title: Aromatic residues in RNase T stack with nucleobases to guide the sequence-specific recognition and cleavage of nucleic acids.
abstract: 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 π-π 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 π-π stacking interactions between nucleobases and protein aromatic residues may guide the sequence-specific activity for DNA and RNA enzymes.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, 11529, Republic of China.|Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan, 112, Republic of China.
authors.forename: Yulander
authors.initials: Y
authors.lastname: Duh
authors.affiliation: Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, 30068, Republic of China.
authors.forename: Yu-Yuan
authors.initials: YY
authors.lastname: Hsiao
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, 11529, Republic of China.
authors.forename: Chia-Lung
authors.initials: CL
authors.lastname: Li
authors.affiliation: Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan, 112, Republic of China.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Huang
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, 11529, Republic of China.|Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei, Taiwan, 10048, Republic of China.
authors.forename: Hanna S
authors.initials: HS
authors.lastname: Yuan
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005095:Exoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013696:Temperature
chemical_list: D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D005095:Exoribonucleases; C042081:exoribonuclease T
keywords: nucleases; protein-DNA interactions; protein-RNA interactions; π-π interactions