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

Abstract

The balance between mRNA synthesis and decay is an important aspect of gene expression in all organisms. The RNases are involved in many functions such as RNA processing, stability and degradation, and their concerted action allows strict regulation of the RNA metabolism [1][2][3][4][5]. The mRNA decay in Escherichia coli is normally initiated by a series of endonucleolytic cleavages catalyzed by RNase E [6][7][8] or RNase III [9,10]. The breakdown products are subsequently degraded by the processive 3¢ to 5¢ exoribonucleases PNPase and ⁄ or RNase II [11][12][13][14]. RNase II and PNPase are the major 3¢ to 5¢ exoribonucleases present in E. coli cells. RNase II accounts for 90% of the exoribonucleolytic activity of E. coli crude extracts, while PNPase is responsible for the remaining 10% [15,16]. It is known that the presence of secondary structures in the RNA is an important determinant for mRNA stability. The processive degradation activity of RNase II is easily blocked by stem-loop structures, while PNPase is able to overcome many of the stem-loop structures that it encounters [17][18][19]. In vivo, RNase II can rapidly degrade some polyadenylated stretches necessary for degradation by PNPase and possibly other exoribonucleases [20,21]. As a consequence, RNase II can paradoxically act as a protector of some RNAs from degradation by blocking the access of other 3¢ to 5¢ exoribonucleases [19][20][21][22][23][24].The use of E. coli strains harboring a defective RNase II has been very useful in the study of the cellular function of this protein, as well as in determining 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.

Determination of Key Residues for Catalysis and RNA Cleavage Specificity

“…The R500A mutation practically inactivated the enzyme, although the protein was still able to bind RNA efficiently. A similar result had previously been obtained when mutating the conserved Asp-209 into an Asn (6,26). Comparable results were obtained with the double mutant D201N/Y313F and the triple mutant D201N/Y313F/E390A, which also have their activities highly impaired.…”

“…This protein degrades RNA hydrolytically in the 3Ј to 5Ј direction, releasing 5Ј-nucleotide monophosphates in a processive manner. Its activity is sequence independent but sensitive to secondary structures, and poly(A) is the preferential substrate for this enzyme (2)(3)(4)(5)(6). In Escherichia coli RNase II is responsible for 90% of the hydrolytic activity in crude extracts (7).…”

The role of the S1 domain in exoribonucleolytic activity: Substrate specificity and multimerization

“…The exoribonucleolytic reactions were carried out in a final volume of 10 mL containing 30 nM of substrate. For (His) 6 -RNase II, (His) 6 -RNase IIDS1, and hybrid proteins the activity buffer contained 20 mM Tris-HCl pH8, 100 mM KCl, 1 mM MgCl 2 , and 1 mM DTT (Amblar and Arraiano 2005). For (His) 6 -RNase R and (His) 6 -PNPase the activity buffers used were the optimal buffers previously described for each protein, i.e., 20 mM TrisHCl pH8, 300 mM KCl, 1 mM MgCl 2 , and 1 mM DTT for RNase R (Cheng and Deutscher 2002) and 50 mM Tris-HCl pH8, 60 mM KCl, 1 mM MgCl 2 , 10 mM Na 2 HPO 4 pH8, and 2 mM DTT for PNPase (Spickler and Mackie 2000;Regonesi et al 2004).…”

“…EMSAs with malE-malF mRNA was performed as described previously (Amblar and Arraiano 2005). Mixtures containing an increasing concentration of each enzyme were incubated for 10 min at 37°C and analyzed in a 5% nondenaturing polyacrylamide gel as previously described.…”

“…RNase II can be divided into several functionally distinct regions (Mian 1997;Amblar and Arraiano 2005;Amblar et al 2006): an N-terminal segment (residues 1-103) containing a cold shock domain (CSD) (Graumann and Marahiel 1998) involved in RNA binding, a central region of z400 residues spanning the catalytic RNB domain (Mian 1997), and a C-terminal segment (residues 533-644) that corresponds to an S1 domain (Bycroft et al 1997). Recently, the 3D structure of E. coli RNase II and its RNA complex have been determined McVey et al 2006).…”