Mutational Analysis of the Length of the J3/4 Domain of Escherichia coli Ribonuclease P Ribozyme

Abstract

We prepared a series of length variants of the J3/4 domain of Escherichia coli ribonuclease P (RNase P) ribozyme: the four-base long J3/4 domain (A⁶²G⁶³G⁶⁴A⁶⁵) was replaced with GGA (denoted ΔA), GA (ΔAG), A (ΔAGG), AAGGA (ΣA), AAAGGA (ΣAA), and AAAAGGA (ΣAAA). The results indicated that truncating and inserting operations of the J3/4 domain drastically reduced ribozyme activity (WT>>ΣAA>ΣA>ΣAAA>>ΔAG>ΔA, ΔAGG), but did not affect the cleavage site selection of a substrate by the ribozyme. The reduced ribozyme activity of each mutant was rescued to some extent by the addition of a high concentration of magnesium ions. Our data indicate that the conserved AGGA sequence was important for efficient ribozyme reactions, and suggested that the length mutations affected ribozyme activity through metal ion binding steps.