A conserved sequence element in ribonuclease III processing signals is not required for accurate in vitro enzymatic...

Ribonuclease III of Escherichia coli is prominently involved in the endoribonucleolytic processing of cell and viral-encoded RNAs. Towards the goal of defining the RNA sequence and structural elements that establish specific catalytic cleavage of RNase III processing signals, this report demonstrates that a 60 nucleotide RNA (R1.1 RNA) containing the bacteriophage T7 R1.1 RNase III processing signal, can be generated by in vitro enzymatic transcription of a synthetic deoxyoligonucleotide and accurately cleaved in vitro by RNase III. Several R1.1 RNA sequence variants were prepared to contain point mutations in the internal loop which, on the basis of a hypothetical 'dsRNA mimicry' structural model of RNase III processing signals, would be predicted to inhibit cleavage by disrupting essential tertiary RNA-RNA interactions. These R1.1 sequence variants are accurately and efficiently cleaved in vitro by RNase III, indicating that the dsRNA mimicry structure, if it does exist, is not important for substrate reactivity. Also, we tested the functional importance of the strongly conserved CUU/GAA base-pair sequence by constructing R1.1 sequence variants containing base-pair changes within this element. These R1.1 variants are accurately cleaved at rates comparable to wild-type R1.1 RNA, indicating the nonessentiality of this conserved sequence element in establishing in vitro processing reactivity and selectivity.