Biochem. J. (2008) 410, 39–48 (Printed in Great Britain)
39
doi:10.1042/BJ20071047
Heterodimer-based analysis of subunit and domain contributions to
double-stranded RNA processing by Escherichia coli RNase III in vitro
Wenzhao MENG and Allen W. NICHOLSON*
Department of Chemistry, Temple University, 1901 North 13th Street, Philadelphia, PA 19122, U.S.A.
Members of the RNase III family are the primary cellular agents
of dsRNA (double-stranded RNA) processing. Bacterial RNases
III function as homodimers and contain two dsRBDs (dsRNAbinding domains) and two catalytic sites. The potential for
functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known.
It is shown that an Escherichia coli RNase III heterodimer that
contains a single functional wt (wild-type) catalytic site and an
inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate
with a single scissile bond with a kcat value that is one-half
that of wt RNase III, but exhibits an unaltered K m . Moreover,
RNase III[E117A/wt] cleavage of a substrate containing two
scissile bonds generates singly cleaved intermediates that are only
slowly cleaved at the remaining phosphodiester linkage, and in
a manner that is sensitive to excess unlabelled substrate. These
results demonstrate the equal probability, during a single binding
event, of placement of a scissile bond in a functional or non-
INTRODUCTION
The enzymatic cleavage of dsRNA (double-stranded RNA) is an
essential event in the maturation and decay of diverse eukaryotic
and bacterial RNAs. Members of the RNase III family are the
primary agents of dsRNA processing and are highly conserved
in eukaryotic and bacterial cells [1–8]. Bacterial RNases III
participate in rRNA and mRNA maturation and also initiate
mRNA decay [1,2,5]. The eukaryotic RNase III orthologues
Dicer and Drosha are involved in microRNA maturation [9–
12], and RNase III polypeptides are essential components of
multisubunit RNA editosomes that catalyse uridine insertion or
deletion in trypanosomatid kinetoplast mRNAs [13,14]. Several
viruses express RNase III orthologues that may be involved
in antagonizing the RNAi (RNA interference)-based antiviral
response [15,16]. RNase III family members can be grouped
into three classes, according to polypeptide structure (Figure 1A).
The Class 3 enzymes include Dicer, whereas Class 2 enzymes
are represented by Drosha. Members of the structurally simplest
Class 1 include the bacterial RNases III, which typically contain
∼ 220 amino acid residues. The N-terminal portion of bacterial
RNase III polypeptides includes the NucD (nuclease domain),
which self-associates to form a single ‘processing centre’ at the
subunit interface that contains two catalytic sites [17,18]. The Cterminal region of the bacterial RNase III polypeptide consists of a
dsRBD (dsRNA-binding domain) that carries a single copy of the
conserved dsRBM (dsRNA-binding motif) [19–21]. As the Class
1 RNase III polypeptides form stable dimers [1,3,5], the bacterial
RNase III holoenzyme contains two dsRBDs (Figures 1B and
functional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of
both phosphodiester linkages by the mutant heterodimer. The rate
of phosphodiester hydrolysis by RNase III[E117A/wt] has the
same dependence on Mg2+ ion concentration as that of the wt
enzyme, and exhibits a Hill coefficient (h) of 2.0 +
− 0.1, indicating
that the metal ion dependence essentially reflects a single catalytic
site that employs a two-Mg2+ -ion mechanism. Whereas an E. coli
RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic
activity. These findings support a reaction pathway involving the
largely independent action of the dsRBDs and the catalytic sites
in substrate recognition and cleavage respectively.
Key words: Dicer, double-stranded RNA (dsRNA)-binding domain (dsRBD), Drosha, Escherichia coli, RNase III,
two-metal-ion catalysis.
1C). Ji and co-workers solved the structure of Aquifex aeolicus
RNase III and specific mutant versions, either bound to dsRNA in
catalytically non-productive modes [22,23] or bound to a cleaved
minimal substrate [24]. These studies revealed the conformational
flexibility of the short linker that connects the dsRBD and NucD
(Figure 1C) and demonstrated the involvement of conserved
NucD residues in bivalent metal cation binding at the catalytic
site, as well as in subunit association.
RNase III family members are phosphodiesterases and require a
bivalent metal ion to hydrolyse phosphodiester linkages, creating
5 -phosphate, 3 -hydroxyl product termini [25,26]. For bacterial
RNases III, Mg2+ is presumably the physiologically relevant
species. However, several other metals, including Mn2+ , Co2+
and Ni2+ , can support catalysis [26–28]. Ongoing biochemical and
structural studies are revealing the basic features of the bacterial
RNase III catalytic mechanism. Each subunit contains a catalytic
site that is responsible for cleaving one of the two phosphodiesters
at a dsRNA target site. Kinetic and inhibitor studies indicate a
two-metal-ion catalytic mechanism for Escherichia coli RNase
III [29]. Since only one metal ion is observed in each catalytic
site in a crystal structure of A. aeolicus RNase III [17,24], it
has been proposed that substrate binding promotes the binding
of the second metal ion [29]. Structural comparisons of A.
aeolicus RNase III with Bacillus halodurans RNase H [24]
and a crystal structure of Giardia intestinalis Dicer with bound
Europium (Eu3+ ) ions [30] have provided a tentative placement of
the second metal-binding site, approx. 4 Å (1 Å = 0.1 nm)
from the other metal, in a manner consistent with a two metal
ion mechanism. Several conserved side chains of one subunit are
Abbreviations used: CBP, calmodulin-binding peptide; dsRNA, double-stranded RNA; dsRBD, dsRNA-binding domain; DTT, dithiothreitol; IPTG,
isopropyl β-D-thiogalactoside; Ni-NTA, Ni2+ -nitrilotriacetate; NucD, nuclease domain; wt, wild-type.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
40
Figure 1
W. Meng and A. W. Nicholson
RNase III family members
(A) Three classes of RNase III polypeptides and their functional domains. Class I orthologues include bacterial (top) and yeast (bottom) RNases III. The Class II orthologues are represented by
Drosha, whereas Class III orthologues are represented by Dicer. DUF, domain of unidentified function; PAZ, Piwi-Ago-Zwille domain; RS, arginine-serine-rich region. (B) Diagram of the homodimeric
structure of a Class I (bacterial) RNase III. (C) Ribbon diagram of a Thermotoga maritima RNase III crystal structure (PDB code: 1O0W). The dsRBD, NucD and linker segment are indicated: the
dsRBD is shown in red–orange–yellow colours, while the NucD is shown in blue–green colours.
near the catalytic site of the other subunit. Thus the possibility
arises that intersubunit interactions may be important for optimal
catalytic site function and perhaps also for regulation of activity.
Although the mutational inactivation of one catalytic site does
not suppress the function of the other catalytic site [31], certain
mutations may confer more subtle changes on the pathway and
kinetics of dsRNA cleavage.
The importance of the dsRBD in bacterial RNase III function
was demonstrated by the in vitro catalytic inactivity of an
E. coli RNase III mutant that lacks both copies of the domain
[32]. The absence of activity reflected an inability to bind
the substrate [32]. Examination of the A. aeolicus RNase III–
dsRNA crystal structures reveals a primary role of the dsRBD
in substrate recognition, in that the domain engages in multiple
contacts with both strands of dsRNA, and with most of the
buried surface area of the substrate involving both dsRBDs [24].
However, it is not known whether both dsRBDs are needed
for catalytic activity or whether the dsRBDs function in a cooperative manner in substrate recognition. The present study
addresses these questions, as well as examining subunit crosstalk through analysis of the biochemical behaviours of specific
mutant heterodimers of E. coli RNase III.
EXPERIMENTAL
Materials
Water was deionized and distilled. Chemicals and reagents were
molecular biology grade and were purchased from Sigma–Aldrich
or Fisher Scientific. Standardized 1 M solutions of MgCl2 and
MnCl2 were obtained from Sigma–Aldrich. Ribonucleoside 5 triphosphates were obtained from Amersham Biosciences. [γ 32
P]ATP (3000 Ci/mmol) and [α-32 P]UTP (3000 Ci/mmol) were
purchased from PerkinElmer. E. coli bulk stripped tRNA was
purchased from Sigma–Aldrich and was further purified by
repeated phenol extraction followed by ethanol precipitation.
T4 polynucleotide kinase was purchased from New England
Biolabs, whereas calf intestine alkaline phosphatase was obtained
from Roche Molecular Biochemicals. Bacteriophage T7 RNA
polymerase was purified in-house as described previously [33].
Oligodeoxynucleotide transcription templates and mutagenic
oligonucleotides were synthesized by Invitrogen, and the
c The Authors Journal compilation c 2008 Biochemical Society
deprotected DNAs were purified by denaturing gel electrophoresis
[28]. Purified DNAs were stored at − 80 ◦C in 10 mM Tris/HCl
and 1 mM EDTA (pH 8).
RNase III heterodimer production
RNase III heterodimers were purified from an E. coli strain
harbouring separate compatible plasmids that contained RNase
III (rnc) genes under control of a T7 promoter. Plasmid pET15b(rnc) was a recombinant form of pET-15b (Novagen) with a
pBR322 replication origin and a copy number of ∼ 30, whereas
pACYC(rnc) was a recombinant form of pACYC184 (New
England Biolabs), with a p15A replication origin and a copy
number of ∼ 12. Mutant rnc genes were cloned into pET15b as described previously [28], using the NdeI and BamHI
sites. To create the pACYC184-rnc plasmid, the E. coli rnc
gene was transferred from plasmid pET-15b(rnc) into plasmid
pCAL-n (Stratagene) by using the NcoI and HindIII restriction
sites. This provided a recombinant plasmid [pCAL-n(rnc)], in
which the rnc gene is fused to an N-terminal CBP (calmodulinbinding peptide) coding sequence [34], directly downstream of
an IPTG (isopropyl β-D-thiogalactoside)-inducible T7 promoter
(T7p-lacO). pCAL-n(rnc) was cleaved with SphI and EcoRV, and
the T7p-CBP-rnc-containing DNA fragment ligated into SphI
and EcoRV-cleaved pACYC184, providing plasmid pACYC184rnc. The construct was verified by DNA sequencing. Mutations
were introduced into the pACYC184 plasmid by a twostep mutagenic PCR protocol employing a single mutagenic
primer [35–37]. To create the E117A mutation, the mutagenic
oligonucleotide used was 5 -ATTAATGCTGCGACGGTGTCG3 , with the mutated base shown in bold and underlined. Correct
introduction of the mutation was verified by DNA sequencing.
Protein purification
His6 -tagged RNase III was purified as described previously [28].
The catalytic properties of His6 –RNase III are essentially the
same as those of the native enzyme [28]. RNase III heterodimers
were overproduced in E. coli BLR(DE3), cells carry (Novagen)
containing pET-15b(rnc) and pACYC184(rnc). BLR(DE3) cells
carry the DE3 gene encoding T7 RNA polymerase under control
of the IPTG-inducible lac promoter and the recA allele serving to
Bacterial RNase III heterodimer analysis
suppress plasmid recombination. Cell growth was at 37 ◦C in LB
(Luria–Bertani) medium (400 ml) containing chloramphenicol
(40 µg/ml) and ampicillin (100 µg/ml). IPTG (1 mM) was added
when cells reached an attenuance of 0.4 (D395 ), and the incubation
continued with aeration for 4 h. Aliquots were removed and
analysed for protein production by SDS/PAGE (12 % gels)
and staining with Coomassie Brilliant Blue R. Cells were collected
by centrifugation at 3500 g for 20 min at 5 ◦C, and stored at
− 20 ◦C until further use. Cells were resuspended in 30 ml
of Buffer A (500 mM NaCl, 5 mM imidazole and 20 mM
Tris/HCl, pH 8) and subjected to repeated sonication on ice.
The following steps were carried out at ∼ 5 ◦C. The sonicated
material was clarified by centrifugation at 3500 g for 20 min
at 5 ◦C. The first chromatographic step employed an Ni-NTA
(Ni2+ -nitrilotriacetate) column (HisBind resin; Novagen; 1.5 ml),
initially washed with 10 column volumes of Buffer A. The
column then was charged using an NiSO4 solution (50 mM).
The clarified cell sonicate (∼ 30 ml) was slowly loaded on to the
column, which was then washed with 20 column volumes of
Buffer A and 10 column volumes of Buffer B (500 mM NaCl,
60 mM imidazole and 20 mM Tris/HCl, pH 8.0). The protein
was eluted with 5 column volumes of Buffer C (1 M NaCl,
400 mM imidazole and 20 mM Tris/HCl, pH 8.0). The protein
was contained mainly in the first three elute volumes. The eluates
were combined and diluted 2-fold in a buffer consisting of 65 mM
Tris/HCl (pH 8.0) and 3 mM CaCl2 and loaded on to a calmodulin
column (Stratagene; 1 ml). Before loading, the column was
prepared by washing with 10 column volumes of Buffer D
(333 mM NaCl, 2 mM CaCl2 and 50 mM Tris/HCl, pH 8.0). After
sample loading, the resin was washed with 20 column volumes of
Buffer D, and the protein was eluted with three 1 ml aliquots
of Buffer E (1 M NaCl, 2 mM EDTA and 50 mM Tris/HCl,
pH 8.0). The eluted protein was dialysed overnight against a
buffer consisting of 1 M NaCl, 60 mM Tris/HCl (pH 8.0), 1 mM
EDTA and 1 mM DTT (dithiothreitol). The purified RNase III
heterodimer preparations were stored at − 20 ◦C in 50 % (v/v)
glycerol, 0.5 M NaCl, 30 mM Tris/HCl (pH 8.0), 0.5 mM EDTA
and 0.5 mM DTT. The purity of the preparation was estimated
by SDS/PAGE (12 % gels) to be ∼ 90 % and the preparation was
free of contaminating nucleases.
The RNase III[NucD/wt] (where wt is wild-type) and RNase
III]NucD/E117A] mutant heterodimers (see the Results section)
were purified as follows. The inclusion body, obtained from lowspeed centrifugation of the sonicated material (see above), was resuspended in 4 ml of Buffer A containing 6 M urea. The solution
was clarified by centrifugation at 3500 g for 20 min at 5 ◦C, and
the supernatant was purified on an Ni-NTA column, followed
by calmodulin column chromatography as described above. The
proteins purified from the soluble fraction and the inclusion body
exhibited essentially the same catalytic activities. However, in
order to provide accurate comparison of activity, cleavage assays
involving the two mutant heterodimers also involved wt enzyme
purified in the same manner. E. coli RNase III carrying a CBP tag
on both subunits was active, but the protein was significantly
less soluble than the His6 -tagged homodimer (W. Meng and
A. W. Nicholson, unpublished work). Finally, the E. coli
BLR(DE3) chromosome carries the rnc gene, which is expressed.
However, the endogenous RNase III polypeptide is not expected
to co-purify either as a homodimer or as a heterodimer, since it
lacks an affinity tag.
Substrate synthesis
RNAs were enzymatically synthesized in vitro by using T7 RNA
polymerase and oligodeoxynucleotide templates according to
41
established protocols [38,39] with modifications as described
previously [28]. The sequences of the oligodeoxynucleotide
transcription templates are available on request. For 5 -32 Plabelling, enzymatically synthesized, non-radioactive RNA
(100 pmol) was treated with calf intestine alkaline phosphatase
(4 units) at 37 ◦C for 1 h using the supplied buffer. The
RNA was purified by phenol/chloroform extraction and ethanol
precipitation. Dephosphorylated RNA (∼ 5 pmol) was incubated
at 37 ◦C for ∼ 30 min with 10 µCi of [γ -32 P]ATP (3000 Ci/mmol)
and T4 polynucleotide kinase (10 units), using the supplied buffer.
The reaction was electrophoresed in a 15 % (w/v) polyacrylamide
gel containing TBE (Tris/borate/EDTA; 1 × TBE = 45 mM
Tris/borate and 1 mM EDTA) buffer and 7 M urea, and the RNA
was isolated as described previously [28]. RNA was stored at
− 20 ◦C in Tris/EDTA buffer (10 mM Tris/HCl and 1 mM EDTA,
pH 7.0).
Substrate cleavage assay
RNase III cleavage assays were performed using internally 32 Plabelled RNA (U-labelled) according to an established protocol
[28]. Specific features of the assays are detailed in the appropriate
Figure or Table legend. In general, assays were conducted by
first incubating the RNA (specific amounts indicated in the
relevant Figure legends) at 37 ◦C in a buffer (160 mM NaCl and
30 mM Tris/HCl, pH 8) for 5 min. RNase III was then added
at the specified concentration, and the sample was incubated
for 1 min at 37 ◦C. Mg2+ was then added at the specified
concentration to initiate the reaction. Reactions were stopped by
adding one-half volume of loading dye that contained 20 mM
EDTA [28]. Aliquots were analysed by electrophoresis in 15 %
(w/v) polyacrylamide gels containing 7 M urea and TBE buffer.
The results were visualized by phosphoimaging (Typhoon 9400
system) and quantified by using ImageQuant software. Curve
fitting for determination of the kinetic parameters was carried out
using Kaleidagraph software (v.3.5) [29,36,37].
Substrate binding assay
Gel mobility-shift assays were performed as described previously
[36,37], using 5 -32 P-labelled R1.1[WC] RNA (see Figure 4A).
Ca2+ (10 mM) was included in the gel shift and electrophoresis
buffers. Electrophoresis was carried out at ∼ 5 ◦C in an 8 %
(w/v) polyacrylamide gel containing 0.5 × TBE buffer and
10 mM CaCl2 . Reactions were visualized by phosphoimaging
and quantified using ImageQuant software. K d values (apparent
dissociation constants) were determined by curve fitting
using Kaleidagraph software (v3.5) and established protocols
[28,40,41].
RESULTS
RNase III heterodimer production involved the expression in vivo
of two RNase III (rnc) genes, carried on separate, compatible
plasmids. Each rnc gene was under control of an IPTG-inducible
T7 promoter, with one of the encoded polypeptides fused to an
N-terminal His6 tag and the other polypeptide fused to an Nterminal, 26-amino-acid CBP. Both polypeptides were produced
in an IPTG-dependent manner, as revealed by SDS/PAGE analysis
of total cellular protein (results not shown). The polypeptides
accumulated to different levels, which in part may reflect the
different plasmid copy numbers. Thus the amount of the RNase
III polypeptide produced from the recombinant pET-15b plasmid
(copy number ∼ 30) was greater than that of the polypeptide
produced from the recombinant pACYC184 plasmid (copy
number ∼ 12). Serial affinity chromatography on Ni-NTA and
c The Authors Journal compilation c 2008 Biochemical Society
42
Figure 2
W. Meng and A. W. Nicholson
Polypeptide profiles of RNase III heterodimers
RNase III heterodimers were purified as described in the Experimental section. Aliquots (∼ 1 µg)
of the purified preparations were electrophoresed in a 12 % (w/v) polyacrylamide gel containing
0.1 % SDS, and polypeptides were visualized by Coomassie Brilliant Blue staining. Lane 1,
molecular-mass markers (M). Molecular masses of the species are given on the left. Lane 2,
His6 -tagged wt homodimer [(His)6 -wt/(H)6 -wt]; lane 3, His6 ,CBP-tagged wt RNase III [(His)6 -wt/
CBP-wt]; lane 4, RNase III[E117A/wt] heterodimer [(His)6 -E117A/CBP-wt]; lane 5,
RNase III[NucD/wt] heterodimer [(His)6 -NucD/CBP-wt]; lane 6, RNase III[NucD/E117A]
heterodimer [(His)6 -NucD/CBP-E117A]. The CBP tag is slightly larger than the His6 tag,
conferring a slower electrophoretic mobility on the polypeptide. The molecular masses (in kDa)
of the RNase III polypeptides are provided on the right-hand side.
calmodulin columns yielded a purified preparation consisting of
two polypeptides of comparable dye staining intensity, consistent
with a heterodimeric form of the holoenzyme (Figure 2, lanes
3–6). While it was shown that the N-terminal His6 tag does not
significantly affect RNase III catalytic activity [28], the effect
of an N-terminal CBP tag is not known. We therefore compared
the steady-state kinetic parameters of RNase III with a CBP tag
on one subunit and a His6 tag on the other subunit, with RNase
III containing His6 tags on both subunits. The substrate is R1.1
RNA (Figure 3A) [42], a 60 nt hairpin based on the bacteriophage
T7 R1.1 RNase III processing signal, which contains a single
primary cleavage site that is recognized in vivo and in vitro, and
a secondary site that is cleaved only under specific conditions
in vitro [1,27,43]. Table 1 shows that the two tagged forms of
wt RNase III exhibit similar K m and kcat values with respect to
cleavage of R1.1 RNA. We conclude that the N-terminal CBP
tag also does not greatly alter catalytic activity. Thus the affinity
tags were retained on the heterodimers in the analyses presented
below, and the mutant heterodimers and wt enzymes all contained
the His6 /CBP affinity tag pair. For convenience, CBP/His6 -tagged
wt RNase III is referred to as RNase III.
RNA processing behaviour of an RNase III heterodimer containing
a single functional catalytic site
The existence of two catalytic sites in bacterial RNase III was
demonstrated by the retained activity of an E. coli RNase III
heterodimer in which one subunit contained the E117K mutation
[31]. E117 binds a catalytically essential Mg2+ ion [24], and
the E117K mutation fully suppresses processing activity when
present in both subunits of E. coli RNase III [44,45]. The
two catalytic sites are symmetrically positioned at the subunit
c The Authors Journal compilation c 2008 Biochemical Society
Figure 3
Catalytic activity of RNase III[E117A/wt]
(A) Structure of R1.1 RNA (60 nt). The primary (1◦) site is cleaved in vivo and in vitro , whereas
the secondary (2◦) site is only efficiently cleaved in vitro , at higher enzyme concentrations or
lowered salt concentration [1,27,43]. (B) Time course for cleavage of internally 32 P-labelled
R1.1 RNA by RNase III and RNase III[E117A/wt]. Enzyme (20 nM) was incubated with R1.1
RNA (63 nM) in cleavage reaction buffer for the specified time as described in the Experimental
section. Reactions were analysed in a 15 % (w/v) polyacrylamide gel containing 7 M urea.
Lanes 1–6, RNase III [(H)6 -wt/CBP-wt] time course. Lanes 7–11, RNase III[E117A/wt]
[(H)6 -E117A/CBP-wt] time course. The positions of the substrate and products are indicated on
the left-hand side.
interface, with each site capable of cleaving one of the two
bonds in a target site phosphodiester pair. Determining the
kinetic behaviour and bivalent metal ion dependence of a singlecatalytic-site RNase III heterodimer could reveal a functional
interdependence of the catalytic sites. We prepared an E. coli
RNase III heterodimer in which one subunit contained the E117A
mutation. This mutation is similar to the E117K mutation, in
that when the E117A mutation is present in both subunits, it
suppresses catalytic activity without affecting substrate binding
[44]. The polypeptide profile of purified RNase III[E117A/wt]
is shown in Figure 2 (lane 4), which reveals two closely spaced
bands of comparable dye staining intensity. The difference in
polypeptide size is attributable to the different affinity tags,
with the CBP tag slightly larger than the His6 tag. An issue
is whether the holoenzyme subunits are stable to exchange, as
such a process would compromise the biochemical analyses.
However, a previous study of an E. coli RNase III heterodimer
provided experimental evidence against such an exchange process
[31]. This finding is also consistent with the extensive subunit
interface observed in protein crystal structures [22–24], which
Bacterial RNase III heterodimer analysis
43
Table 1 Steady-state kinetic parameters, apparent dissociation constants
and Hill coefficients for E. coli RNase III mutant heterodimers
Kinetic parameters were determined using internally 32 P-labelled R1.1 RNA as the substrate. The
K d values were determined by gel mobility-shift assays, using 5 -32 P-labelled R1.1[WC] RNA
as the ligand (see the Results section). The data for the E. coli RNase III homodimer (His6 /His6 )
are taken from previous studies [26,29] and are given here for comparison with the E. coli RNase
III heterodimer (His6 /CBP) kinetic parameters. n.d., Not determined.
K m (nM)
k cat (min−1 )
k cat /K m (M−1 · min−1 )
K d (nM)
h
wt/wt (His6 /His6 )
wt/wt (His6 /CBP)
E117A/wt
NucD/wt
42
1.2
2.8 × 107
5.4 +
− 0.6
2.0 +
− 0.1
48 +
−2
2.9 +
− 0.37
6.0 × 10
8.6 +
− 0.6
2.0 +
− 0.1
41 +
−5
1.2 +
− 0.37
2.9 × 10
12.0 +
− 1.3
2.0 +
− 0.1
157 +
− 29
1.1 +
− 0.26
7.0 × 10
141 +
− 13
n.d.
is stabilized by multiple hydrophobic interactions and hydrogen
bonds, and therefore is expected to provide a strong kinetic as
well as thermodynamic barrier to dimer dissociation.
A representative cleavage assay using RNase III[E117A/wt]
is shown in Figure 3(B), which reveals that the heterodimer is
catalytically active and cleaves R1.1 RNA with the same
specificity as RNase III (Figure 3B, compare lanes 2–6 with
lanes 7–11). Given the asymmetry of R1.1 RNA and RNase
III[E117A/wt], the binding of this RNA to the mutant heterodimer
can occur in either of two ways: one binding mode would place
the single scissile bond in the functional catalytic site, leading
to cleavage, whereas the other binding mode would place the
scissile bond in the inactive site, disallowing cleavage. Assuming
comparable affinities for both binding modes, there is expected to
be an equal probability of forming productive and non-productive
enzyme–substrate complexes. As such, at saturating substrate
concentrations, the V max (and thus also the kcat ) for the reaction
involving RNase III[E117A/wt] would be expected to be onehalf that of the reaction involving RNase III, whereas the K m
values would be predicted to be essentially the same. In fact,
Table 1 shows that the kcat value for RNase III[E117A/wt] cleavage
−1
of R1.1 RNA is 1.2 +
− 0.3 min , whereas that of RNase III is
−1
2.9 +
0.3
min
.
In
addition,
the
K m values are similar (41 +
−
−5
and 48 +
2
nM
respectively).
−
Gel shift assays were performed to determine the affinity of
RNase III[E117A/wt] for R1.1 RNA. In this assay, Mg2+ was
replaced by Ca2+ , which promotes substrate binding but does not
support cleavage [44]. The K d values are provided in Table 1,
where it is seen that the RNase III[E117A/wt] · R1.1[WC] RNA
complex has a K d value of 12.0 +
− 1.3 nM, which is only modestly
higher than that of the RNase III · R1.1[WC] RNA complex
(K d = 8.6 +
− 0.6 nM). The comparable K d values are consistent
with the comparable K m values. The kinetic and substrate binding
data support a model of interaction of a single-catalytic-site
heterodimer with a single-scissile-bond substrate that involves
alternative productive/non-productive binding modes. In addition,
the inactivation of a catalytic site does not affect the steadystate catalytic parameters above and beyond that which would be
expected for the productive/non-productive binding model.
The RNase III[E117A/wt] heterodimer is also predicted to
exhibit an altered manner of cleavage of substrates with two
scissile bonds at the target site. If each catalytic site cleaves only
one phosphodiester during a single binding event, then the release
and rebinding of the singly cleaved product would be required
for cleavage of the remaining bond. The release–rebinding event
may be reflected in the accumulation of singly cleaved species,
especially if the species exhibit a weaker affinity for enzyme than
uncleaved substrate. R1.1[WC] RNA is a fully base-paired variant
of R1.1 RNA and contains two scissile bonds at the target site
Figure 4 RNase III[E117A/wt] processing of R1.1[WC] RNA and the
generation of singly cleaved intermediates
(A) Structure of R1.1[WC] RNA. The two scissile bonds are indicated by the arrows. (B) Comparison of RNase III [(H)6 -wt/CBP-wt] and RNase III[E117A/wt] [(H)6 -E117A/CBP-wt] action in
a time course cleavage assay. Internally 32 P-labelled R1.1[WC] RNA (57 nM) was incubated at
37 ◦C with RNase III (10 nM) for the indicated times. Aliquots were electrophoresed in a 15 %
(w/v) polyacrylamide gel containing 7 M urea. Reactions were visualized by phosphoimaging.
Lane 1, R1.1[WC] RNA incubated with RNase III in the absence of Mg2+ ; lanes 2–7, RNase III
incubated with substrate and Mg2+ for the indicated times; lanes 8–13, RNase III[wt/E117A]
incubated with the substrate and Mg2+ for the indicated times. The positions of R1.1[WC] RNA
and its cleavage products are shown on both sides of the gel image.
(Figure 4A) [46]. A time course assay using RNase III[E117A/wt]
reveals the production of singly cleaved intermediates, which
are slowly converted into fully cleaved products (Figure 4B,
lanes 8–13). In contrast, efficient generation of the fully cleaved
products occurs with RNase III, with negligible amounts of
single cleaved species (Figure 4B, lanes 2–7). If the singly
cleaved species are released from RNase III[wt/E117A] prior to
cleavage of the second bond, then the addition of excess unlabelled
substrate would be expected to block the second cleavage step.
Figure 5(B) shows an experiment in which a 10-fold molar
excess of unlabelled R1.1[WC] RNA was added shortly after
initiation of a cleavage reaction involving RNase III[E117A/wt].
The presence of excess substrate blocks further cleavage of the
intermediates, as well as preventing cleavage of unchanged 32 Plabelled substrate (Figure 5B, compare lanes 7–12 with lanes 1–6).
The specificity of inhibition is demonstrated by the lack of effect
of a 10-fold molar excess of tRNA (Figure 5B, lanes 13–18).
For RNase III, the addition of an excess of R1.1[WC] RNA also
blocked further cleavage of unchanged, radiolabelled substrate,
but did not cause an accumulation of singly cleaved intermediates
(Figure 5A, compare lanes 8–13 with lanes 1–7). Thus, in contrast
with RNase III[wt/E117A], wt RNase III efficiently cleaves both
phosphodiesters of R1.1[WC] RNA during a single binding event.
Bivalent metal ion dependence of RNase III[wt/E117A] catalytic
activity
Kinetic, inhibitor and structural studies on RNase III and Dicer
[24,29,30] indicate a two-metal-ion catalytic mechanism [47,48].
If the two-metal-ion mechanism is strictly a function of a single
catalytic site, then RNase III[wt/E117A] would exhibit the same
metal dependence as the wt enzyme. We measured the Mg2+
concentration dependence of RNase III[E117A/wt] cleavage of
R1.1 RNA, using single-turnover conditions in which phosphodiester cleavage is the rate-limiting step [26,29]. The measured Hill coefficient (h) is 2.0 +
− 0.1 (Table 1), which is the same
value as that obtained for the wt enzyme, and indicates that the
two-Mg2+ -ion mechanism in fact reflects the function of a single
catalytic site.
c The Authors Journal compilation c 2008 Biochemical Society
44
W. Meng and A. W. Nicholson
Figure 5 Effect of excess unlabelled substrate on RNase III heterodimer
cleavage of R1.1[WC] RNA
The assay used internally 32 P-labelled R1.1[WC] RNA (57 nM). Enzyme (10 nM) was incubated
with R1.1[WC] RNA as described in the Experimental section. Reactions were electrophoresed in a
15 % (w/v) polyacrylamide gel containing 7 M urea and visualized by phosphoimaging. Reaction
times (min) are indicated above the lanes. (A) Chase experiments involving RNase III [(H)6 wt/CBP-wt]. Lanes 1–7, control time course (no chase); lanes 8–13, unlabelled R1.1[WC]
RNA chase (1 µM). Lanes 14–19, Chase using tRNA (1 µM). The asterisk indicates the time
point of addition. (B) Chase experiments involving RNase III[wt/E117A] [(H)6 -E117A/CBP-wt].
Lanes 1–6, control time course assay (no chase). Lanes 7–12, unlabelled R1.1[WC] RNA chase
(1 µM). Lanes 13–18, tRNA chase (1 µM). Asterisks indicate the time of excess RNA addition.
E. coli RNase III catalytic activity is supported by Mn2+
ion in the 0.1–1 mM concentration range, with concentrations
above ∼ 1 mM causing inhibition of cleavage [27,49]. The
inhibition is proposed to be due to Mn2+ binding to a site near
the catalytic site [36]. To determine whether Mn2+ inhibition
is a function of a single catalytic site, the cleavage of R1.1
RNA by RNase III[E117A/wt] was measured as a function of
the Mn2+ concentration. Figure 6 shows that Mn2+ supports
RNase III[E117A/wt] cleavage of R1.1 RNA at low concentrations, but inhibition is observed at higher concentrations,
similar to what is observed with RNase III. The heterodimer has
maximal activity at the same Mn2+ concentration as that of the
wt enzyme, but is inhibited to a greater degree. The retained
sensitivity of RNase III[E117A/wt] to Mn2+ ion indicates that the
inhibition is not a strict function of both catalytic sites.
Processing activity of an RNase III heterodimer containing a single
dsRBD
An E. coli RNase III mutant that lacks both dsRBDs is inactive
under standard reaction conditions in vitro [32]. It is not known
whether a single dsRBD is sufficient to support activity, and if
so, how removal of a dsRBD may alter the kinetic parameters.
c The Authors Journal compilation c 2008 Biochemical Society
Figure 6
Inhibition of RNase III[E117A/wt] by Mn2+ ion
(A) Mn2+ titration experiment. Cleavage reactions employed RNase III[E117A/wt]
[(H)6 -E117A/CBP-wt] heterodimer (50 nM) and internally 32 P-labelled R1.1 RNA (110 nM).
Reactions were initiated by addition of Mn2+ , and incubated at 37 ◦C for 3 min. Reactions were
electrophoresed in a 15 % (w/v) polyacrylamide gel containing 7 M urea and visualized by
phosphoimaging. Lane 1, incubation without Mn2+ . Lanes 2–9, 0.1, 0.25, 0.5, 0.75, 1, 2, 5
and 10 nM Mn2+ respectively. (B) Graph showing the fraction of R1.1 RNA cleaved by RNase
III (wt/wt), RNase III[E117A/wt] and RNase III[NucD/wt] as a function of Mn2+ concentration.
For RNase III[E117A/wt], the points correspond to the concentrations in the experiment shown
in (A).
For example, if strong functional co-operativity between the
dsRBDs is necessary for activity, then a single-dsRBD RNase III
heterodimer would be severely defective. We prepared an E. coli
RNase III heterodimer containing a single dsRBD (RNase
III[NucD/wt]). A representative cleavage assay using internally
32
P-labelled R1.1 RNA is shown in Figure 7(A). The assay shows
that RNase III[NucD/wt] is catalytically active and cleaves R1.1
RNA at the canonical site (Figure 7A, lanes 7–11). Thus a single
dsRBD is sufficient for activity, and site-specificity is not altered.
However, RNase III[NucD/wt] cleaves R1.1 RNA more slowly
than RNase III (Figure 7A, compare lanes 7–11 with lanes 1–6).
To determine the cause(s) of the reduced rate, the K m and kcat
values were determined (Table 1). The K m of 157 +
− 29 nM and
−1
the kcat of 1.1 +
− 0.2 min for RNase III[NucD/wt] are ∼ 3-fold
greater and ∼ 2-fold lower respectively than the values for RNase
III. We conclude that removal of a dsRBD weakens substrate
affinity, as indicated by the larger K m , and that it also perturbs one
or more steps in the enzyme–substrate complex, as indicated by
the lower kcat value.
Bacterial RNase III heterodimer analysis
45
contrast, RNase III formed a discrete shifted product, with little
aggregation of RNA. By measuring the fraction of unbound RNA
as a function of enzyme concentration, a K d of ∼ 140 nM can be
estimated. In contrast, the complex involving RNase III has a K d
of 8.6 nM (Table 1). The higher K d value for RNase III[NucD/wt]
reflects a weakened affinity for the substrate and is consistent with
the larger K m value (see above).
Severely defective RNA processing activity of the RNase
III[NucD/E117A] double-mutant heterodimer
Figure 7
Catalytic activity of RNase III with a single dsRBD
(A) Time course cleavage assay using internally 32 P-labelled R1.1 RNA. Enzyme (10 nM)
and substrate (43 nM) were incubated in reaction buffer as described in the Experimental
section. Reactions were electrophoresed in a 15 % (w/v) polyacrylamide gel containing
7 M urea and visualized by phosphoimaging. Lane 1, R1.1 RNA incubated in the absence
of Mg2+ . Lanes 2–6, complete reaction involving RNase III [(H)6 -wt/CBP-wt], incubated
for the indicated times. Lanes 7–11, complete reaction involving RNase III[NucD/wt]
[(H)6 -NucD/CBP-wt], incubated for the indicated times. (B) Time course cleavage assay
using internally 32 P-labelled R1.1[WC] RNA. Lane 1, incubation in the absence of Mg2+ .
Lanes 2–6, complete reaction involving RNase III (wt/wt). Lanes 7–11, complete
reaction involving RNase III[NucD/wt]. Lanes 12–16, complete reaction involving RNase
III[NucD/E117A]. Reaction times are indicated. The singly cut intermediates are indicated by the
arrows on the right-hand side.
The source of the K m and kcat effects was investigated further by
examining the ability of RNase III[NucD/wt] to cleave R1.1[WC]
RNA. A time course assay shows that R1.1[WC] RNA is cleaved,
albeit more slowly compared with the reaction involving RNase
III (Figure 7B, compare lanes 7–11 with lanes 1–6). In addition,
singly cleaved intermediates are observed (Figure 7B, lanes 8–11).
The addition of excess unlabelled R1.1[WC] RNA blocked further
cleavage of the intermediates, as well as preventing cleavage of
unchanged substrate (results not shown). The greater amount of
the shorter intermediate product relative to the longer intermediate
product suggests a greater affinity of the longer intermediate
product for RNase III, allowing more efficient cleavage at the
remaining bond (see the Discussion section). The presence of
these species is indicative of a lower stability of the enzymecleaved intermediate and also suggests that the subunit lacking the
dsRBD is catalytically defective. The latter possibility is examined
further below.
A gel shift assay involving RNase III[NucD/wt] and 5 -32 Plabelled R1.1[WC] RNA did not yield a specific complex. Instead,
there occurred a protein-concentration-dependent disappearance
of the unbound RNA, accompanied by an accumulation of the
RNA at the bottom of the gel well (results not shown). This
suggests protein aggregation accompanying RNA binding. In
It is possible that the diminished activity of RNase III[NucD/wt]
reflects a specifically compromised function of the subunit lacking
the dsRBD, rather than a generalized effect of dsRBD removal
on holoenzyme activity. In such a model, the dsRBD would
function primarily in a co-ordinate manner with the catalytic
domain of the same subunit. If this model were true, and if
the dsRBD of one subunit cannot functionally substitute for
the other dsRBD, then inactivation of the catalytic site of the
full-length subunit in RNase III[NucD/wt] would be expected to
severely reduce activity, since both subunits would be catalytically
impaired. RNase III[NucD/E117A] was prepared to test this
model. Here, one subunit lacks the dsRBD, while the fulllength subunit carries the E117A mutation. Time course and
enzyme titration assays reveal that, in comparison with RNase
III[NucD/wt] or RNase III[E117A/wt] (see above), the RNase
III[NucD/E117A] heterodimer cleaves R1.1[WC] RNA only
at high enzyme concentrations, or at extended reaction times
(Figure 7B, lanes 12–16). Thus point mutational inactivation of
the catalytic site in one subunit further impairs activity over that
caused by removal of the dsRBD from the opposing subunit. The
relation of this result to the RNase III mechanism of action is
discussed below.
DISCUSSION
The present study used mutant heterodimers of E. coli RNase
III to assess the consequences of single catalytic site inactivation
or dsRBD deletion on substrate recognition and cleavage. RNase
III heterodimer production was accomplished using an in vivo
system that co-produces two RNase III polypeptides with different
affinity tags. Serial affinity chromatography provided RNase
III heterodimers free of the corresponding homodimers. As
discussed, the E. coli RNase III heterodimers do not measurably
engage in subunit exchange, thus allowing enzymological assays
uncompromised by contaminating homodimers. The catalytic
activity of RNase III[E117A/wt] demonstrates the functional
independence of the two catalytic sites and confirms the previous
report of retained processing activity of RNase III[E117K/wt]
[31]. The rate constant for phosphodiester cleavage by RNase
III[E117A/wt] under single-turnover conditions has the same
Mg2+ concentration dependence as the wt enzyme (h = 2.0).
Thus the probable two-Mg2+ -ion mechanism for phosphodiester
hydrolysis by RNase III [24,29] can be attributed to the operation
of a single catalytic site, rather than reflecting a contribution of
metal ions bound to both catalytic sites. Whereas there is no
description yet of a bacterial RNase III structure with two Mg2+
ions in both catalytic sites, a crystal structure of G. intestinalis
Dicer [30] exhibits two Europium (Eu3+ ) ions bound to each
catalytic site, in positions consistent with a two-metal-ion catalytic
mechanism. The single metal ion observed in the catalytic sites of
the bacterial RNase III structures [17,24] is probably responsible
for activating the water nucleophile, while the second metal ion
(whose binding may accompany substrate binding) may facilitate
c The Authors Journal compilation c 2008 Biochemical Society
46
Figure 8
W. Meng and A. W. Nicholson
Proposed pathway of dsRNA recognition and cleavage by RNase III and comparison with the RNase III[NucD/wt] pathway
The two schemes incorporate the original proposal by Ji and co-workers that the first step involves dsRNA recognition by only one of the two dsRBDs [23]. (A) Pathway for wt RNase III. Note the
alternative pathways in step 1, which includes three elementary steps and which affords the catalytically competent complex, which converts into the product complex in step 2. (B) Pathway for RNase
III[NucD/wt]. In this scheme, there is only one pathway leading to the catalytically competent complex, and only one phosphodiester is cleaved per substrate binding event (step 2). The alteration in
step 2 may reflect (i) the selective inactivity of the subunit lacking the dsRBD (see the Results section), (ii) a weakened binding affinity for the singly cleaved intermediate or (iii) a combination of
both effects.
departure of the ribose 3 -oxygen. Thus a pre-catalytic RNase
III–substrate complex would contain four Mg2+ ions, with each
catalytic site containing two Mg2+ ions. The inhibition of E.
coli RNase III by Mn2+ at concentrations > 1 mM was revealed
in the initial characterization of the enzyme [49]. The retained
sensitivity of RNase III[E117A/wt] to Mn2+ indicates that the
inhibitory mechanism does not require two functional catalytic
sites. Determination of the mechanism of Mn2+ inhibition requires
further kinetic and structural studies.
The inactivation of a single catalytic site has specific effects
on the steady-state rate constants and pattern of cleavage of
substrates with either one or two scissile bonds. While RNase
III[E117A/wt] exhibits an unaltered K m for cleavage of a substrate
with a single scissile bond, it has a kcat that is one-half that of
the wt enzyme. The 2-fold difference in kcat values most likely
reflects the two possible modes of substrate binding to the mutant
heterodimer, which determines whether the scissile bond is placed
in a functional or non-functional catalytic site. For a substrate with
two scissile bonds, the pattern of cleavage indicates that a given
catalytic site can cleave only one phosphodiester during a single
binding event. Thus RNase III[E117A/wt] cleavage of R1.1[WC]
RNA generates significant levels of singly cleaved intermediates,
and these species must dissociate and rebind in order for the
remaining phosphodiester to be cleaved. On dissociation of the
singly cleaved species, the shorter of the two cleavage products
may spontaneously disengage from the larger product, creating
a modified larger product carrying either a 5 or 3 single-strand
extension, depending on which site was cleaved. The new species
may be only weakly bound by the enzyme, leading to a much
slower second cleavage step, as observed. The action of singlecatalytic-site E. coli RNase III heterodimers towards dsRNA is
formally similar to that of Type II restriction enzyme heterodimers
that carry a single functional catalytic site and that cleave only
one DNA strand during a binding event [50,51]. We note that
c The Authors Journal compilation c 2008 Biochemical Society
while E. coli RNase III can be used to create short dsRNAs as
gene silencing reagents [52], a single-catalytic-site heterodimer
can provide a source of nicked dsRNAs for functional analyses of
dsRNA recognition and processing.
The present study has shown that a single dsRBD can support
substrate cleavage by E. coli RNase III under standard conditions
in vitro. However, both dsRBDs are required for full activity. The
∼ 3-fold higher K m for E. coli RNase III[NucD/wt] cleavage of
R1.1 RNA, compared with that of RNase III, and the observation
of singly cleaved intermediates in reactions involving R1.1[WC]
RNA, suggest that the mutant heterodimer–substrate complex
is less stable than the complex involving the wt enzyme. The
higher K m probably incorporates a statistical factor, in that RNase
III[NucD/wt] is only half as likely to recognize substrate as the wt
enzyme. The 2-fold lower kcat for RNase III[NucD/wt] indicates
that a step associated with the enzyme–substrate complex is
perturbed. One possibility is that the subunit that lacks the dsRBD
is specifically defective in catalysis (see below).
Do the two dsRBDs bind the substrate in an independent or cooperative manner? A thermodynamic analysis of protein–RNA
recognition [53] may provide some insight. If binding of the two
dsRBDs to the substrate is fully co-operative, then the free energies of binding of each dsRBD would be additive, and the measured apparent binding affinity (K a ) for RNase III would be
the product of the K a values of the individual dsRBD. If the
binding of the two dsRBDs is fully non-co-operative, then
the measured K a for RNase III would be the sum of the K a
values of the individual dsRBDs. The K d for the complex of
R1.1[WC] RNA bound to the isolated dsRBD is ∼ 800 nM [54].
Thus, if the two dsRBDs function in a fully co-operative manner,
the predicted K a (1/K d ) for RNase III would be 1.6 × 1012 M−1 .
If instead the two dsRBDs are non-co-operative, the predicted
K a for RNase III would be 2.5 × 106 M−1 . The measured K a
for RNase III is 2.5 × 108 M−1 , indicating that RNase III binds
Bacterial RNase III heterodimer analysis
the substrate ∼ 6000 times more weakly than expected for full
co-operativity, and only ∼ 100 times more strongly than expected
for full non-co-operativity. Thus the analysis indicates a largely
independent (non-co-operative) behaviour of the two dsRBDs in
recognizing the substrate.
These considerations, along with the structural features of
non-catalytic [22,23] or post-catalytic [24] RNase III · dsRNA
complexes, suggest a pathway for substrate recognition by
bacterial RNase III (Figure 8A). This model is based on a previous
proposal by Ji and co-workers for bacterial RNase III recognition
of dsRNA [23]. The pathway involves initial recognition of the
substrate by a dsRBD, followed by NucD engagement of the substrate, with an accompanying protein conformational change.
The other dsRBD then engages the substrate to stabilize the
catalytically competent complex (Figure 8A). As discussed
previously [23], the two steps are dependent on the flexibility
of the linker connecting the dsRBD and NucD. This model is
also consistent with a stopped-flow kinetic analysis that detected
an RNase III conformational change associated with substrate
binding [26]. The measured second-order rate constant (K a ) is
smaller than the diffusion-controlled rate constant, suggesting that
the event reflects a protein conformational change immediately
following initial binding of the substrate [26]. The presence of
only a single dsRBD would provide a single pathway in step 1
(see Figure 8B), which would be reflected in a higher K m
(see the Results section). The presence of only one dsRBD
would also affect the catalytic step, if the dsRBD participates
in catalysis. In this regard, the severely defective activity of
RNase III[NucD/E117A] indicates that the dsRBD preferentially
supports the action of the catalytic site of the same subunit.
This functional coupling would explain the 2-fold lower kcat of
RNase III[NucD/wt] cleavage of R1.1 RNA, and the generation of
singly cleaved R1.1[WC] RNA intermediates, compared with the
action of the wt enzyme. The behaviour of RNase III[NucD/wt]
may shed light on the mechanisms of eukaryotic RNase III
orthologues that contain a single dsRBD. Thus the mammalian
Drosha polypeptide, which contains a single dsRBD, is only
catalytically active in the presence of DGCR8 protein, which
contains two dsRBDs [55]. One possibility would be that one
of the dsRBDs of DGCR8 binds the substrate, while the second
dsRBD may functionally complement the single Drosha dsRBD
to provide optimal catalytic activity.
DNA sequencing analyses were performed by the DNA Sequencing Facility of the University
of Pennsylvania. We thank Dr R. H. Nicholson (Department of Biology, Temple University,
Philadelphia, PA, U.S.A.) for helpful comments on this paper, and other members of the
laboratory for their support and encouragement. This research was supported by NIH
(National Institutes of Health) grant number GM56457.
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