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PMID: 1709490 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Nucleic acids research ·Vol. 19 ·No. 8 ·1991-04-25 ·Pages 1759-66

Chelladurai BS, Li H, Nicholson AW

Abstract

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.

MeSH Terms
Base Sequence DNA, Bacterial Endoribonucleases/metabolism Escherichia coli/enzymology,genetics Escherichia coli Proteins Molecular Sequence Data Mutagenesis Nucleic Acid Conformation RNA Processing, Post-Transcriptional RNA, Bacterial/metabolism Ribonuclease III Substrate Specificity T-Phages/genetics Transcription, Genetic
Chemicals
DNA, Bacterial Escherichia coli Proteins RNA, Bacterial Endoribonucleases Ribonuclease III ribonuclease III, E coli
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Chelladurai B S
Department of Biological Sciences, Wayne State University, Detroit, MI 48202.
Li H
Nicholson A W
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/19.8.1759
Published
1991-04-25
Pages
1759-66
Language
English
Region
England
NLM ID
0411011
PMCID
PMC328101
Subset
IM
Grants
NIGMS NIH HHS · GM-41283 · United States
External Links
PubMed source DOI: 10.1093/nar/19.8.1759 PMC Full Text View journal details
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