| DExH/D-box proteins,the largest family of the RNA helicase,belong to the helicase superfamily 2(SF2).Nearly all aspects of RNA metabolism,from transcription and translation to mRNA decay,involve DExH/D-box proteins.DEAD-box protein family was a typical representative of DExH/D-box protein family.The structure of DEAD-box proteins contains two conserved RecA-like domains,including 13 characteristic sequence motifs.The name "DEAD-box" is derived from the Asp-Glu-Ala-Asp sequence in motif?,reading D-E-A-D.DEAD-box proteins catalyze ATP-driven unwinding of RNA duplexes by a mode distinct from canonical helicases,which based on translocation of the enzymes along the nucleic acid.DEAD-box proteins loaded on the duplex region and then pry only the short RNA-RNA and DNA-RNA duplexes apart in an ATP dependent fashion,and this process does not need ATP hydrolysis,ATP hydrolysis promote recycling of the enzymes.For many DEAD-box proteins,loading onto the duplex is facilitated by single-stranded regions adjacent to the duplex,and the polarity of the single-stranded regions is not critical for unwinding.This type of unwinding mechanism is termed local strand separation,and this mode was thought to be the unique way for the localized rearrangement of RNA and RNA-protein complex by the DEAD-box proteins catalyze in the cell.Dedlp comes from Saccharomyces cerevisiae,and it was a typical representative of DEAD-box RNA helicase.Dedlp was first isolated as an essential domain in the chromosomal region,which encoding the HIS3 gene,so it was pertinently dubbed Dedl-Defines Essential Domain 1.The research found that Dedlp worked as oligomer.Single molecule fluorescence resonant energy transfer(sm-FRET)method was used to detect the conformation changes by detecting the energy transfer efficiency between fluorescence donor and acceptor in single molecule.Compared with the traditional ensemble averages methods,sm-FRET detection can analyze the single molecule in the system,get the histogram distribution of the molecules and also analyze the kinetics of the biomolecule reaction.sm-FRET has rapidly developed to answer fundamental questions about DNA replication and recombination,RNA transcription,protein translation,RNA folding and catalysis,protein folding and conformational changes,various motor proteins,membrane fusion proteins,ion channels,signal transduction,and helicases.In this study,the full-length Dedlp was used as the research protein,we first using 0.5 mM IPTG induce BL21(DE3)expressed the soluble Dedlp.And purified Dedlp with two-step purification,which combined with the Ni-NTA agarose affinity chromatography and the Capto-DEAE weak anion exchange chromatography.Then verified the helicase enzyme activity of Dedlp through the PAGE gel.Then prepared the fluorescent labelled four dsRNA,which have different double chain length and ssRNA direction.And then used sm-FRET technology to explore the unwinding mechanism of Dedlp.The results showed that,first of all,Dedlp can unwind RNA duplex on single molecule level,and had ATP concentration dependence,the unwinding rate was slower with longer dsRNA,and had no dependence on the direction of the single-standed RNA,but the unwinding rate of 3' overhang was slightly faster than the 5' overhang.Second,Dedlp major unwound the dsRNA in one-step,and about 30%of the dsRNA were unwound in two steps.Moreover,we observed one or more nonproductive small peaks before RNA duplex were completely unwound,which has no length and polarity dependence.Through analyzing the kinetics of small peaks,there were two or three hidden kinetic steps in it,consistent with the process of ATP binding and hydrolysis.Through the FRET distribution of the small peaks and the middle state of two steps,they were consistent with prying apart 6-10 base pairs.All of these results were consistent with local strand separation mode.So we direct confirmed that Dedlp unwound RNA duplexes by a local strand separation mode with pre-destabilization on single molecule level. |
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