Purification and characterization of the Escherichia coli exoribonuclease RNase R. Comparison with RNase II

Zhuan Fen Cheng, Murray P. Deutscher

Research output: Contribution to journalArticle

137 Scopus citations

Abstract

Escherichia coli RNase R, a 3′ → 5′ exoribonuclease homologous to RNase II, was overexpressed and purified to near homogeneity in its native untagged form by a rapid procedure. The purified enzyme was free of nucleic acid. It migrated upon gel filtration chromatography as a monomer with an apparent molecular mass of ∼95 kDa, in close agreement with its expected size based on the sequence of the rnr gene. RNase R was most active at pH 7.5-9.5 in the presence of 0.1-0.5 mM Mg2+ and 50-500 mM KCI. The enzyme shares many catalytic properties with RNase II. Both enzymes are nonspecific processive ribonucleases that release 5′-nucleotide monophosphates and leave a short undigested oligonucleotide core. However, whereas RNase R shortens RNA processively to di- and trinucleotides, RNase II becomes more distributive when the length of the substrate reaches ∼10 nucleotides, and it leaves an undigested core of 3-5 nucleotides. Both enzymes work on substrates with a 3′-phosphate group. RNase R and RNase II are most active on synthetic homopolymers such as poly(A), but their substrate specificities differ. RNase II is more active on poly(A), whereas RNase R is much more active on rRNAs. Neither RNase R nor RNase II can degrade a complete RNA-RNA or DNA-RNA hybrid or one with a 4-nucleotide 3′-RNA overhang. RNase R differs from RNase II in that it cannot digest DNA oligomers and is not inhibited by such molecules, suggesting that it does not bind DNA. Although the in vivo function of RNase R is not known, its ability to digest certain natural RNAs may explain why it is maintained in E. coli together with RNase II.

Original languageEnglish (US)
Pages (from-to)21624-21629
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number24
DOIs
StatePublished - Jun 14 2002

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this