Study On Enzymatic Property Of DDX21 RNA Helicase

DEAD-box helicase is a kind of molecular motor protein that catalyze dsRNA or dsRNA/DNA unwinding utilizing the energy derived from the hydrolysis of nucleotriglyceride(NTP,generally ATP).DEAD-box helicases are involved in various stages of RNA metabolism(from the transcription to the decline of RNA),the structures are extremely conservative,but the functions are different?DDX21 belongs to the DEAD-box protein family,DDX21 has been reported to work in ribosomal RNA biogenesis,RNA editing and RNA transcription,and play a crucial role in the occurrence of different tumors and various inflammatory reactions.Although there have been many studies on DDX21,the enzymatic and functional characteristics of DDX21 are still unclear due to the immaturity of the research technology or the difficulty of obtaining the materials of DDX21 and RNA.In the paper,we systematically studied the physicochemical properties of DDX21 and its truncated mutants,it lays the foundation for the follow-up study of function and structure.In this paper,we used the new biochemical and biophysical new technique such as gel filtration chromatography,the dynamic laser scattering,fluorescence anisotropy value analysis and Stopped-flow to inllustrate the aggregation state and the depolymerization state caused by nucleic acid,and the biochemical characteristics of human full-length DDX21 in detail.We further examined the function of the GUCT_RHII domain and four FRGQR repeats near the C-terminus of DDX21,and found that this region induces the dissociation of the multimeric form,and is associated with the binding affinity,unwinding activity,and annealing activity of DDX21 RNA helicase.The main achievements are as follows:Firstly,prokaryotic expression vectors of human full-length DDX21 RNA helicase and various truncated proteins were constructed using in vitro recombinant technology.The recombinant protiens were overexpressed in E.coli.And high-quality and high-purity proteins were obtained through a series of optimization and purification methods.Analysis of the oligomeric form of each protein using dynamic light scattering and gel filtration,the study found that the full-length DDX21 RNA helicase are polymer(about pentamer)in solution.In the presence of nucleic acid RNA,dsDNA and special structure(G-quadruplex),the polymer physically depolymerized to low molecular mass compound.It was found that the oligomeric form of DDX21 was significantly regulated by the N-terminal non-functional region(181 amino acids at the N-terminal)and the four FRGQR repeat domains at the C-terminal: when 181 amino acids at the N-terminal are missing,DDX21 will turn into dimer protein;when the four FRGQR repeat domains at the C-terminal are deleted,it will be transformed into a superpolymer protein with poor homogeneity.Secondly,the binding reactions between proteins and nucleic acids were compared and analyzed by fluorescence polarization technique.It was found that when full-length DDX21 protein combined with ssDNA,dsDNA and RNA substrates of various structural,the binding ability increased with the increase of the length of nucleic acid substrates,and finally reached a stable trend.Its affinity to ssDNA was much lower than to dsDNA and RNA substrates.Affinity,however,is relatively strong for DNA with special sequence(For example,G-quadruplex).The ssRNA binding ability of truncated proteins lacking N-terminal 181 aa is basically the same as that of full-length proteins.While the ssRNA binding activity of truncated proteins lacking only four repetitive FRGQR domains in C-terminal will be severely reduced.Then,the unwinding characteristics of DDX21 were analyzed by stopped-flow spectrophotometry.The results showed that the protein had bi-directional unwinding activity to dsRNA and hybrid dsRNA/DNA.It was found that the unwinding amplitude of DDX21 RNA helicase depended significantly on the length of the tail of the substrate.The longer the tail chain is,the higher the amplitude of unwinding is,and the faster the rate of unwinding is.Similarly,the stopped-flow spectrophotometry technique was used to analyze the unwinding of truncated proteins,it was found that the three domains of DEADc,HELICc and GUCT_RHII jointly participate in the unwinding function of DDX21.Finally,the annealing activity of DDX21 RNA helicase was analyzed by stopped-flow spectrophotometry technology.It was found that the full-length protein not only annealed dsRNA and hybrid dsRNA/DNA,but also annealed two single-stranded DNA,and its annealing activity was stronger than that of pure-stranded RNA and hybrid-stranded RNA/DNA.However,DDX21 C-terminal four FRGQR repeating domains play a major role in regulating its annealing activity,the lacking of four FRGQR repetitive domains at C-terminal causeed the loss of annealing ability.This study revealed that the GUCT_RHII domain of DDX21 and the four FRGQR repetitive domains at C-terminal play an important role in its structure and function,which provides an important theoretical basis for the future study of the structure and cell function of DDX21.In conclusion,this paper systematically studied the enzymatic properties of DDX21,the regulation mechanism of DDX21 oligomerization and its unwinding and annealing activities were described.