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 |