Expression, Purification And Activity Analysis Of A DEAH-box RNA Helicase AtRH59in Arabidopsis

RNA helicases are enzymes that utilize NTP energy derived from the hydrolysis tounwind double-strand RNA and modify RNA structures. RNA helicases are proved to beinvolved in every step of the activities related with RNA, including nuclear transcription,pre-mRNA splicing, translation, RNA degradation, ribosome biogenesis and assembly. Now,the research involved in the functions of RNA helicases has been a focus in life sciences.In our previous research, we isolated an Arabidopsis SALK T-DNA insertion mutant, inwhich the development of female gametophytes was delayed. The TAIL-PCR result revealedthat there was a T-DNA insertion in RNA Helicase gene AtRH59. Amino acid sequenceanalysis showed that AtRH59encoded a DEAH-box protein of RNA helicases. AtRH59had ahigh homology with yeast Prp22p, which involved in the splicing of the small ribosomalsubunit synthesis and18s rRNA precursors. These results suggested that AtRH59may beinvolved in rRNA splicing, protein synthesis and the development of the gametophytes. Atpresent, the activity of some DEAD-box RNA helicases have been reported, but have no studyin DEAH-box RNA helicase. In this paper, we cloned, expressed and purified a putative RNAhelicase containing DEAH box, AtRH59from Arabidopsis. We analyzed its activity. Themain results are as follows:（1） AtRH59possessed eight conserved motifs Ⅰ, Ⅰa, Ⅰb, Ⅱ, Ⅲ, Ⅳ, Ⅴ and Ⅵ in N terminal,which indicated that AtRH59protein belonged to DEAH-box RNA helicase.（2） The protein activity assay showed that AtRH59had the ATPase activity and couldunwind the dsDNA and dsRNA with the hydrolysis of ATP. Further analysis showed that theactivity of AtRH59helicase was affected by Mg²⁺concentration.（3） We detected the activity of AtRH59by designing different structures of substrates,which formatted by dsRNA and dsDNA. The results showed that AtRH59had strong helicaseactivity to unwind the nucleotide double chain with free3’ and5’ end but almost had no activity to unwind the nucleotide double chain with blunt-end. Further study found thatAtRH59had a weak polarity of unwinding activity and it was preferential to unwind dsRNAalong the3’-5’direction.