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.. Abbreviations Hi
Trang 1inactivates 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; KD , 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.
Trang 2the 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.
Trang 3that 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[8-3H]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 Ni2+ 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[8-3H]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)1of protein)
BL21(DE3)
BL21(DE3) [pFCT6.9]
BL21(DE3) [pMAA]
After 2 h of
IPTG induction
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.
Trang 4no 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[8-3H]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 [3H]AMP in
15 min at 30C)
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
Mg2+ 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 Mg2+ 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 MgCl2 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 Mg2+ concentra-tions (Fig 4B) The enzyme was highly active at all MgCl2 concentrations tested, although the maximum activity was obtained between 5 lm and 1 mm 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 MgCl2 concentrations of ‡ 500 lm, being the major product at ‡ 1 mm MgCl2 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 MgCl2 to the reaction
Trang 5mixture 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 Mg2+
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 Mg2+
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 15C to
37C) The results obtained showed that, even at
15C, 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.
Trang 6correspond to RNA–protein complexes, indicating that,
despite the mutation, the D209N protein is able to
bind RNA
To investigate the role of MgCl2in formation of the
RNA–protein complex, binding assays were performed
with the malE-malF substrate at 37C 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 Mg2+
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 (KD) 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 KDvalue for the mutant protein was 330 nm The KDof 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 Mg2+ 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 colicell [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 eupro-karyotes [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 MgCl2 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.
Trang 7protein 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,
5 nm 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 funcorganiza-tional 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.
Trang 8the 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 abspres-ence 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 Mg2+, 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 Mg2+
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 Mg2+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 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.
Trang 9structures 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 MgCl2,
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 Mg2+, 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 MgCl2 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 Mg2+ 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+lacIqD(lacZ)M15 ⁄ D(lac-proAB) glnV44 e 14–gyrA96
recA1 relA1 endA1 thi hsdR17] [40] for cloning experiments
and BL21(DE3) (F–rB–mB–gal ompT(int::PlacUV5T7
-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 (Gfi 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 carryresult-ing 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
37C 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 insolsol-uble 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
Na2HPO4, 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]
Trang 10In 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 [32P]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[8-3H]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 MgCl2 and the protein concentration
(crude extracts or purified proteins) indicated above The
mixtures were incubated at 30C for 5 min and the
reac-tions were stopped by cooling at 4C Trichloroacetic acid
(10%, v⁄ v) was added to the mixture to precipitate the
undegraded substrate and, after centrifugation (15 min,
20 000 g, 4C), 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 [3H]AMP in
15 min at 30C
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 37C
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 MgCl2in 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 90C in the Tris component of the assay buffer and
allowed to reanneal at 37C for 20 min prior to the
addi-tion of the other buffer components The reacaddi-tion 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 90C 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 containload-ing 30% (v⁄ v) glycerol, 0.25% (v ⁄ v) xylene cyanol, and 0.25% (v⁄ v) bromophenol blue, and analyzed in
a 5% (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 KD 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 KD (K) of wild-type and of D209N proteins was calculated from the equation log[K]¼
nlog[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 Cieˆncia e Tecnologia, Portugal
References
1 Arraiano CM & Maquat LE (2003) Post-transcriptional control of gene expression: effectors of mRNA decay Mol Microbiol 49, 267–276
2 Deutscher MP (1993) Ribonuclease multiplicity, diver-sity, and complexity J Biol Chem 268, 13011–13014