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

A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding Mo ´ nica Amblar and Cecı ´lia M. Arraiano Instituto de Tecnologia Quı ´ mica e Biolo ´ gica ⁄ Universidade Nova de Lisboa, Oeiras, Portugal 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 [1–5]. The mRNA decay in Escherichia coli is normally initiated by a series of endonucleolytic cleavages catalyzed by RNase E [6–8] or RNase III [9,10]. The breakdown products are subsequently degra- ded by the processive 3¢ to 5¢ exoribonucleases PNPase and ⁄ or RNase II [11–14]. 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 [17–19]. In vivo, RNase II can rapidly degrade some polyadenylated stretches necessary for degradation by PNPase and possibly other exoribonuc- leases [20,21]. As a consequence, RNase II can paradox- ically act as a protector of some RNAs from degradation by blocking the access of other 3¢ to 5¢ exo- ribonucleases [19–24]. The use of E. coli strains harboring a defective RNase II has been very useful in the study of the cellu- lar function of this protein, as well as in determining Keywords RNase II; exoribonuclease; RNA degradation; RNA binding; RNR family Correspondence C. M. Arraiano, Instituto de Tecnologia Quı ´ mica e Biolo ´ gica ⁄ Universidade Nova de Lisboa, Apartado 127, 2781–901 Oeiras, Portugal Fax: +351 21 4411277 Tel: +351 21 4469547 E-mail: cecilia@itqb.unl.pt (Received 30 July 2004, revised 29 October 2004, accepted 11 November 2004) doi:10.1111/j.1742-4658.2004.04477.x 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 evi- dence 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 per- formed for any protein of the RNR family of exoribonucleases. Abbreviations His(6)-RNase II, RNase II with a six-His-Tag fused at the N-terminal end; His(6)-RNase IID209N, His(6)-RNase II with the amino acid substitution D209N; IPTG, isopropyl thio-b-d-galactoside; nt, nucleotides; pol, polymerase; K D , equilibrium dissociation constant; ss, single strand; PAA, polyacrylamide; UE, units of enzymatic activity, namely the amount of protein required for the release of 10 nmol of [ 3 H]AMP in 15 min at 30 °C. FEBS Journal 272 (2005) 363–374 ª 2004 FEBS 363 the role of other ribonucleases [11,21,24–29]. For instance, the rnb500 temperature-sensitive mutant strain demonstrates that the absence of both RNase II and PNPase activity leads to cell death [25]. Despite the wide use of these mutant strains during the last 20 years, nothing is known about the mutations responsible for such phenotypes. The study of the molecular basis of the absence of RNase II activity in these strains will highlight some important information in the knowledge of RNase II proteins. To date, there are no structural or mutational data available from any other proteins of the family. The SK4803 strain is par- ticularly interesting since the mutant gene (rnb296) encodes an inactive RNase II enzyme [25]. In this report we demonstrate that the single amino acid sub- stitution Asp209fiAsn in RNase II is able to cause the total inactivation of the enzyme without affecting its RNA binding capability. In addition, metal ions seem to be required for activity but not for substrate bind- ing, suggesting the involvement of Asp209 in metal binding at the active site of the enzyme. Results Cloning of the rnb296 mutant gene and overexpression of the mutant protein The E. coli SK4803 strain deficient in RNase II acti- vity, which carries the rnb296 allele, has been previ- ously described [25]. Crude extracts from this strain were totally inactive in the degradation of polyadenylic acid [poly(A)] but this seemed to have no detrimental effect on cell viability or mRNA degradation rate [25]. In order to identify the mutation(s) responsible for this phenotype, we sequenced the gene encoding the RNase II protein of this mutant strain. To achieve this, the chromosomal DNA of E. coli SK4803 was used as a source in the PCR amplification of the rnb296 gene and the DNA sequence of the PCR product was deter- mined. The results revealed that the rnb296 gene differs only in one base from the wild-type rnb gene: G1148 was replaced with A in the rnb296 mutant gene and this single mutation leads to the substitution of Asp209 with Asn in the RNase II sequence. RNase II is the prototype of the widely distributed RNR family of ribonucleases. It has been hypothesized that the cata- lytic activity of the RNR proteins resides in their cen- tral domain, named RNB. Asp209 of RNase II lies in this RNB domain, more specifically in the highly con- served motif I of this domain (Fig. 1), and is present in almost all members of the RNR family of exoribonuc- leases [30,31]. All of these data suggest that Asp209 might have a key role in the RNase II enzyme. In order to analyze the function of this residue, the rnb296 mutation was cloned into the previously described pFCT6.9 plasmid [29]. This plasmid contains the wild- type rnb gene from E. coli cloned into the pET15 fusion vector (Novagen). Under isopropyl thio-b-d-galactoside (IPTG) induction, the pFCT6.9 plasmid directs the expression of the six-histidine-tagged RNase II protein [His(6)-RNase II] that was previously shown to be act- ive [29]. The 996-bp NheI fragment from the rnb296 gene containing the corresponding mutation (G1148A) was cloned into plasmid pFCT6.9 obtaining pMAA (see Experimental procedures). The presence of the cor- rect 296 mutation was confirmed by DNA sequencing, and the pMAA plasmid was further transferred to E. coli BL21(DE3) to overproduce the corresponding six-histidine-tagged RNase IID209N mutant protein [His(6)-RNase IID209N]. The suitability of E. coli BL21(DE3)[pMAA] as a source of His(6)-RNase IID209N was tested by induc- tion of the corresponding cells with IPTG at 37 °C. Samples were taken at different induction times and the protein content was analyzed by SDS ⁄ PAGE (Fig. 2). The results revealed that, after 2 h of IPTG treatment, His(6)-RNase IID209N was the major pro- tein in cell extracts, corresponding to  14% of the total protein content (Fig. 2A). The solubility of the protein upon induction was also analyzed and the results revealed that more than 70% of the His (6)-RNase IID209N was soluble (Fig. 2B), similar to Fig. 1. Schematic representation of the structure of RNase II. Three different domains can be proposed for RNase II: the N-terminal cold shock domain (CSD), the central RNB domain (RNB), and the C-terminal S1 domain (S1). The four sequence motifs of the RNB domains are depicted (I–IV) and the sequence pattern of the motif I is shown. The syntax of the pattern follows that used in PHI-BLAST searches (http://bioweb.pasteur.fr/seqanal/interfaces/ phiblast.html#pattern). Residues conserved in > 80% of the ana- lyzed sequences are shown as bold letters. The position corres- ponding to the D209N mutation is indicated with an arrow. RNase II mutant with RNA binding but no activity M. Amblar and C. M. Arraiano 364 FEBS Journal 272 (2005) 363–374 ª 2004 FEBS that obtained with His(6)-RNase II protein from BL21(DE3) cells containing pFCT6.9 (data not shown). These results revealed that, as with the wild-type enzyme [29], the maximum induction of His(6)-RNase IID209N is reached after 2 h of IPTG treatment, and these were the conditions used for fur- ther purification of the protein. The exoribonucleolytic activity of the crude extracts, before and after induction with IPTG, was also ana- lyzed using poly[8- 3 H]adenylic acid as a substrate and the results obtained are summarized in Table 1. The activity levels of the extracts from cells overexpressing His(6)-RNase IID209N protein (BL21(DE3)[pMAA]) after 2 h of induction were similar to those obtained with the BL21(DE3) without plasmid. In contrast, the presence of plasmid pFCT6.9, encoding the wild-type His(6)-RNase II enzyme, produced a 60-fold induction of RNase II activity after 2 h of IPTG treatment. Therefore, the His(6)-RNaseII-D209N protein pro- duced upon IPTG induction was totally inactive, and this inactivation was exclusively caused by the substitu- tion of Asp209 with Asn. Purification and properties of His(6)-RNase IID209N A purification procedure for the His(6)-RNase II pro- tein has been previously described [29]. Based on this initial protocol, a new purification procedure for His (6)-RNase IID209N protein and the wild-type enzyme was standardized by the introduction of several modifi- cations. Cultures of 100 mL of BL21(DE3) cells con- taining the pMAA or pFCT6.9 plasmids, respectively, were grown at 37 °C and induced with IPTG for 2 h. Then, the cells were disrupted and treated as described in the Experimental procedures, and the extracts obtained were applied to chelating sepharose high per- formance columns previously charged with Ni 2+ ions. Different concentrations of imidazol-eluting agent were tested to develop the purification procedure described in the Experimental procedures. Following this procedure, His(6)-RNase IID209N and His(6)-RNase II proteins were obtained with > 90% purity. The exoribonucleolytic activity of purified enzymes was first tested by using the linear substrate, poly[8- 3 H]adenylic acid. The D209N mutant protein was unable to degrade the polyribonucleotide and had Table 1. Specific exoribonucleolytic activity in crude extracts from BL21(DE3) overproducer strains on poly(A) substrate. The exoribo- nuclease activity was measured before and after 2 h of isopropyl thio-b- D-galactoside (IPTG) induction of BL21(DE3) containing the indicated plasmid. BL21(DE3) cells without plasmid were used as a control. Each value is the mean of at least three independent experiments. UE, the amount of protein required for the release of 10 nmol of [ 3 H]AMP in 15 min at 30 °C. Specific exoribonuclease activity (UEÆlg )1 of protein) BL21(DE3) BL21(DE3) [pFCT6.9] BL21(DE3) [pMAA] Before IPTG induction 0.12 0.37 0.03 After 2 h of IPTG induction 0.22 22.42 0.17 AB Fig. 2. Overexpression of RNase IID209N by induction with isopropyl thio-b-D-galactoside (IPTG). (A) Crude extracts from BL21(DE3) cells harboring the pMAA plasmid were induced by IPTG. Samples were withdrawn at the time-points indicated in the figure, after the addition of IPTG, and the total protein content was analyzed by electrophoresis in a 0.1% SDS, 10% acrylamide gel. (B) The soluble (S) and insoluble (I) protein fraction from cultures induced for 2 h with IPTG were analyzed in a 0.1% SDS, 10% polyacrylamide gel. The His(6)-RNase IID209N (RNase IID209N) protein is indicated with an arrow. St, molecular mass maker. M. Amblar and C. M. Arraiano RNase II mutant with RNA binding but no activity FEBS Journal 272 (2005) 363–374 ª 2004 FEBS 365 no detectable activity, even when higher amounts of protein were used (23 lg per reaction). By contrast, the purified His(6)-RNase II was highly active in the degra- dation of poly[8- 3 H]adenylic acid, with an activity of > 325 UEÆlg )1 of protein (results not shown) (UE: units of enzymatic activity, namely the amount of pro- tein required for the release of 10 nmol of [ 3 H]AMP in 15 min at 30 °C). Previous studies on the RNase II enzyme revealed that its activity is blocked by double-stranded structures on the RNA molecule [12,13,18,19]. Various mRNA transcripts harboring stem–loop structures have been tested as RNase II substrates [13,18,19,32–34] and in all cases the enzyme catalyzed the degradation of the sin- gle-stranded (ss) portion of the RNA molecule from its 3¢ end until it reached the double-stranded region. In order to analyze the effect of the D209N mutation on the exoribonucleolytic activity of RNase II on struc- tured substrates, we tested the degradation ability of both His(6)-RNase II and the D209N mutant enzyme by using two different mRNAs, namely SL9A [13] and malE-malF [18]. The SL9A substrate is a small RNA molecule of 83 nucleotides (nt), consisting of an ss 3¢- extension (of 41 nucleotides), which mimics a typical bacterial poly(A) tail, plus a stem–loop structure (9 bp stem and four-residue loop) and a short 5¢-single stran- ded arm (of 19nt). The exonuclease assays performed revealed that His(6)-RNase II, similarly to that previ- ously reported for RNase II [13], degrades the SL9A RNA substrate in a two-step process (Fig. 3A). The enzyme initially catalyzes a rapid shortening of the RNA molecule from its 3¢ end, generating a set of inter- mediates, followed by the further degradation of these intermediates at a slower rate. As shown in Fig. 3A, the SL9A substrate was totally converted into shorter inter- mediate products in only 30 s by 2 nm purified His(6)- RNase II. These intermediates, partially resistant to degradation, presumably correspond to the stem–loop structure with a 3¢ ss extension of  6–9 nucleotides [13]. Longer reaction times (up to 30 min) resulted in the diminution of the intermediate length as a result of limited digestion by the enzyme. By contrast, the D209N mutant enzyme was unable to degrade the SL9A RNA. As shown in Fig. 3A, 100% of the full- length starting material remained intact, even after 30 min of incubation with 540 nm of the mutant enzyme. Similar results were obtained with the longer mRNA transcript corresponding to the intergenic region of the malE-malF operon. This substrate consists of a 375 nucleotides RNA molecule containing two stem–loop structures: a large secondary structure formed by the two inverted palyndromic REP sequences; and a smaller and weaker secondary structure at the 3¢ end of the mRNA [18,35]. As with the other substrates tested, no exoribonuclease activity was detected with the D209N mutant on the malE-malF transcript, even after 30 min of incubation (Fig. 3B), confirming the complete inactivation of the enzyme caused by the mutation. However, His(6)-RNase II was highly active in the degradation of this substrate and in only 30 s 70% of the full-length product disappeared (Fig. 3B). In agreement with data previously reported for the wild-type RNase II enzyme [18], digestion of the malE-malF transcript by the fusion derivative His(6)- RNase II rendered two main intermediate products: P1 and P2 (Fig. 3). Such intermediates presumably corres- pond to the stalling of the enzyme in the vicinity of the two secondary structures of the mRNA. Metal dependency of the exoribonuclease activity of RNase II The above results pointed out the importance of Asp209 for RNase II, as its substitution with Asn leads to a total inactivation of the enzyme. RNase II, like other exoribonucleases, requires the presence of Mg 2+ in the reaction to catalyze the degradation of RNA. The acidic nature of Asp209, together with its conservation in RNase II-like enzymes, suggests that this residue is one of the metal ligands at the active site of RNase II. If this assumption is correct, a reduced affinity for the metal ion should be expected in the D209N mutant protein. Consequently, the use of a higher Mg 2+ concentration might allow us to detect some nuclease activity with the mutant enzyme. To test this hypothesis, the exoribonuclease activity of His(6)- RNase II and the D209N mutant protein was tested in the presence of different concentrations of the metal ion. MgCl 2 concentrations ranging from 0.5 lm to 10 mm were assayed by using the malE-malF tran- script (Fig. 4A). Quantification of the reaction prod- ucts revealed that His(6)-RNase II was able to degrade the mRNA within a wide range of Mg 2+ concentra- tions (Fig. 4B). The enzyme was highly active at all MgCl 2 concentrations tested, although the maximum activity was obtained between 5 lm and 1 m m. Inter- estingly, the rate between the P1 and P2 products var- ied depending on the metal ion concentration. The P2 intermediate was the main product obtained at lower metal concentrations (from 0 to 100 lm), while P1 was only observed at MgCl 2 concentrations of ‡ 500 lm, being the major product at ‡ 1mm MgCl 2 . These results suggest different properties of the exoribo- nucleolytic activity of RNase II depending on the metal ion concentration. Surprisingly, activity assays performed without adding MgCl 2 to the reaction RNase II mutant with RNA binding but no activity M. Amblar and C. M. Arraiano 366 FEBS Journal 272 (2005) 363–374 ª 2004 FEBS mixture revealed a residual activity of His(6)-RNase II that only disappeared after the addition of 10 mm EDTA. This fact indicates that some essential Mg 2+ atoms are bound to the protein and that they cannot be easily removed from the protein structure simply by buffer-exchange. In the case of the D209N mutant pro- tein, no exoribonuclease activity was detected at any metal ion concentration tested (data not shown), indi- cating that the loss of activity in the mutant protein cannot be restored by increasing the Mg 2+ concentra- tion. RNA binding ability of RNase II and the D209N mutant The data presented above demonstrate that the D209N amino acid substitution leads to a loss in the exoribo- nucleolytic activity of RNase II. Such inactivation can be caused by a defect in the catalytic reaction, by a decrease in substrate affinity, or both. To investigate whether the D209N mutation impairs the RNA bind- ing, band-shift assays were performed with the radio- actively labeled transcripts SL9A and malE-malF. In order to reduce the degradation of the substrate upon binding of the wild-type enzyme, the incubation was performed at different temperatures (from 15 °Cto 37 °C). The results obtained showed that, even at 15 °C, the incubation of His(6)-RNase II with either SL9A or malE-malF transcripts resulted in bands with higher gel mobility than the free substrate (Fig. 5). Such bands corresponded to degradation products, and the intensity increases with the protein concentra- tion. By contrast, the D209N protein generated only retardation bands with both transcripts. These bands Fig. 3. Exoribonuclease activity of RNase II and the D209N mutant enzyme on mRNA transcripts. The exoribonuclease activity of His(6)-RNase II (wild-type, WT) and His(6)-R- Nase IID209N (D209N) enzymes was assay- ed by using SL9A mRNA (A) or malE-malF mRNA (B) transcripts. Reactions were per- formed as described in the Experimental procedures using 2 n M WT enzyme or 540 n M D209N mutant protein. Samples were taken at the time-points indicated in the figure, and the reaction products were analyzed in 8% polyacrylamide (PAA) (A) or 6% PAA (B), 7 M urea gels. A schematic representation of the substrates and reac- tion products is depicted. M. Amblar and C. M. Arraiano RNase II mutant with RNA binding but no activity FEBS Journal 272 (2005) 363–374 ª 2004 FEBS 367 correspond to RNA–protein complexes, indicating that, despite the mutation, the D209N protein is able to bind RNA. To investigate the role of MgCl 2 in formation of the RNA–protein complex, binding assays were performed with the malE-malF substrate at 37 °C in the presence or in the absence of EDTA. As shown in Fig. 6A, incubation of the wild-type protein with the RNA sub- strate in the absence of EDTA resulted in the expected degradation bands. However, when 10 mm EDTA was added to the binding reaction, the degradation of the substrate was inhibited and only retarded bands cor- responding to RNA–protein complexes were detected. These results indicate that RNase II requires Mg 2+ ions for catalysis but not for substrate binding. RNA– protein complexes were detected from 5 nm of wild- type enzyme when the incubation was performed in the presence of EDTA.With the D209N mutant enzyme, retarded bands were observed either in the presence or absence of EDTA, and in both conditions RNA–protein complexes were also observed from 5 to 10 nm of protein (Fig. 6B). The equilibrium dissoci- ation constant (K D ) values of both wild-type and mutant proteins were estimated from gel-shift assays. The values obtained in the presence of EDTA were 382 nm for the wild-type protein and 344 nm for the D209N mutant. In the absence of EDTA, the K D value for the mutant protein was 330 nm. The K D of the two proteins was analogous, showing that both enzymes have similar affinity for this substrate. These results clearly indicate that the D209N mutation does not affect the ability of RNase II to form stable RNA– protein complexes and that the presence or the absence of Mg 2+ does not influence the substrate binding of the mutant protein. Discussion Eight different 3¢ to 5¢ exoribonucleases have been char- acterized in E. coli and this group of enzymes accounts for all the exoribonucleolytic activities present in an E. coli cell [36]. These enzymes have been grouped into six superfamilies and various subfamilies based on extensive sequence analysis and catalytic properties [31]. The RNase II belongs to the RNR family of exo- ribonucleases and, together with RNase R, has been considered as the prototype of the RNR-like enzymes. This family is widely distributed among all organisms, and RNase II homologs are found in almost all pro- karyotes and eukaryotes [30,31]. Many in vitro studies of the 3¢ to 5¢ exoribonucleolytic activity of E. coli RNase II have been performed [12,18,19,37] and its implications in prokaryotic mRNA decay in vivo have been well characterized [11,20,21,24,27,28]. However, to date no structural or mutational analysis have been performed for E. coli RNase II or for any other RNR family member. An E. coli strain deficient in RNase II activity [25] has been widely used for many years in the study of RNase II. This strain (SK4803) carries the rnb296 allele and it was previously demonstrated that the crude extracts were unable to degrade the polyadenylic acid [11,25]. Although this strain has been extensively used, nothing is known about the mutation responsible for the synthesis of an inactive RNase II. In this report, we determined the DNA sequence of the rnb296 gene and we demonstrated that the single substitution of Asp209 by Asn in RNase II (D209N) is responsible for the loss of RNase II activity. Our stud- ies on the purified His(6)-RNase IID209N mutant B A Fig. 4. Metal dependence of the exoribonuclease activity of RNase II. Exoribonuclease activity of His(6)-RNase II in the presence of different concentrations of MgCl 2 . Assays were performed as described in the Experimental procedures using 1 n M enzyme and the metal ion concentration indicated in the figure. (A) The reaction products were analyzed in a 6% PAA ⁄ 7 M urea gel. (B) The percent- age of exonuclease activity was estimated from the gel by quanti- fication of the band intensities. The RNA degradation was determined by calculating the ratio of the reaction products (P1 and P2) and the substrate (S) on the respective lane. Each value is the mean of three independent experiments. RNase II mutant with RNA binding but no activity M. Amblar and C. M. Arraiano 368 FEBS Journal 272 (2005) 363–374 ª 2004 FEBS protein demonstrated that Asp209 is absolutely essen- tial for the exoribonucleolytic activity of RNase II but does not seem to be involved in substrate binding. No cleavage activity was detected either with the linear polyadenylic acid substrate or with stem–loop contain- ing RNAs (SL9A or malE-malF RNAs) in the pres- ence of 540 nm His(6)-RNase IID209N. However, 5nm of the mutant protein was sufficient to form sta- ble RNA–protein complexes. Moreover, we also dem- onstrate that RNase II requires metal ions for catalysis but not for substrate binding. These findings provide the first identification of a key residue for catalysis in RNase II and support the hypothesis of the organiza- tion in independent functional domains for these enzymes. It has been previously proposed that the cat- alytic ability of RNR proteins resides in a central region of  400 residues, termed the RNB domain [31]. Multiple sequence alignments of this catalytic domain revealed the presence of four highly conserved sequence motifs (I–IV) containing some invariant carb- oxylate residues [30,38]. Figure 1 shows the sequence pattern of motif I inferred from a multiple sequence alignment that includes 27 prokaryotic and eukaryotic RNR-like enzymes (http://www.sanger.ac.uk/cgi-bin/ Pfam/getacc?PF00773). Asp209 of RNase II lies in motif I of the RNB domain. This position is occupied by an acidic residue (aspartate or glutamate) in 86% of the RNR proteins aligned (Fig. 1), and in 75% of the enzymes corresponds to an aspartate. The presence of carboxylate residues is common in proteins that cat- alyze phosphoryl transfer reactions, such as nucleic acid polymerases or nucleases, and they are required for the co-ordination of divalent metal ions that are essential for catalysis [39]. Given its high degree of conservation, Asp209 seems to be a good candidate for being one of the metal ligands at the active site of A B Fig. 5. Gel retardation assay of mRNA transcripts with His(6)-RNase II and His(6)-RNase IID209N. SL9A (2 fmol) (A) or malE-malF (2 fmol) (B) mRNA transcripts were incubated at 15 °C with the His(6)-RNase II (wild-type; WT) and His(6)-RNase IID209N (D209) mutant enzyme under the conditions described in the Experimental procedures. The enzyme concentration used is indicated in the figure. After electrophor- esis, the mobility of free RNA as well as the RNA–protein complexes was detected by autoradiography. M. Amblar and C. M. Arraiano RNase II mutant with RNA binding but no activity FEBS Journal 272 (2005) 363–374 ª 2004 FEBS 369 the enzyme. Under this hypothesis, its replacement with Asn would lead to a loss of the metal co-ordina- tion with the subsequent loss in activity, without affecting substrate binding. Our band-shift experiments demonstrate that the RNA binding of the D209N mutant is not reduced and not influenced by the pres- ence or absence of a metal binding inhibitor (10 mm EDTA). In some cases, these metal ligands can be sub- stituted with a water molecule or by another acidic residue in the vicinity, allowing the metal binding even in the absence of these residues. This normally results in a decrease in metal affinity, indicating that the activ- ity of the mutant protein could be restored, at least in part, by increasing the metal concentration. Despite our efforts to detect any exoribonucleolytic activity at different concentrations of Mg 2+ , we were unable to observe digestion by the D209N mutant protein. Nev- ertheless, these results do not rule out that Asp209 is involved in the binding of metal ions at the active site. The substitution of this aspartate with asparagine can induce conformational changes in the metal binding pocket that totally prevent the co-ordination of Mg 2+ to the active site. However, another hypothesis must be taken into account. Instead of a metal ligand, Asp209 may act as a general base during the reaction, generating the nucleophilic hydroxide group that will attack the scissile phosphate of the RNA. Under this hypothesis, Asp209 would be directly responsible for the catalytic event in RNase II. Based on the results presented here we cannot exclude the possibility that inactivation of the enzyme is caused by subtle con- formational changes owing to the amino acid replace- ment. However, the dramatic effect on RNase II catalysis caused by the substitution of only one residue strongly suggests a crucial role of this amino acid in the RNase II enzyme. The analysis of the exoribonuclease activity with dif- ferent Mg 2+ concentrations revealed that the wild-type enzyme is active within a wide range of the metal ion. However, different kinds of products are released depending on the concentration of this metal ion. The malE-malF RNA molecule contains two stem–loop Fig. 6. Effect of EDTA in RNA binding. malE-malF mRNA transcripts (2 fmol) were incubated at 37 °C with His(6)-RNase II (A) or His(6)- RNase IID209N (B) mutant enzyme in the absence of EDTA (– EDTA) or in the presence of 10 m M EDTA (+ EDTA). The reaction was per- formed as described in the Experimental procedures. The enzyme concentration used is indicated in the figure. RNA–protein complexes were detected and quantified by using the PhosphorImager system from Molecular Dynamics. RNase II mutant with RNA binding but no activity M. Amblar and C. M. Arraiano 370 FEBS Journal 272 (2005) 363–374 ª 2004 FEBS structures. RNase II may catalyze the nibbling of this RNA from the 3¢ end until it reaches the first secon- dary structure, rendering the P1 product, or the degra- dation may continue until the second stem–loop (a more stable secondary structure) is reached, generating the P2 product. At lower concentrations of MgCl 2 , RNase II seems to be able to easily overcome the first stem–loop and the degradation only stops in the vicin- ity of the second structure (P2 is the major product). However, at higher concentrations of Mg 2+ , the deg- radation is blocked by the first stem–loop structure. These phenomena may respond to alterations in the strength of the stem–loop structure caused by the increase of MgCl 2 concentration, becoming more resistant to degradation, or to different cleavage properties of RNase II, depending on the metal ion concentration. The hypothesis that RNase II activity can be regulated by the Mg 2+ concentration is very interesting. Aspects such as processivity of the RNase II enzyme or its ability to overcome weak secondary structures during degradation could be adequate to the physiological requirements by changes in concentra- tions of free divalent metal ions. Experimental procedures Materials Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Hertfordshire, UK), T7 RNA polym- erase was obtained from Promega (Charbonnie ` res-les-Bains, France), and Pfu DNA polymerase was obtained from Fer- mentas (Vilnius, Lithuania). Oligonucleotide primers were synthesized by Sigma Genosys (Cambridge, UK). Bacterial strains, plasmids, and RNA substrates The E. coli strains used were JM109 [F¢ (traD36 proA +- B + lacI q D(lacZ)M15 ⁄D(lac-proAB) glnV44 e 14 – gyrA96 recA1 relA1 endA1 thi hsdR17] [40] for cloning experiments and BL21(DE3) (F – r B – m B – gal ompT (int::P lacUV5 T7 - gen1 imm21 nin5) [41] for expression and purification of enzymes. The rnb296 mutant gene was obtained from the SK4803 E. coli strain deficient in RNase II activity [25]. The plasmids used for in vitro transcription reactions were pCH77 [18] and pSL9A [13]. Cloning of the rnb296 mutation The rnb296 mutant gene was amplified from the chromoso- mal DNA of E. coli SK4803 by using a standard PCR reac- tion with Pfu DNA polymerase. The primers used for the amplification matched perfectly 45 nucleotides upstream of the initiation codon (5¢-GCGTAAAACTGTCAGCCGCT- 3¢) and 47 nucleotides downstream of the stop codon (5¢-CTGGATATAACGAAGGTAGAGC-3¢) of RNase II, respectively. The DNA sequence of the 2048 bp PCR prod- uct was determined (STABvida, Oeiras, Portugal). To ensure that the mutation(s) detected were not introduced during amplification, three independent PCR reactions were performed and both strands of each PCR product were sequenced. A point mutation (G fi A) at the 1148 position of the rnb gene was detected and further inserted into the previously described pFCT6.9 plasmid [29] that contains the wild-type rnb gene cloned into the pET15 vector (Novagen, Lisbon, Portugal). The insertion was performed by digestion of the rnb296 PCR product and pFCT6.9 with NheI followed by ligation with T4 DNA ligase. The result- ing plasmid carrying the 296 mutation, named pMAA, was transferred to the E. coli BL21(DE3) host strain. Overexpression and protein purification The expression of His(6)-RNase II and its mutant deriv- ative carrying the substitution of Asp209 with Asn [His(6)- RNase IID209N] was achieved by IPTG induction of BL21(DE3) [42] containing either pFCT6.9 or pMAA plas- mids, respectively. Cells were grown in LB (Luria–Bertani) medium, supplemented with 150 lgÆmL )1 of ampicillin, at 37 °C. After reaching an attenuance (D) of 0.4 at 600 nm, the cultures were induced by adding 1 mm IPTG. Samples were withdrawn at different induction times, and crude extracts were prepared as previously described [43] to ana- lyze the exoribonucleolytic activity and the total protein content. The solubility of both wild-type and mutant pro- teins during induction was tested by separation of the sol- uble and insoluble protein fractions as previously described [44], followed by fractionation in SDS ⁄ PAGE. The purification of His(6)-RNase II and His(6)-RNase IID209N proteins was performed by histidine affinity chromatography using the HiTrap Chelating HP system (Amersham Biosciences, Buckinghamshire, UK). For this purpose, 100 mL of IPTG-induced cultures were harvested by centrifugation, washed with 20 mL of buffer A (20 mm Na 2 HPO 4, 0.5 m NaCl), and suspended in 4 mL of lysis buffer (20 mm imidazol, 1 mm phenylmethanesulfonyl fluoride, 1 mg mL )1 of lysozyme in buffer A). Cell lysis was performed as previously described [43] and the clarified extracts were added to a HiTrap Chelating Sepharose 1 mL column equilibrated in buffer A plus 20 mm imidazol. After a washing step with 70 mm imidazol in buffer A, the pro- tein was eluted from the column with buffer A containing 0.5 m imidazol. The sample buffer was changed by 20 mm Tris pH 8 and 100 mm KCl through ion-exchange chroma- tography, and 50% (v ⁄ v) glycerol was added prior to stor- age at )20 °C. The protein concentration was determined by using the Lowry method [45]. M. Amblar and C. M. Arraiano RNase II mutant with RNA binding but no activity FEBS Journal 272 (2005) 363–374 ª 2004 FEBS 371 In vitro transcription of RNAs SL9A and malE-malF RNA molecules were obtained by in vitro transcription using the pSL9A plasmid linearized with XbaI [13] or the pCH77 plasmid linearized with EcoRI [18] as templates, respectively. The transcription reactions were performed by using the Riboprobe kit from Promega following the instructions given by the manufacturers, in a 20 lL volume, containing 20 lCi of [ 32 P]dUTP[aP] (Amer- sham Biosciences). Radioactively labeled RNA transcripts were purified on a 6% polyacrylamide ⁄ 7 m urea gel, as pre- viously described [46]. Activity assays The exoribonucleolytic activity on poly[8- 3 H]adenylic acid (Amersham Biosciences) was assayed essentially as des- cribed previously [11] except for the introduction of some modifications in order to make the experiment quantitative. RNase II activity was determined by measuring the release of acid-soluble radioactivity from 6 nmol of substrate. The reactions were performed in a 60 lL volume of activity buf- fer (100 mm KCl, 20 mm Tris pH 8, 0.5 mgÆmL )1 BSA) containing 1 mm MgCl 2 and the protein concentration (crude extracts or purified proteins) indicated above. The mixtures were incubated at 30 °C for 5 min and the reac- tions were stopped by cooling at 4 °C. Trichloroacetic acid (10%, v ⁄ v) was added to the mixture to precipitate the undegraded substrate and, after centrifugation (15 min, 20 000 g,4°C), the soluble [ 3 H]AMP was measured in a scintillation counter. One UE is defined as the amount of protein required for the release of 10 nmol of [ 3 H]AMP in 15 min at 30 °C. The exonucleolytic activity of purified proteins was also assayed on the in vitro-transcribed mRNAs SL9A [13] and malE-malF [18]. Cleavage assays were performed at 37 °C in 15 lL of cleavage buffer containing 20 mm Tris ⁄ HCl, pH 8, 2 mm dithiothreitol, 100 mm KCl, and 0.5 mgÆmL )1 BSA. The concentration of MgCl 2 in the reaction mixture was varied in order to determine its influence on cleavage activity (see figure legends). The RNA substrate (10 000 counts per minute per reaction) was denatured for 10 min at 90 °C in the Tris component of the assay buffer and allowed to reanneal at 37 °C for 20 min prior to the addi- tion of the other buffer components. The reaction was initi- ated by the addition of 2 nm His(6)-RNase II or 540 nm His(6)-RNase IID209N purified proteins. Samples were withdrawn at the time-points indicated in the figure legends and quenched in 3 volumes of formamide-containing dye. Reaction products were incubated at 90 °C for 5 min and analyzed on a 6% (w ⁄ w) or an 8% (w ⁄ w) polyacryl- amide ⁄ 7 m urea gel (for SL9A or malE-malF substrates, respectively). Bands were detected by autoradiography and the exonucleolytic activity was calculated by quantification of the relative intensities of the bands. Band-shift assays The RNA binding ability of purified enzymes was analyzed through band-shift experiments by using the in vitro tran- scribed mRNAs SL9A and malE-malF. The reaction mixture (10 lL) contained 2 fmol of the mRNA substrate (10 000 counts per minute), 100 mm KCl, 2 mm dithiothreitol, 20 mm Tris ⁄ HCl, pH 8, and 10% (v ⁄ v) glycerol. Appropriate amounts of BSA were added to the reaction in order to obtain a final concentration of 0.1 lg of protein per assay. No divalent metal ions were added to the mixture and, when indicated, 10 mm EDTA was used. The mRNA substrates were denatured ⁄ renatured prior adding to the mixture, as described for the activity assays. Protein was added last to the final concentration specified in the figure legends, and incubations were performed at different temperatures for 10 min. The reactions were stopped by adding 2 lL of load- ing buffer containing 30% (v ⁄ v) glycerol, 0.25% (v ⁄ v) xylene cyanol, and 0.25% (v ⁄ v) bromophenol blue, and analyzed in a5%(w⁄ v) nondenaturing polyacrylamide gel. Electrophor- esis was performed with 89 mm Tris ⁄ borate, 8 mm EDTA, pH 8.5 (Tris ⁄ borate ⁄ EDTA) buffer at 20 mA and 4 °C. After 5 h of electrophoresis the gel was fixed by incubation in 7% (v ⁄ v) acetic acid for 5 min and further dried. The RNA–protein complexes were detected by using the phos- phorImager system from Molecular Dynamics. The K D value of wild-type– and D209N–RNA complex formation was estimated from the gel by quantification of the bands using the imagequant software (Molecular Dynamics). The values obtained for the RNA–protein complex [C], free RNA [R] and protein concentration [P], were plotted using the Hill representation (log([C] ⁄ ([R]–[C])) vs. log[P]). The Hill coefficient n for the protein tested ranged from 1.02 to 1.06. The apparent K D (K) of wild-type and of D209N proteins was calculated from the equation log[K] ¼ n log[P] – log ([C] ⁄ ([R]–[C])), as described previously [43]. Acknowledgements We thank Dr G. Rivas and Dr P. Go ´ mez-Puertas for helpful discussions, and Dr P. Lo ´ pez for critical read- ing of the manuscript. M. Amblar was a recipient of a FCT Postdoctoral fellowship. The work at the ITQB was supported by FCT-Fundac¸ a ˜ o para a Cieˆ ncia e Tecnologia, Portugal. 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Arraiano Instituto de Tecnologia Quı ´ mica e. demonstrate that the single amino acid sub- stitution Asp209fiAsn in RNase II is able to cause the total inactivation of the enzyme without affecting its RNA binding capability. In addition, metal. indi- cating that the loss of activity in the mutant protein cannot be restored by increasing the Mg 2+ concentra- tion. RNA binding ability of RNase II and the D209N mutant The data presented above