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(Received for publication, August 25, 1995) From the
Ribonuclease II (RNase II) is a major exonuclease in Escherichia coli that hydrolyzes single-stranded
polyribonucleotides processively in the 3` to 5` direction. To
understand the role of RNase II in the decay of messenger RNA, a strain
overexpressing the rnb gene was constructed. Induction
resulted in a 300-fold increase in RNase II activity in crude extracts
prepared from the overexpressing strain compared to that of a
non-overexpressing strain. The recombinant polypeptide (Rnb) was
purified to apparent homogeneity in a rapid, simple procedure using
conventional chromatographic techniques and/or fast protein liquid
chromatography to a final specific activity of 4,100 units/mg.
Additionally, a truncated Rnb polypeptide was purified, solubilized,
and successfully renatured from inclusion bodies. The recombinant Rnb
polypeptide was active against both [
Because the rate of synthesis of any given protein is directly
proportional to the concentration of its message, regulating the
balance between mRNA decay and its synthesis is an important aspect of
gene expression. In Escherichia coli, it is widely accepted
that mRNA decay is initiated by a series of endonucleolytic cleavages
catalyzed by RNase E (1, 2, 3) or
occasionally by RNase III (4, 5) followed by
processive exonucleolytic degradation of the message to oligo- and
mononucleotides(1, 2, 3) . Two
3`-exonucleases have been implicated in this process: ribonuclease II
(RNase II) ( RNA structure, known
to be an important determinant of mRNA stability, can protect upstream
sequences from digestion by the 3`-exonucleases. The Rho-independent
terminator sequence (trp t) of the tryptophan operon (15) and the intergenic (malE-malF) REP sequence of
the maltose operon (16) are classic examples of secondary
structures that protect upstream RNA from 3`-exonucleolytic degradation
both in vitro and in vivo. These investigations
implied that the observed protection by 3`-stem-loop structures was the
result of an impediment to the processive activities of RNase II and,
to a lesser extent, of PNPase (15, 16, 17) .
Recent observations of the decay of RNA-OUT, the antisense RNA that
regulates Tn10/IS10 transposition, demonstrate that
the higher the thermal stability of the RNA structure, the larger the
barrier to degradation by RNase II(18) . Degradation by PNPase
is much less affected by the relative stability of the RNA-OUT
structure(18) . Interestingly, RNA-OUT appears to be stabilized
approximately 3-fold against PNPase attack by RNase II(18) . In
addition, the rpsO mRNA is also stabilized significantly by
the presence of RNase II(19) . Although the mechanism by which
RNase II shelters upstream sequences from further exonucleolytic attack
is not understood, the observed protection was attributed to the
formation of a stable RNase II-RNA complex, which sequesters the 3`-end
of the transcript(18, 19) . As part of the
investigation of the functional and biophysical properties of RNase II
and its role in the overall decay of mRNA, we have overexpressed RNase
II and developed a rapid and simple purification of the enzyme free of
other nucleases. The purified enzyme was used to investigate the
mechanisms by which stem-loop structures impede exonucleases and the
ability of RNase II to act as a repressor of PNPase activity.
Figure 1:
3`-Exonucleolytic degradation of a
partial duplex RNA substrate. Panel a presents a schematic
diagram of the 3`-exonucleolytic degradation of the 92-nucleotide RNA
substrate on the left to the 77-nucleotide product on the right. A time course digestion of the synthetic RNA transcript
t40B (a) as described under ``Experimental
Procedures'' is shown in panels b and c. Crude
extracts (S-150 fraction) prepared from strain CF881 (b) or
strain 18-11 (c) were added to a final concentration of
0.5 µg/ml and incubated with substrate at 37 °C. Aliquots were
removed from the reaction mixture at the times indicated (in minutes)
as described under ``Experimental Procedures.'' The digestion
products were analyzed by electrophoresis through a 10% polyacrylamide
gel under denaturing conditions. NP denotes a control lane
containing substrate incubated in the absence of protein for 60 min.
The 92-nucleotide substrate (S) and the stable degradative
intermediate (P) (77 nucleotides) are indicated with arrows.
Figure 2:
PNPase is unable to attack partially
digested t40B. a, crude extracts (S-30 fraction) prepared from
strain 18-11 at a final concentration of 0.2 mg/ml were incubated
with t40B in the absence (lanes 1-4) or presence (lanes 5-8) of 10 mM phosphate at 37 °C. b, extracts (S-30 fraction) prepared from strain 18-11
at a final concentration of 0.2 mg/ml were incubated in the presence of
10 mM phosphate at 37 °C with t40B, which had been
previously digested to 77 nucleotides with purified Rnb polypeptide,
extracted with phenol/chloroform, and precipitated with ethanol.
Aliquots were removed from the reaction mixture at the times indicated
(in minutes) as described under ``Experimental Procedures.''
The digestion products were analyzed by electrophoresis through a 10%
polyacrylamide gel under denaturing conditions. The 92-nucleotide
substrate (S), previously digested substrate (S*),
and the 77-nucleotide degradative intermediate (P) are
indicated with arrows. NP denotes a control lane
containing substrate incubated in the absence of protein for 30
min.
Figure 3:
Strategy for cloning and overexpression of rnb. Panel a illustrates a linear representation of
the rnb gene of E. coli and its flanking sequences.
Plasmids pGC100 (b) and pGC101 (c) were constructed
by polymerase chain reaction as described in the text. The solid
box represents the cloned sequence, and the line represents sequences derived from the vector pET-11. Coordinates
of the rnb sequence and the predicted start and stop codons
are shown in a. The Shine-Dalgarno sequence is represented by
an enclosed SD, and B denotes the position of the BamHI restriction sites. The open box represents
regions 3` to the rnb gene also cloned into plasmid pGC100 (b). Plasmid pGC101 (c) lacks 29 nucleotide residues
deleted from the 3`-end of the rnb coding sequence. This
deletion has extended the open reading frame 66 nucleotide residues
into the T7 terminator region of pET-11 shown by the hatched
box. The ``TGA'' stop codon at position 2078, predicted
by the original rnb sequence(23) , is not in frame in
the corrected sequence.
Upon induction
of cultures of GC100 or GC101 with IPTG, the Rnb polypeptide was
expressed to the extent that it represented the most abundant
polypeptide in whole cell extracts and a significant fraction of the
total cellular protein (Fig. 4, lane 3). When assayed
against poly(A), crude extracts (S-30) from strain GC100 displayed a
specific activity of 1,184 units/mg (Table 1), 300-fold higher
than that obtained from crude extracts prepared from the haploid strain
CF881 (specific activity = 3.9 units/mg). An efficient method of
purification was developed in part by exploiting several effective
steps from previously published
methods(12, 13, 14) . The initial step relies
on Cibacron blue-agarose chromatography to remove the bulk of the
nucleic acids and contaminating proteins while the majority (>90%)
of the Rnb polypeptide remains bound to the column. Considerable
efficiency was gained by loading the 3 M NaCl eluate from this
column directly onto a hydroxylapatite column. This proved to be an
invaluable step in the purification method since concentration,
desalting, and significant purification of the Rnb polypeptide could
take place in a single step. The apparent loss of activity after
hydroxylapatite chromatography (Table 1) may have been due to the
inhibition by Ca
Figure 4:
Purity of the recombinant Rnb polypeptide.
Cultures of GC100 were induced with IPTG and grown for 5 h at 30 °C
prior to harvest, lysis, and purification as described under
``Experimental Procedures.'' The following samples were
denatured, separated in a 13% SDS-polyacrylamide gel, and stained with
Coomassie Blue: lane 1, molecular mass standards (Bio-Rad); lane 2, boiled cell extract from a noninduced culture of
GC100; lane 3, boiled cell extract from an induced culture of
GC100; lane 4, S-30 extract (10 µg); lane 5,
pooled fractions obtained from chromatography of the S-30 fraction on
blue-agarose (BA) (2.5 µg); lane 6, pooled
fractions from chromatography of the BA fraction on
hydroxylapatite (HTP) (1.75 µg); lane 7, pooled
fractions from chromatography of the HTP fraction on Resource Q (Q) (1.5 µg); and lane 8, pooled fractions from
chromatography of the HTP fraction on heparin-agarose (HA)
(1.5 µg).
If recoveries from the heparin-agarose chromatography step
in Table 1are extrapolated to include all the material in pool A
from the hydroxylapatite column, the overall yield is 29%. This
apparent overall yield is low for two reasons. First, the activity in
crude extracts represents the sum of activities of a number of endo-
and exonucleases and overstates the activity of RNase II. Second,
fractions were pooled to maximize purity rather than yield particularly
after hydroxylapatite chromatography. The addition of 22 amino acid
residues, derived from the vector pET-11, to the C terminus of the
truncated Rnb* polypeptide, resulted in the formation of insoluble
inclusion bodies upon induction of cultures of GC101 with IPTG. The
inclusion bodies were subsequently purified to near homogeneity by
differential centrifugation in the presence of detergent. Authentic
RNase II activity was recovered following solubilization, reduction,
and refolding of the truncated Rnb* polypeptide from the inclusion
bodies. Further purification of the renatured truncated Rnb*
polypeptide from most contaminants could be achieved by ion exchange
chromatography (FPLC). The truncated Rnb* polypeptide eluted from the
Resource Q column over a broad range of NaCl concentrations likely
reflecting the several different populations of misfolded and inactive
polypeptides present in the preparation. RNase II activity eluted from
the column as a sharp peak at a NaCl concentration of 220 mM.
Although a significant amount of activity could be recovered from the
inclusion bodies, the specific activity of this preparation was quite
poor, 54 milliunits/mg, a small fraction of that obtained for the
full-length Rnb polypeptide.
Figure 5:
a, degradation of the partial duplex RNA
substrate by purified Rnb polypeptide. In lanes 2-6, the
t40B transcript was incubated with the purified Rnb polypeptide (1.2
milliunits; 5 ng/ml) for the indicated times (in minutes) at 37 °C
as described under ``Experimental Procedures.'' Lanes 7 and 8 show a 60-min digestion of the t40B transcript
incubated with the purified Rnb polypeptide (2.0 milliunits and 4.1
units; 8.3 ng/ml and 16 µg/ml, respectively). The digestion
products were subsequently analyzed by gel electrophoresis. b,
thermal inactivation of the recombinant Rnb polypeptide. Rnb
polypeptide (0.5 µg/ml) was incubated in the absence of substrate
for the indicated times (in minutes) prior to addition to a complete
reaction mixture. Incubation was continued for an additional 15 min at
a final concentration of Rnb polypeptide of 8.3 ng/ml. The digestion
products were analyzed by gel electrophoresis. The substrate (S) and the degradative intermediate (P) are
indicated by arrows, while the shorter degradation product
shown in lane 8 is indicated by an arrowhead. NP denotes control lanes containing substrate incubated in the
absence of protein for 60 min.
Digestion of the
t40B transcript is complete after a 60-min incubation with 2.0
milliunits of RNase II activity (Fig. 5a, lane
7). Approximately 20% of the substrate is resistant to degradation
by the Rnb polypeptide even after addition of 200 milliunits of fresh
enzyme (data not shown). A fraction of the substrate appears to form
concatemers and as a result does not have free 3`-ends accessible to
the enzyme. Interestingly, digestion of t40B for 60 min at 37 °C
with 4 units of enzyme resulted in a further shortened (73 nt) but
stable degradation intermediate depicted by the arrowhead (Fig. 5a, lane 8). This experiment
suggests that at high concentrations, the Rnb polypeptide can remove
three to four additional unpaired residues in the t40B duplex remaining
from a previous round of digestion. Several previously published
reports have suggested that RNase II, of varying degrees of purity, is
readily inactivated by
heat(10, 11, 12, 13, 28) .
We have tested the purified Rnb polypeptide and found that the
recombinant enzyme is also susceptible to thermal inactivation (Fig. 5b). A comparison of Fig. 5b, lanes 2 and 3, shows that less than 1% of the
activity remains after a 5-min incubation of the Rnb polypeptide in the
absence of substrate at 37 °C. Interestingly, the enzyme is
stabilized in the presence of substrate and can remain active up to 60
min at 37 °C (Fig. 5a). Activity can also be
stabilized by the addition of substrate to Rnb polypeptide, which has
been partially inactivated by a brief incubation in buffer at 37
°C. Once activity has been lost to thermal inactivation, however,
it cannot be regained upon addition of substrate (data not shown). The
77-nt product also stabilized the enzyme against heating. Rnb
polypeptide was incubated in the presence of 1.5 pmol of partially
digested t40B for 5 min at 37 °C prior to incubation with
full-length t40B transcript. The 77-nt product not only protected the
Rnb polypeptide against thermal inactivation but also appeared to
stimulate the activity of the enzyme for the full-length substrate by
approximately 2-fold (data not shown). The apparent stimulation may be
attributable to a decreased rate of thermal inactivation. Taken
together, the data demonstrate that the enzyme can be stabilized by
both substrate and product. In contrast, both a single-stranded DNA
oligonucleotide (33-mer) and double-stranded plasmid DNA inhibited the
activity of RNase II but were unable to provide significant protection
from heating (data not shown) unlike oligonucleotides of
deoxy(C) Stabilization of RNase II by the digested
t40B transcript implies that in the absence of any free
3`-single-stranded ends, the Rnb polypeptide can bind RNA even if it is
not a substrate. To test this hypothesis, t40B was incubated briefly
with a large excess of Rnb polypeptide, sufficient to digest it to 73
nt, and then subjected to UV photocross-linking. Fig. 6, lane 1, shows labeling of a band of 70 kDa, the size expected
for the Rnb polypeptide. In addition, there is label associated with a
band of 14 kDa, which we believe to be RNase A. The Rnb polypeptide is,
therefore, able to bind its product (Fig. 6, lane 1) in
the absence of any other proteins or cofactors. A 70-kDa protein,
corresponding to the molecular mass of RNase II, was also labeled in
crude extracts prepared from strain CF881 (Fig. 6, lane
3). All bands were sensitive to proteinase K treatment (Fig. 6, lanes 2 and 4). A comparison of Fig. 6, lanes 1 and 3, also demonstrates that
UV cross-linking can provide an important assessment of the purity of
the enzyme preparation in light of the affinity chromatography
techniques utilized in the purification. Since there are a large number
of RNA binding proteins in crude extracts prepared from E. coli that have a significant affinity for the t40B transcript, the
presence of even a small percentage these contaminants would be readily
detected in the purified material (Fig. 6, compare lane 3 to lane 1).
Figure 6:
UV cross-linking of t40B to purified
recombinant Rnb polypeptide and proteins in the S-150 fraction prepared
from strain CF881. Labeled t40B was incubated with purified recombinant
Rnb polypeptide (2.1 units, 50 µg/ml) or with 10 µg of an S-150
fraction prepared from strain CF881 (33 milliunits) at a concentration
of 1 mg/ml, irradiated with UV, digested with ribonucleases, and then
separated by SDS-PAGE as described under ``Experimental
Procedures.'' A duplicate sample was treated with proteinase
K(
We have also tested whether the 77-nt
product would inhibit the activity of the Rnb polypeptide in subsequent
rounds of digestion. In the first experiment, the Rnb polypeptide (3.3
milliunits) was incubated with 25 pmol of unlabeled t40B (2.5-fold
molar excess over labeled t40B) for 2.5 min at 37 °C prior to
addition of labeled t40B. The kinetics of digestion of labeled t40B
over a 60-min time course were identical to those in an incubation in
which the same amount of enzyme was incubated directly with labeled
t40B (data not shown). In the second experiment, the t40B transcript,
which had been previously digested with Rnb polypeptide, extracted with
phenol/chloroform, and ethanol precipitated, was used in a competition
experiment. Equimolar amounts of digested t40B did not alter the
kinetics of disappearance of the 92-nt substrate and thus were unable
to compete effectively for the Rnb polypeptide (Fig. 7).
Although the 77-nt product can protect the enzyme from thermal
inactivation, it cannot inhibit its activity.
Figure 7:
Competition between partially digested
t40B and complete t40B. Rnb polypeptide (2.0 milliunits, 8.3 ng/ml) was
incubated with intact t40B in the presence (
Recent observations
have suggested that RNase II can protect ``upstream'' RNA
sequences from PNPase attack through the formation of a stable
RNA-RNase II complex(18, 19) . We have further
investigated this hypothesis by incubating the 77-nt product, produced
by the action of the Rnb polypeptide (see above), with crude extracts
prepared from strain 18-11 in the presence of 10 mM sodium phosphate. The data demonstrate that the 77-nt product is
resistant to digestion by a PNPase-like activity (Fig. 2b, lanes 1-4). As discussed
above, the 92-nt substrate is rapidly shortened to approximately 77 nt
in the presence of phosphate over a 30-min time course of digestion (Fig. 2a, lanes 5-8).
The linear kinetics observed for the reaction
demonstrate that RNase II stalls at regions of secondary structure,
however briefly, but can disengage from the ``stalled''
substrate and reassociate with a new free 3`-end. This is substantiated
by the demonstration that the Rnb polypeptide can cycle from an
unlabeled to a labeled substrate. Our finding of dissociation from a
substrate with 9 unpaired protruding nucleotides at the 3`-end of the
77-nt product is in good agreement with the 10-15-nt digestion
limit product obtained for RNase II acting on
homopolymers(28) . Interestingly, RNase II can participate in
the processing of some tRNAs in vitro by degrading long
trailing sequences but must be able to dissociate from the precursor to
allow final maturation of the tRNA by other processing
exonucleases(29) . Two lines of evidence suggest that the
Rnb polypeptide can also reassociate with the 77-nt product of
digestion. First, at high concentrations of enzyme the Rnb polypeptide
can remove three to four additional unpaired residues remaining from a
previous round of digestion. Second, the Rnb polypeptide can bind its
73-77-nt product as evidenced by UV cross-linking and protection
from thermal inactivation. Although partially digested t40B can bind to
the Rnb polypeptide, it does not compete with the full-length
substrate, indicating that the preferred substrate for RNase II has an
extended free 3`-end. Moreover, the lack of competition by product
implies that product binds to a site distinct from that of the
substrate.
These observations suggest a possible model for the control of mRNA
degradation at the 3`-end. As RNase II encounters a region of secondary
structure, it stalls. If the structure is unstable, the enzyme may
advance through the stem-loop in the 3` to 5` direction. However, if
the structure is a stable REP sequence or a Rho-independent terminator,
RNase II will dissociate from the transcript before the duplex opens.
We propose that loss of the single-stranded 3` overhang, which reduces
the affinity of RNase II for the stalled transcript, may also reduce
the ability of the much larger PNPase to bind and degrade transcripts.
It could also reduce the affinity of putative RNA helicases for such
structures. Addition of a new 3`-end by PcnB followed by the action of
PNPase, known to be less susceptible to RNA secondary structure than
RNase
II(15, 16, 17, 18, 19) ,
would be required for the degradation of strong REP and terminator
sequences. Thus, a competition between removal of a 3`-overhanging
sequence by RNase II and extension-degradation by PcnB and PNPase,
respectively, would develop at the 3`-end of extended RNA secondary
structures and may account for the heterogeneity in the 3`-ends of
oligoadenylated RNA I(33) .
Note Added
in Proof-We have discovered that an A
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 1048-1053
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]poly(A) as
well as a novel (synthetic partial duplex) RNA substrate. The data show
that the Rnb polypeptide can disengage from its substrate upon stalling
at a region of secondary structure and reassociate with a new free
3`-end. The stalled substrate formed by the dissociation event cannot
compete for the Rnb polypeptide, demonstrating that duplexed RNAs
lacking 10 protruding unpaired nucleotides are not substrates for RNase
II. In addition, RNA that has been previously trimmed back to a region
of secondary structure with purified Rnb polypeptide is not a substrate
for polynucleotide phosphorylase-like activity in crude extracts. The
implications for mRNA degradation and the proposed role for RNase II as
a repressor of degradation are discussed.
)and polynucleotide phosphorylase
(PNPase)(6) . RNase II, which is responsible for the majority
of the exonucleolytic activity in E. coli extracts (7) , hydrolyzes RNA to release 5`-mononucleotides(8) ,
while PNPase phosphorylyzes RNA to mononucleoside
diphosphates(9) . Although RNase II activity was first
described over three decades ago (10, 11) and purified
from whole cells several years
later(12, 13, 14) , details of its role in
mRNA degradation are still poorly understood.
Bacterial Strains and Plasmids
The E. coli strain 18-11 (rna, rnb
, rnd
, rbn
, rnt
) (20) was obtained from Dr. M. P. Deutscher (University of
Connecticut Health Center, Farmington), while the strain CF881 F
lac argA trp recB1009 (xthA-pnc)
rna was obtained from Dr. M. Cashel (National Institutes
of Health). The vector pET-11 and its host stain BL21(DE3) (21) were obtained from Novagen. The plasmid pRP40 (22) was obtained from Dr. N. Sonenberg (McGill University,
Montreal). The following oligonucleotide primers were synthesized based
on the previously published rnb sequence(23) : fP1
(5`-GCGAGGATCCAGGAGGTGACAATTATGTTTCAGGACAAC) and rP1
(5`-GCGAGGATCCTTTCCATGCGGACTTCGGCATTA). An additional reverse primer
rP2 (5`-GCGAGGATCCATCGACGGTCAGACTCATCATCA) was constructed based on the
partial DNA sequences of pRZA17 and pRZA18 obtained from Dr. C. M.
Arraiano (Centro de Tecnologia Química e
Biológica, University of Lisbon, Portugal) which
contain the 3`-untranslated region of the rnb gene. The
predicted coding sequence of the rnb gene of E. coli was amplified from genomic DNA of strain MV1190 by the polymerase
chain reaction. The products were cleaved with BamHI and
ligated into the unique BamHI site of pET-11. The orientation
of the 2.4- (fP1-rP2) and 1.9-kilobase pair (fP1-rP1) BamHI
fragments in the recombinant plasmids was verified by restriction
mapping and DNA sequencing of the entire rnb gene. The
resulting plasmids, pGC100 and pGC101, were used to transform BL21(DE3)
to yield strains GC100 and GC101, respectively.RNase II Assays
The 92-nucleotide (nt)
partial duplex RNA substrate, which we call t40B (previously called RNA
I) (22) , was generated from the plasmid pRP40 linearized with
the restriction enzyme BamHI. Synthesis of uniformly labeled
t40B was directed from an SP6 promoter in the presence of
[
-P]CTP as described
previously(24) . Assays for RNase II activity were assembled in
a 70-µl reaction volume containing 10 pmol of labeled t40B in a
reaction buffer containing 17 mM HEPES
NaOH, pH 7.5, 0.5
mM MgAc
, 100 mM KCl, 2 mM DTT,
5% glycerol, and 10 µg/ml acetylated bovine serum albumin (New
England Biolabs). Protein was added last to the final concentration
specified in the figure legends, and incubations were performed at 37
°C. Samples were withdrawn at various times and quenched in 3
volumes of loading buffer containing 90% deionized formamide, 22 mM Tris, 22 mM boric acid, 0.5 mM EDTA, 0.1% xylene
cyanol FF, and 0.1% bromphenol blue. The products were resolved by
electrophoresis on 10% polyacrylamide gels containing 8 M urea
and visualized by autoradiography or with a Molecular Dynamics
PhosphorImager system. Activity was also determined by release of
acid-soluble radioactivity from
[H]poly(A)(25) . 1 unit of RNase II
activity is defined as the release of 1 µmol of AMP/h.Preparation of Crude Extracts
Cultures of
CF881 and 18-11 grown in 1 liter of rich medium (21) to
late logarithmic phase were harvested by centrifugation and frozen at
-70 °C until use. The thawed cells were resuspended in 3
volumes of buffer A (60 mM TrisHCl, pH 7.5, 10 mM MgCl, 60 mM NH
Cl, 0.05 mM EDTA, 1 mM DTT) and ruptured by passage through an Aminco
French pressure cell at 15,000 p.s.i. The cell lysate was centrifuged
at 30,000 
g for 30 min in a Beckman JA-20 rotor at 4
°C. The supernatant (S-30) was then centrifuged at 150,000 g in a Beckman Ti70.1 rotor for 2 h at 4 °C. The
supernatants, S-30 and S-150, were the source of crude extracts for
subsequent experiments.Preparation of RNase II (Rnb) from an Overexpressing
Strain
Cultures of GC100 were grown in a rich medium (21) at 30 °C to early logarithmic phase and induced with
0.4 mM isopropyl 
-thiogalactopyranoside (IPTG) for 5 h.
The cultures were chilled, and the cells were harvested by
centrifugation at 4,000 g for 10 min. All subsequent
procedures were performed at 4 °C. Cell pellets were resuspended in
20-25 ml of buffer B containing 50 mM HEPESNaOH,
pH 7.5, 500 mM NaCl, 1 mM MgCl, 0.1
mM EDTA, 5 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 0.8 µg/ml leupeptin, and 2
µg/ml aprotinin. The cells were ruptured by passage through an
Aminco French pressure cell at 15,000 p.s.i. The lysate was centrifuged
at 30,000 g for 60 min in a Beckman JA-20 rotor to
pellet unbroken cells and insoluble material. Approximately 60 mg of
the S-30 was loaded onto a column of Affi-Gel blue (Bio-Rad) (1.25
21.5 cm) previously equilibrated with 3 column volumes of
buffer C (25 mM HEPESNaOH, pH 7.5, 5% glycerol, 2 mM DTT, 1 mM MgCl, 0.1 mM EDTA)
containing 500 mM NaCl. The column was washed with 3-5
column volumes of this buffer at a flow rate of 8.3 ml/h (6.75 cm/h)
driven by a P1 peristaltic pump (Pharmacia Biotech Inc.). The Rnb
polypeptide was eluted with 5 column volumes of buffer C containing 3 M NaCl. The eluent was pumped directly onto a column of
hydroxylapatite (Bio-Rad) (0.75 8.5 cm) at a flow rate of 6.7
ml/h (15 cm/h). After washing with 5 column volumes of buffer C
containing 1 mM sodium phosphate, pH 7.5, the Rnb polypeptide
was eluted with a 50-ml gradient of sodium phosphate, pH 7.5
(1-250 mM), in buffer C at a concentration of 75 mM sodium phosphate. Fractions containing the Rnb polypeptide were
divided into pool A or pool B based on the contaminants present in the
fractions. A portion of pool A was loaded onto a column of Affi-Gel
heparin (Bio-Rad) (0.75 8.0 cm). The column was washed with
3-5 column volumes of buffer C at a flow rate of 7.2 ml/h (16
cm/h). The Rnb polypeptide was eluted from the column with a 50-ml
gradient of NaCl (0-400 mM) in buffer C at a
concentration of 130-140 mM NaCl. Alternatively,
chromatography (FPLC) of pool A on a Resource Q column (Pharmacia) was
substituted for the Affi-Gel heparin step. After loading the sample and
washing it with 5 column volumes of buffer C containing 150 mM
NaCl, the Rnb polypeptide was eluted from this resin with a 50-ml
gradient of NaCl (100-400 mM) in buffer C at a
concentration of 220 mM NaCl. The presence of the Rnb
polypeptide in various fractions was monitored qualitatively by
polyacrylamide gel electrophoresis and quantitatively by enzyme assay
(see above). The pooled fractions obtained from heparin-agarose
chromatography were the source of purified Rnb polypeptide in all
subsequent experiments.UV Photocross-linking
Assay mixtures were
prepared as described above with 160 fmol of t40B substrate. After
incubation on ice for 2-5 min, the sample was subjected to a
single 2-6-ns pulse (40-50 mJ) with a 266-nm UV laser
(Spectra Physics) as described previously(26) . The sample was
then incubated with 5 µg of RNase A and 5 units of RNase T1 at 37
°C for 45 min to remove excess RNA. Each digested sample was boiled
in an equal volume of SDS sample buffer and separated
electrophoretically on a 15% SDS-polyacrylamide gel. The cross-linked
proteins were visualized by autoradiography.
Exonucleolytic Activity in Crude Extracts From E.
coli
A partially duplexed RNA substrate (Fig. 1a) was used to assay extracts generated from
various E. coli strains for putative RNA helicase activities.
Instead of detecting an activity that could unwind the duplexed RNA to
monomers, we observed the partial degradation of the synthetic
substrate in extracts that are wild type for RNase II activity but not
in extracts deficient for a number of exonucleases including RNase II (Fig. 1, compare b and c). Complete conversion
of the 92-nt substrate to a relatively stable 77-nt degradative
intermediate was observed in crude extracts prepared from strain CF881
over a 60-min time course (Fig. 1b). The exact size of
the product was determined on a sequencing gel (data not shown). In
contrast, crude extracts prepared from strain 18-11 were unable
to digest the substrate (Fig. 1c). Several additional
experiments were undertaken to confirm that the 77-nt degradation
product (shown in Fig. 1a) corresponds to the product
of RNase II stalling 9 nucleotides 3` to the double-stranded region of
the substrate. First, the denatured 77-nt product retains a 5`-end
label (data not shown). Second, the partial duplex substrate is
resistant to digestion by the purified Ams/Rne/Hmp-1 polypeptide, the
catalytic subunit of RNase E(27) , under conditions where
authentic substrates would be processed to completion (data not shown).
Third, incubation of the 92-nt substrate under conditions where PNPase,
the other major exonucleolytic activity in E. coli, would be
active also generates a 77-nt product but only in the presence of 10
mM sodium phosphate (Fig. 2a). In this case,
however, the 77-nt product can be degraded further in prolonged
incubations (data not shown). Moreover, extracts prepared from a strain
containing the mutant pnp-7 allele, which largely lacks PNPase
activity but does contain RNase II activity, also generate the 77-nt
product in the presence or absence of phosphate (data not shown).
Although contributions from other exonucleases cannot be excluded
completely, the phosphate-independent formation of the 77-nt product is
most consistent with RNase II activity. This was confirmed (see below)
using purified recombinant RNase II.
Overexpression and Purification of RNase II
(Rnb)
The predicted coding sequence of the rnb gene of E. coli was amplified by the polymerase chain
reaction as described under ``Experimental Procedures.'' All
primers contained BamHI restriction sites, and fP1 also
contains a Shine-Dalgarno sequence 5` to the rnb start codon
such that the amplified product could be cloned into the unique BamHI site of pET-11 and subsequently overexpressed using the
T7 RNA polymerase encoded by BL21(DE3)(21) . The partial
structures of plasmids containing all or part of the rnb gene
are depicted in Fig. 3, b and c. Due to errors
in the previously published rnb sequence, which predicted a
stop codon at position 2078(23) , plasmid pGC101 (Fig. 3c) contains most of the rnb coding
sequence except for a deletion of 26 nucleotide residues, which is
replaced by 66 nucleotide residues of vector-derived sequence at the
3`-end of the construct. Plasmid pGC100 (Fig. 3b)
contains the entire predicted 1932-nucleotide residue open reading
frame, the 3`-untranslated region including the putative
Rho-independent terminator, and approximately 400 nucleotide residues
of intercistronic spacer under the control of the T7-lac promoter-operator region in pET-11(21) .
ions leached from the column at high
ionic strength, as Ca
has been reported to inhibit
RNase II activity(10, 11) . Final purification of Rnb
from most contaminants could be achieved by affinity chromatography on
heparin-agarose or by ion exchange chromatography (FPLC). A sample of
the purified Rnb polypeptide is shown in Fig. 4, lanes 7 and 8. Based upon Coomassie Blue or silver staining of
overloaded polyacrylamide gels, the preparation was judged to be about
95% pure with a few faint minor contaminating bands. The specific
activity of the Rnb polypeptide purified to the end of the
heparin-agarose step was determined to be 4,100 units/mg, which is
nearly 2-fold greater than that reported by others for the enzyme
purified from whole cells(11, 12, 13) .
However, the specific activity of this preparation is approximately
2.5-fold lower than the best reported purification(14) . It is
quite possible that not all of the overexpressed Rnb polypeptide is
properly folded or fully active. Nonetheless, this method provides a
more rapid and facile purification of RNase II with good yields and
activity.
Properties of the Rnb Polypeptide
The
purified Rnb polypeptide was active against the partial duplex t40B
substrate in a manner similar to the activity originally detected in
crude extracts from strain CF881 (Fig. 5a, lanes
2-6). Under conditions in which enzyme is limiting (molar
ratio of substrate to enzyme 2300:1), the 3`-single-stranded tails are
removed from the substrate during a 60-min incubation at 37 °C to
generate a degradative intermediate, which has been shortened by about
15 nucleotides. The appearance of the degradative product is linear for
30 min, after which the rate declines gradually (Fig. 5a, lanes 2-6). Thus, each enzyme
molecule is turning over more than 30,000 times.
, which can reduce the rate of thermal
inactivation(28) .
) (PROT K) prior to electrophoresis (lanes 2 and 4). Lanes 1 and 2, purified
recombinant Rnb polypeptide; lanes 3 and 4, crude
extract prepared from strain CF881.
) or absence
(
) of 10 pmol of t40B that had been previously digested to yield
a 77-nucleotide product with the purified Rnb polypeptide. The products
were resolved by gel electrophoresis, and the relative amounts of t40B
were quantified with a PhosphorImager, expressed as picomoles of RNA
remaining, and plotted as a function of
time.
The Mechanism of Action of RNase II on a Novel
Substrate
We envisage that the action of RNase II on t40B
can be described by the following sequential steps: 1) binding to a
free 3`-end on the 92-nt substrate, 2) processive hydrolysis of 15
phosphodiester bonds, 3) stalling of the enzyme approximately 9
unpaired nucleotides from the 10-bp G-C-rich stem, 4) dissociation of
the enzyme from the substrate, and (5) thermal inactivation of
a fraction of the dissociated enzyme. The duration of each such cycle
at steady state can be calculated from the apparent turnover number,
which we estimate as 9 nts based on a rate of
0.16 pmol of product formed per min at 4.3 fmol of enzyme. This yields
a cycle time of 1.67 s, the time to remove 15 nucleotides from each
3`-end (15 nt/9 nt
s). The time actually
required for hydrolysis of 15 phosphodiester bonds (step 2 in the
cycle) is only 0.21 s, however, as the reported turnover number for
RNase II acting on poly(A) is 70
nt
s(28) . If we assume that this
turnover number also applies to the 15 residues removed from t40B and
that no enzyme is lost to thermal inactivation (step 5), then steps 1,
3, and 4 account for 1.46 s (1.67 - 0.21 s) of each cycle. As a
consequence, RNase II cannot remain bound to a substrate once
processive hydrolysis has ceased any longer than 1.46 s. The latter
represents a maximum value for step 3 in the proposed cycle, as binding
(step 1), dissociation (step 4), and thermal inactivation (step 5) are
not negligible.
A Model for the Control of mRNA Degradation at the
3`-End
As discussed in the Introduction, 3`-stem-loop
structures have been shown to protect upstream RNA sequences from
digestion by 3`-exonucleases(15, 16, 17) .
The observed protection of upstream sequences was originally attributed
to the impeding of the processive activities of RNase II or PNPase by
RNA structure. Our results, however, demonstrate that the Rnb
polypeptide loses its apparent processivity nine residues 3` to a
region of strong RNA secondary structure, where it leaves the substrate
rapidly and reassociates with a new free 3`-end. The data imply that
the recently observed stabilization of the Tn10/IS10 antisense RNA-OUT (18) and the stabilization of rpsO mRNA (19) by RNase II are probably due to the removal of
the 3`-overhang rather than to the formation of a stable RNA-RNase II
complex, which blocks access of PNPase to the 3`-end of mRNAs. However,
it should be noted that dissociation and/or binding events could be
retarded in vivo if free 3`-ends are limiting or if stem-loop
binding proteins stabilize an RNase II-product
complex(16, 30, 31) . It has been suggested
that mRNAs with an immediate 3`-stem-loop structure, analogous to the
77-nt product, are poor substrates for PNPase(32) . Our data
demonstrate that a PNPase-like activity in crude extracts can degrade
the t40B substrate in a phosphate-dependent manner while the 77-nt
product, produced by the action of the purified Rnb polypeptide, is not
an efficient substrate. Conceivably, extension of the 3`-end by poly(A)
polymerase (PcnB)(32, 33, 34, 35) could provide a necessary single-stranded platform for
PNPase to overcome the apparent indirect inhibition by RNase II. Utility of t40B as a Substrate for RNase II
Activity
This partially duplexed RNA is an effective
substrate for investigating the properties of RNase II and offers at
least four significant advantages over assays previously utilized for
detecting RNase II activity. First, the t40B transcript resembles
natural mRNA substrates more closely than the homopolymeric substrates
utilized in traditional assays as it contains both 3`-unpaired
extensions of essentially random composition and a stable duplex
mimicking stem-loop structures found in natural mRNAs. Second, the
stalling of the enzyme at the duplexed region reflects the known
behavior of RNase II on RNAs containing regions of extensive secondary
structure(15, 16, 17, 18) . Third,
the formation of a stable degradative intermediate provides an internal
control that distinguishes RNase II activity from single and double
strand-specific endonucleases. Finally, the high specific activity of
the synthetic transcript increases the sensitivity of the assay and
allows for the detection of activity at low substrate concentrations
(10-10
) closer to the
physiological range.
)-thiogalactopyranoside.
We thank Drs. Murray Deutscher and Michael Cashel for
providing strains 18-11 and CF881, respectively, and Dr. Nahum
Sonenberg for the plasmid pRP40. We also thank Dr.
Cecília Arraiano and Rita
Zilhão for sending plasmids containing the 3`
region of the rnb gene prior to publication. G transition,
resulting in a single amino acid change, Ser
Gly
, was inadvertantly incorporated into plasmic pGC100.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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