Overexpression, Purification, and Properties of Escherichia coli Ribonuclease II

Glen A Coburn,George A Mackie

doi:10.1074/jbc.271.2.1048

Glen A Coburn, George A Mackie

Open Access

https://doi.org/10.1074/jbc.271.2.1048

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Abstract

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.

Highlights

Because the rate of synthesis of any given protein is directly proportional to the concentration of its message, regulating the balance between mRNA decay and its synthesis is an important aspect of gene expression
In Escherichia coli, it is widely accepted that mRNA decay is initiated by a series of endonucleolytic cleavages catalyzed by RNase E [1,2,3] or occasionally by RNase III [4, 5] followed by processive exonucleolytic degradation of the message to oligo- and mononucleotides [1,2,3]
Several additional experiments were undertaken to confirm that the 77-nt degradation product corresponds to the product of RNase II stalling 9 nucleotides 3Ј to the double-stranded region of the substrate

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids—The E. coli strain 18 –11 (rnaϪ, rnbϪ, rndϪ, rbnϪ, rntϪ) [20] was obtained from Dr M. 60 mg of the S-30 was loaded onto a column of Affi-Gel blue (Bio-Rad) (1.25 ϫ 21.5 cm) previously equilibrated with 3 column volumes of buffer C (25 mM HEPES1⁄7NaOH, pH 7.5, 5% glycerol, 2 mM DTT, 1 mM MgCl2, 0.1 mM EDTA) containing 500 mM NaCl. The column was washed with 3–5 column volumes of this buffer at a flow rate of 8.3 ml/h (6.75 cm/h) driven by a P1 peristaltic pump (Pharmacia Biotech Inc.). After loading the sample and washing it with 5 column volumes of buffer C containing 150 mM NaCl, the Rnb polypeptide was eluted from this resin with a 50-ml gradient of NaCl (100 – 400 mM) in buffer C at a concentration of 220 mM NaCl. The presence of the Rnb polypeptide in various fractions was monitored qualitatively by polyacrylamide gel electrophoresis and quantitatively by enzyme assay (see above).

RESULTS

DISCUSSION

Methods