Ribonuclease T1

I.U.B.: 3.1.27.3

Ribonuclease T1 is an endoribonuclease, highly specific in its mode of attack. The enzyme cleaves ribonucleic acid (or deaminated ribonucleic acid) between 3'-guanylic residues (or inosine 3'-phosphate and xanthosine 3'-phosphate) and the 5'-hydroxy residues of adjacent nucleotides with the formation of the corresponding intermediate 2',3'-cyclic phosphates. Thorough reviews of properties, structure and function are provided by Uchida and Egami (1971) and Egami et al. (1964). Ribonuclease T1, because of its specificity, has been a commonly used cleavage enzyme for the determination of structure, nearest neighbor frequencies, and sequence of RNA (Miura and Egami 1960; Rushizky and Sober 1962; Miura 1964; Holley et al. 1965; Madison and Kung 1967; Neelon et al. 1967). The enzyme has further application in the preparation of nucleoside 2',3'-cyclic phosphates, the synthesis of oligonucleotides and the removal of RNA from DNA preparations (Saito and Miura 1963). Conditions under which ribonuclease T1 forms exclusive 3',5'-phosphodiester linkages have been described (Rowe and Smith 1970).

Characteristics of Ribonuclease T1 from Aspergillus oryzae:

Enzymatic Reaction (image will open in a new window)

Molecular weight: 11,000 (Egami et al. 1964).

Optimum pH: 7.5 (Egami et al. 1964).

Composition: The complete covalent structure of the enzyme has been reported by Takahashi (1971). A glutamate residue has been identified as part of the active site (Takahashi et al. 1967). See also Sander and Ts'o (1971) and Eftink and Ghiron (1975) and Rüterjans and Pongs (1971).

Extinction coefficient: = 19 (Egami et al. 1964).

Isoelectric point: pH 2.9 (Egami et al. 1964).

Inhibitors and activators: Ag⁺, Zn²⁺, Cu²⁺, and Hg²⁺ at 1 X 10^-3 M are strongly inhibitory. The stimulatory effects of both histidine and EDTA are attributed to chelation of contaminating inhibitor cations.

Specificity: For guanine residues of RNA (Sato-Asano 1959). See also, opening paragraph and reviews noted therein. Irie (1965) has raised questions about the absolute specificity of RT1.

Stability: It was observed by Takahashi (1966) that ribonuclease T1 was resistant to trypsin and chymotrypsin digestion. Studies have shown that this is only true in the presence of phosphate ions. (Shiozawa, 1969). It is hydrolyzed by pepsin with progressive loss of activity (Takahashi 1966). Relatively, RT1 is a stable enzyme. It is not inactivated by brief heating (100°C, 10 minutes at pH 6) a fact to be noted in attempting to terminate catalysis. It is increasingly unstable above pH 9 but is fairly stable to acid conditions. The enzyme is stable near neutrality in the frozen state and in ammonium sulfate suspension for years at 4°C and in solution at 4°C for weeks.