標題: Aromatic residues in RNase T stack with nucleobases to guide the sequence-specific recognition and cleavage of nucleic acids
作者: Duh, Yulander
Hsiao, Yu-Yuan
Li, Chia-Lung
Huang, Jason C.
Yuan, Hanna S.
生物科技學系
Department of Biological Science and Technology
關鍵字: protein-DNA interactions;protein-RNA interactions;nucleases;pi-pi interactions
公開日期: 十二月-2015
摘要: RNase T is a classical member of the DEDDh family of exonucleases with a unique sequence preference in that its 3\'-to-5\' exonuclease activity is blocked by a 3\'-terminal dinucleotide CC in digesting both single-stranded RNA and DNA. Our previous crystal structure analysis of RNase T-DNA complexes show that four phenylalanine residues, F29, F77, F124, and F146, stack with the two 3\'-terminal nucleobases. To elucidate if the pi-pi stacking interactions between aromatic residues and nucleobases play a critical role in sequence-specific protein-nucleic acid recognition, here we mutated two to four of the phenylalanine residues in RNase T to tryptophan (W mutants) and tyrosine (Y mutants). The Escherichia coli strains expressing either the W mutants or the Y mutants had slow growth phenotypes, suggesting that all of these mutants could not fully substitute the function of the wild-type RNase T in vivo. DNA digestion assays revealed W mutants shared similar sequence specificity with wild-type RNase T. However, the Y mutants exhibited altered sequence-dependent activity, digesting ssDNA with both 3\'-end CC and GG sequences. Moreover, the W and Y mutants had reduced DNA-binding activity and lower thermal stability as compared to wild-type RNase T. Taken together, our results suggest that the four phenylalanine residues in RNase T not only play critical roles in sequence-specific recognition, but also in overall protein stability. Our results provide the first evidence showing that the pi-pi stacking interactions between nucleobases and protein aromatic residues may guide the sequence-specific activity for DNA and RNA enzymes.
URI: http://dx.doi.org/10.1002/pro.2800
http://hdl.handle.net/11536/133636
ISSN: 0961-8368
DOI: 10.1002/pro.2800
期刊: PROTEIN SCIENCE
Volume: 24
Issue: 12
起始頁: 1934
結束頁: 1941
顯示於類別:期刊論文