A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding

Mónica Amblar, Cecília M. Arraiano

Research output: Contribution to journalArticlepeer-review

38 Citations (Scopus)

Abstract

Exoribonuclease II (RNase II), encoded by the rnb gene, is a ubiquitous enzyme that is responsible for 90% of the hydrolytic activity in Escherichia coli crude extracts. The E. coli strain SK4803, carrying the mutant allele rnb296, has been widely used in the study of the role of RNase II. We determined the DNA sequence of rnb296 and cloned this mutant gene in an expression vector. Only a point mutation in the coding sequence of the gene was detected, which results in the single substitution of aspartate 209 for asparagine. The mutant and the wild-type RNase II enzymes were purified, and their 3′ to 5′ exoribonucleolytic activity, as well as their RNA binding capability, were characterized. We also studied the metal dependency of the exoribonuclease activity of RNase II. The results obtained demonstrated that aspartate 209 is absolutely essential for RNA hydrolysis, but is not required for substrate binding. This is the first evidence of an acidic residue that is essential for the activity of RNase II-like enzymes. The possible involvement of this residue in metal binding at the active site of the enzyme is discussed. These results are particularly relevant at this time given that no structural or mutational analysis has been performed for any protein of the RNR family of exoribonucleases.

Original languageEnglish
Pages (from-to)363-374
Number of pages12
JournalFEBS Journal
Volume272
Issue number2
DOIs
Publication statusPublished - 1 Jan 2005

Keywords

  • Exoribonuclease
  • RNA binding
  • RNA degradation
  • RNase II
  • RNR family

Fingerprint Dive into the research topics of 'A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding'. Together they form a unique fingerprint.

Cite this