TAILIEUCHUNG - Báo cáo khoa học: A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding

Exoribonuclease II (RNase II), encoded by thernb gene, is a ubiquitous enzyme that is responsible for 90% of the hydrolytic activity in Escherichia colicrude extracts. The E. colistrain SK4803, carrying the mutant allelernb296, has been widely used in the study of the role of RNase II. We determined the DNA sequence ofrnb296 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 | ềFEBS Journal A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding Monica Amblar and Cecilia M. Arraiano Institute de Tecnologia Química e Biológica Universidade Nova de Lisboa Oeiras Portugal Keywords RNase II exoribonuclease RNA degradation RNA binding RNR family Correspondence C. M. Arraiano Instituto de Tecnologia Quimica e Biologica Universidade Nova de Lisboa Apartado 127 2781-901 Oeiras Portugal Fax 351 21 4411277 Tel 351 21 4469547 E-mail cecilia@ Received 30 July 2004 revised 29 October 2004 accepted 11 November 2004 doi 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. The balance between mRNA synthesis and decay is an important aspect

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