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.

Keywords: nucleases; protein-DNA interactions; protein-RNA interactions; π-π interactions.

Figure 1

Crystal structures of RNase T in complex with the cleavable (AA complex) and noncleavable (CC complex) single‐stranded DNA. (A) Overall structure of the dimeric RNase T bound with a DNA segment with a sequence of 5′‐ACC‐3′. (B) In the CC complex (PDB entry: 3V9Z), the DNA with a 3′‐end CC is bound in an inactive conformation with only one Mg²⁺ ion (red sphere) in the active site. (C) In the AA complex (PDB entry: 3V9X), a DNA with a 3′‐end AA is bound in an active conformation with two Mg²⁺ ions in the active site. The grey spheres represent water molecules bound to Mg²⁺. (D) Schematic diagram showing that the 3′‐end AA bases stack with the four phenylalanine side chains in RNase T. The scissor indicates the phophodiester bond that is cleaved by RNase T.

Figure 2

Transformation rescue experiments of the RNase T‐knockout E. coli K12 strains (Δrnt) by expression of RNase T mutants. The RNase T deletion strain (K12‐Δrnt) had a slow‐growth phenotype that can be fully rescued by introducing a plasmid expressing wild‐type RNase T as shown by the colony size variations of E. coli K12 strains on LB plates. The negative control of the “Binding” mutant (F29A/E73A/F77A) could not rescue the slow growth phenotype. The 2W1A and 2Y1A mutants moderately rescued, whereas the 4W, 4W1A, 2W3A, 4Y, 4Y1A, and 2Y3A mutants could not rescue, the slow growth phenotype of the RNase T deletion strain.

Figure 3

The recombinant RNase T mutants share similar secondary structures to that of wild‐type RNase T. (A) The purified recombinant RNase T mutants, 2W1A, 2W3A, 2Y1A, 4Y, and 4Y1A, had a high structural homogeneity as shown by SDS‐PAGE. (B) The circular dichroism (CD) spectra of RNase T mutants 2W3A, 4Y, and 4Y1A are similar to that of wild‐type RNase T. The spectra were represented by mean residue ellipticity (θ) in deg⋅cm²⋅dmol⁻¹.

Figure 4

Mutating phenylalanine residues alters the cleavage preferences of RNase T. (A) RNase T was incubated with a single‐stranded DNA with a different sequence at 3'‐end: 5'‐AGTTATGAXX−3', XX = AA, GG, or CC. The exonuclease activity of the wild‐type RNase T is inhibited by the single‐stranded DNA with a 3'‐end CC, with a cleavage order of preference of AA > GG ≫ CC. (B) The RNase T mutant 2W3A (F29W/F77W/E73A/F124A/F146A) had weaker exonuclease activity but a similar sequence‐specific exonuclease activity to wild‐type RNase T. (C,D) The 4Y (F29Y/F77Y/F124Y/F146Y) and 4Y1A (F29Y/F77Y/F124Y/F146Y/E73A) mutants had an altered sequence‐dependent exonuclease activity compared to wild‐type RNase T with a cleavage preference of A > G ≃ C.

Figure 5

The DNA binding activity and thermal stability of wild type and phenylalanine mutation in RNase T. (A) Gel shift assay revealed that wild‐type RNase T had a higher binding affinity to the single‐stranded DNA (5'‐AACCTTACAAA‐3') than the mutated enzymes, including 2W3A, 4Y, and 4Y1A. (B) The thermal stability of wild‐type and mutated RNase T was monitored by circular dichroism from 1 to 90°C at a wavelength of 220 nm. The melting temperature was 50°C for wild‐type RNase T, 42°C for 2W3A, 40°C for 4Y, and 49°C for 4Y1A.