Insights into the sequence specificity of RNase T in digestion of nucleic acids

• Main Authors: Yulander Duh, 杜莉雲
• Other Authors: Hanna S. Yuan
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/05281701565007438829

碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 100 === RNase T is a 3'-to-5' DEDDh-family exonuclease, participating in the 3' maturation of stable RNA, including tRNA and rRNA, in E. coli. RNase T has a unique sequence preference where its exonuclease activity is inhibited by the substrates with a 3'-terminal cytosine (C), referred to as the C effect. Our previous crystal structural analysis of RNase T-DNA complexes suggests that four phenylalanine residues, F29, F77, F124 and F146 stack with the two 3'-terminal bases and probably dominate the C effect of RNase T. It has been shown that phenylalanine prefers not to make pi-pi stacking interactions with C, whereas tyrosine prefers A but not G, and tryptophan has no specific sequence preference. To elucidate the structural basis of the sequence specificity of RNase T, here we mutated the four phenylalanine residues to tryptophan (W mutants) and tyrosine (Y mutants). We expected that the engineered Y mutants should be inhibited by the substrates with 3'-terminal G, and the W mutants should be less sequence specific favoring all four types of nucleotides. We found that the E. coli strains expressing the W mutants or Y mutants had slow growth phenotypes similar to that of the RNast T knockout strain. The DNA digestion assays showed that the Y mutants had altered sequence preference as it was inhibited by the ssDNA with 3'-terminal C and G. The W mutants had weaker exonuclease activities but a similar sequence specificity as that of wild-type RNase T. DNA binding assays and thermal stability monitored by circular dichroism (CD) further showed that the W and Y mutants all had reduced DNA-binding affinity and reduced thermal stability as compared to that of wild-type enzyme. Our results suggest that the four phenylalanines in RNase T not only play critical roles in substrate binding and selection, but also in overall protein stability. The RNase T with the four Phe mutated to Tyr indeed had different sequence specificity. Nevertheless, these RNase T mutants are not stable, with reduced substrate-binding activities thus not functional in cells. These results imply that the hydrophobic residues in the DEDDh family of exonucleases, that are matched at the same positions as those of the four phenylalanine residues in RNase T, are likely important for substrate binding and selection.