Overexpression, Purification, and Properties of Escherichia coli Ribonuclease II
| Autor: | Glen A. Coburn, George A. Mackie |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Exonuclease
Messenger RNA Base Sequence RNase P Molecular Sequence Data Repressor RNA Fast protein liquid chromatography Cell Biology Biology medicine.disease_cause Biochemistry Molecular biology Recombinant Proteins RNA Bacterial Polynucleotide Exoribonucleases Escherichia coli biology.protein medicine Molecular Biology |
| Zdroj: | Journal of Biological Chemistry. 271:1048-1053 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.271.2.1048 |
| Popis: | Ribonuclease II (RNase II) is a major exonuclease in Escherichia coli that hydrolyzes single-stranded polyribonucleotides processively in the 3' to 5' direction. To understand the role of RNase II in the decay of messenger RNA, a strain overexpressing the rnb gene was constructed. Induction resulted in a 300-fold increase in RNase II activity in crude extracts prepared from the overexpressing strain compared to that of a non-overexpressing strain. The recombinant polypeptide (Rnb) was purified to apparent homogeneity in a rapid, simple procedure using conventional chromatographic techniques and/or fast protein liquid chromatography to a final specific activity of 4,100 units/mg. Additionally, a truncated Rnb polypeptide was purified, solubilized, and successfully renatured from inclusion bodies. The recombinant Rnb polypeptide was active against both [3H]poly(A) as well as a novel (synthetic partial duplex) RNA substrate. The data show that the Rnb polypeptide can disengage from its substrate upon stalling at a region of secondary structure and reassociate with a new free 3'-end. The stalled substrate formed by the dissociation event cannot compete for the Rnb polypeptide, demonstrating that duplexed RNAs lacking 10 protruding unpaired nucleotides are not substrates for RNase II. In addition, RNA that has been previously trimmed back to a region of secondary structure with purified Rnb polypeptide is not a substrate for polynucleotide phosphorylase-like activity in crude extracts. The implications for mRNA degradation and the proposed role for RNase II as a repressor of degradation are discussed. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|