title: A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding.
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.
authors.affiliation: Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal.
authors.forename: Mónica
authors.initials: M
authors.lastname: Amblar
authors.forename: Cecília M
authors.initials: CM
authors.lastname: Arraiano
mesh_terms: D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005095:Exoribonucleases / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D012313:RNA / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; D012313:RNA; D004492:Edetic Acid; D005095:Exoribonucleases; C022535:exoribonuclease II; D008274:Magnesium