TY - JOUR
T1 - A conserved sequence element in ribonuclease III processing signals is not required for accurate in vitro enzymatic cleavage
AU - Chelladurai, Bhadrani S.
AU - Li, Honglin
AU - Nicholson, Allen W.
N1 - Funding Information:
We thank Dr. Robin Hood of the Wayne State Central Instrumentation Facility (Department of Chemistry) and Dr. June Snow of the Wayne State Macromolecular Facility (Department of Biochemistry) for synthesis of deoxyoligonucleotides. A generous initial provision of RNase HI from Hugh Robertson (Cornell University Medical College) is gratefully acknowledged. We also thank Drs. J.Dunn, F.W.Studier and A.Rosenberg (Brookhaven National Laboratory) for providing plasmids pAR1219 and pAR2637. The efforts of J.Butler in assembling the manuscript, and comments by Dr. A.Siegel are much appreciated. This work was supported by Grant GM-41283 to A.W.N. from the National Institutes of Health, and by the Center for Molecular Biology, Wayne State University.
PY - 1991/4/25
Y1 - 1991/4/25
N2 - 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 17 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.
AB - 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 17 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.
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U2 - 10.1093/nar/19.8.1759
DO - 10.1093/nar/19.8.1759
M3 - Article
C2 - 1709490
AN - SCOPUS:0025829785
SN - 0305-1048
VL - 19
SP - 1759
EP - 1766
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 8
ER -