities in genomic stability. This arrangement comprises a helicase motor domain, a RecQ C-terminal (RecQ-C) region including a winged-helix (WH) domain, and a ‘Helicase and RNase D C-terminal’ (HRDC) domain. Single-molecule real-time translocation and DNA unwinding by full-length Escherichia coli RecQ and variants lacking either the HRDC or both the WH and HRDC domains was analyzed. RecQ operated under two interconvertible kinetic modes, ‘slow’ and ‘normal’, as it unwound duplex DNA and translocated on single-stranded (ss) DNA. Consistent with a crystal structure of bacterial RecQ bound to ssDNA by base stacking, abasic sites blocked RecQ unwinding. Removal of the HRDC domain eliminates the slow mode while preserving the normal mode of activity. Unexpectedly, a RecQ variant lacking both the WH and HRDC domains retains weak helicase activity. The inclusion of E. coli ssDNA-binding protein (SSB) induces a third ‘fast’ unwinding mode four times faster than the normal RecQ mode and enhances the overall helicase activity (affinity, rate, and processivity). SSB stimulation was, furthermore, observed in the RecQ deletion variants, including the variant missing the WH domain. Our results support a model in which RecQ and SSB have multiple interacting modes.

Moreover, a pronounced second peak in the pair distribution function has been observed in fully conjugated chain, at larger length scales. This feature indicates that the chain segments tend to self-assemble as the conjugation along the chain increases. Xylene enhances the rigidity of PPV backbone to yield extended structures, while tetrahydrofuran solvates the side groups to form compact coils in which the l p is much shorter.

No abstract

DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.