Study And Comparison On The Unwinding Mechanism Of DExH/D-box RNA Helicase RHA And Dedlp

RNA helicases which are common in organisms can unwind double stranded RNA or remodel nucleic acid-protein complex by using energy from binding and hydrolyzing NTPs.DExH/D-box RNA proteins,as the largest member of RNA helicases,play key roles in all aspects of RNA metabolism,including pre-mRNA splicing,mRNA export,ribosome biogenesis and translation,RNA degradation,etc.It is well known that the unwinding feature is an important basis for the helicase to remodel the ribonucleoprotein complex and to play key roles in their biochemical functions.At present,the studies on the unwinding mechanism of DExH/D-box RNA helicases have shown that,DExH/D-box RNA helicases can be mainly characterized by two large subfamilies:the DEAD-box helicases and the DExH-box helicases,which exert helicase activity in the "directional translocation"mechanism and the "local strand separation" mechanism,respectively.RNA helicase A(RHA)is a typical member of DExH-box helicases.It utilizes NTPs to unwind duplexes through translocating rapidly,processively and directionally from 3' to 5'.Unlike RHA,Ded1p as a typical member of DEAD-box helicases,was reported to unwind duplex without polarity.Ded1p promotes unwinding initiation by directly binding to the double-stranded region of the substrate with opening a limited number of base pairs.Although some unwinding characteristics of RHA and Dedlp have been revealed,their physical mechanism of nucleic acid recognition and translocation remain obscure.Here,we chose the full length RHA and Ded1p as research object,and used biochemical approaches and smFRET technology to explore the specific unwinding mechanism of these two helicases respectively.1.Expression and purification of RHA in vitroIn this study,293 f cells in suspension culture and sf9 cells were successively selected as host cells to express the full-length RHA protein.The results were as follows:(1)The full-length RHA protein could be expressed in both cells.For 293f cells,the highest expression level for intracellular expression and secretory expression exhibited at 48 h and 120 h hours after transfection respectively.(2)Although the pure recombinant RHA proteins were obtained after the Ni2+-NTA-agarose and Capto DEAE sepharose chromatography purification,the expression level in sf9 cells was significantly higher than that in 293 f cells.2.Unwinding mechanism study on RHAIn this study,using biochemical approaches,we probed the unwinding reactions of RHA on some chemical and structural modified substrates.The results were as follows:(1)RHA translocates efficiently along the 3' overhang of RNA,but not DNA,with a requirement of covalent continuity.Ribose-phosphate backbone lesions on both strands of the nucleic acids,especially on the 3' overhang of the loading strand,affect RHA unwinding significantly.(2)RHA requires RNA on the 3' overhang which directly or indirectly connects with the duplex region to mediate productive unwinding,and the unwinding efficiency increases with increasing the RNA component length.(3)The junction region in the 3'-overhang which is immediately adjacent to the duplex region of the substrates plays a critical role in RHA unwinding reaction.When the RNA component is placed at the junction,even 4 nt can significantly improve the unwinding efficiency of RHA.Collectively,these findings propose a basic loading and translocation mechanism of RHA backbone tracking and unwinding.3.Comparative study on unwinding mechanisms of RHA and DedlpIn this study,using single molecule fluorescence resonance assays,we probed the detailed dynamics of duplexes unwinding for Dedlp at single molecule level.The results were as follows:(1)Several pre-destabilization cycles which have consistent FRET values with separating 6 or 10 base pairs were observed before the actual strand separation.And the frequency of these pre-destabilization cycles are independent on ATP concentration,duplexs length and single-stranded tail polarity.(2)About 75%Dedlp unwinds RNA duplexs in a one-step manner,while the rest in a two-step manner.Collectively,these findings propose a two-pathway unwinding model for Ded1p,in which the "secondary unwinding way" is consistent with the canonical "local strand separation" mechanism.Finally,the comparison of the unwinding mechanism between RHA and Ded1p shows that,both RHA and Ded1p unwind RNA duplex in an ATP-dependent manner.The ssRNA tail of RNA duplex is required for RHA unwinding,but not for Ded1p.RHA promotes unwinding initiation by binding to the ssRNA tail,while Dedlp directly binds to the double-stranded region.In addition,although RHA is a typical polar helicase,it exhibits similar unwinding characteristic with Dedlp for utilizing ssRNA tail that not directly connected with the double-stranded region to facilitate unwinding initiation.