RNA: STRUCTURE METABOLISM AND

CATALYSIS:
Purification and Characterization of the
Escherichia coli Exoribonuclease RNase R:
COMPARISON WITH RNase II

Zhuan-Fen Cheng and Murray P. Deutscher

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J. Biol. Chem. 2002, 277:21624-21629.
doi: 10.1074/jbc.M202942200 originally published online April 10, 2002

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THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of the Escherichia coli
Exoribonuclease RNase R
COMPARISON WITH RNase II*
Published, JBC Papers in Press, April 10, 2002, DOI 10.1074/jbc.M202942200
Zhuan-Fen Cheng and Murray P. Deutscher‡
From the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine,
Miami, Florida 33101
Escherichia coli RNase R, a 3ⴕ 3 5ⴕ exoribonuclease
homologous to RNase II, was overexpressed and puri-
fied to near homogeneity in its native untagged form by
a rapid procedure. The purified enzyme was free of nu-
cleic acid. It migrated upon gel filtration chromatogra-
phy as a monomer with an apparent molecular mass of
⬃95 kDa, in close agreement with its expected size based
on the sequence of the rnr gene. RNase R was most
active at pH 7.5–9.5 in the presence of 0.1– 0.5 mM Mg2ⴙ
and 50 –500 mM KCl. The enzyme shares many catalytic
properties with RNase II. Both enzymes are nonspecific
processive ribonucleases that release 5ⴕ-nucleotide
monophosphates and leave a short undigested oligonu-
cleotide core. However, whereas RNase R shortens RNA
processively to di- and trinucleotides, RNase II becomes
more distributive when the length of the substrate
reaches ⬃10 nucleotides, and it leaves an undigested
core of 3–5 nucleotides. Both enzymes work on sub-
strates with a 3ⴕ-phosphate group. RNase R and RNase II
are most active on synthetic homopolymers such as
poly(A), but their substrate specificities differ. RNase II
is more active on poly(A), whereas RNase R is much
more active on rRNAs. Neither RNase R nor RNase II
can degrade a complete RNA-RNA or DNA-RNA hybrid
or one with a 4-nucleotide 3ⴕ-RNA overhang. RNase R
differs from RNase II in that it cannot digest DNA oli-
gomers and is not inhibited by such molecules, suggest-
ing that it does not bind DNA. Although the in vivo
function of RNase R is not known, its ability to digest
certain natural RNAs may explain why it is maintained
in E. coli together with RNase II.
Escherichia coli contains eight distinct exoribonucleases that
play important roles in every aspect of RNA metabolism (1, 2).
One of these enzymes, now termed RNase R, was originally
identified and partially purified and characterized as a nonspe-
cific residual exoribonuclease present in strains lacking RNase
II (3, 4). It was subsequently rediscovered in our laboratory as
a nuclease active against rRNA and given the name RNase R
(5, 6). The enzyme accounts for ⬃2% of poly(A)-degrading ac-
tivity remaining in crude extracts of cells lacking RNase I and
RNase II (7). Partially purified RNase R is a 3⬘ 35⬘ exoribo-
* This work was supported by Grant GM16317 from the National
Institutes of Health. The costs of publication of this article were de-
frayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence and reprint requests should be addressed:
Dept. of Biochemistry and Molecular Biology, University of Miami
School of Medicine, P. O. Box 016129, Miami, FL 33101. Tel.: 305-243-
3150; Fax: 305-243-3955; E-mail: mdeutsch@med.miami.edu.
21624
Vol. 277, No. 24, Issue of June 14, pp. 21624 –21629, 2002
Printed in U.S.A.
Received for publication, March 27, 2002
nuclease that releases 5⬘-nucleoside monophosphates, and cat-
alytically, it resembles RNase II (3, 4).
Downloaded from http://www.jbc.org/ at Technion-Israel Institute of Technology on March 4, 2014
RNase R is encoded by the rnr gene located at 95 min on the
E. coli chromosome (7). This gene was originally termed vacB;
and in Shigella and in enteroinvasive E. coli, it is necessary for
expression of virulence (8). In laboratory strains of E. coli,
RNase R is dispensable for cell viability (7). Moreover, multiple
mutant strains lacking RNases R and T, RNases R and PH, and
RNases R, II, D, and BN grow essentially normally on rich
media (7). On the other hand, a double mutant strain devoid of
RNase R and polynucleotide phosphorylase is inviable (7). This
observation suggests that RNase R and polynucleotide phos-
phorylase serve some overlapping essential function(s) in E.
coli that cannot be satisfied by any of the other cellular exori-
bonucleases. Preliminary data indicate that at least one of
these functions is an RNA quality control process that elimi-
nates defective rRNA.1
RNase R, together with RNase II, is a member of the RNR
superfamily of exoribonucleases (1). As might be expected from
their similarity in catalytic properties, RNase R and RNase II
also share structural properties, including ⬃60% sequence ho-
mology. Interestingly, RNase R is even more widespread
among eubacteria than is RNase II (1). In fact, RNase R is the
only one of the eight known bacterial exoribonucleases to be
present in Mycoplasma (1).
As part of our continuing efforts to determine the physiolog-
ical role of RNase R, we have developed a simple procedure to
purify the enzyme to near homogeneity. In this study, we
describe the purification of RNase R and provide a detailed
characterization of its structure, catalytic properties, and sub-
strate specificity. In view of its high degree of similarity to
RNase II, for many experiments, we have directly compared
the properties of RNase R with those of a homogeneous prep-
aration of RNase II (9).
EXPERIMENTAL PROCEDURES
Materials—Restriction endonucleases, T4 DNA ligase, T4 polynucle-
otide kinase, and the Klenow fragment of DNA polymerase I were
obtained from New England Biolabs Inc. Calf intestine alkaline phos-
phatase was from Promega. The QIAEX II gel extraction kit was pur-
chased from QIAGEN Inc. [32P]Orthophosphate and [␥-32P]ATP (6,000
Ci/mmol) were obtained from PerkinElmer Life Sciences. Polyvinyli-
dene difluoride membranes were a product of Pall Corp. [3H]Poly(A)
and Ultrogel AcA44 were obtained from Amersham Biosciences.
Poly(A), DNase I, and bacterial alkaline phosphatase (from E. coli) and
its substrate p-nitrophenyl phosphate were from Sigma. Affi-Gel blue-
agarose (100 –200 mesh) and protein size markers were obtained from
Bio-Rad. SequaGel, used to make urea-polyacrylamide gels, was pur-
chased from National Diagnostics, Inc. All other chemicals were re-
agent-grade. RNA oligomers were synthesized by Dharmacon Research
1
Z.-F. Cheng and M. P. Deutscher, unpublished data.
This paper is available on line at http://www.jbc.org
 Escherichia coli RNase R
FIG. 1. Cloning of the E. coli gene rnr for overexpression. A
shows the sequence immediately upstream of rnr. The residues shown
in boldface are the two possible initiation codons for rnr; underlined is
the stop codon of yjeB; and the italicized sequence is a possible Shine-
Dalgarno sequence assuming that the downstream ATG is the actual
initiation codon. B shows a linear representation of the rnr gene of E.
coli and its flanking genes. C shows plasmid pETR used to overexpress
RNase R. Plasmid pETR was constructed as described under “Experi-
mental Procedures.” It contains the rnr gene and a portion of the
upstream gene yjeB cloned immediately after the lac operator and the
Shine-Dalgarno sequence of the vector.
Inc., and DNA oligomers were synthesized by the DNA Core Facility of
our department. The RNA oligomers used for substrate specificity stud-
ies were 5⬘-CCCCACCACCAUCACUU-3⬘ (17-mer), its 17-mer comple-
ment, and the homo-oligomers A6, A8, A10, and A17. The DNA oligomers
used were 5⬘-GATGGTGGTGGGG-3⬘ (13-mer) and 5⬘-AAGTGATGGT-
GGTGGGG-3⬘ (17-mer).
Bacterial Strains and Plasmids—The E. coli K12 strain CMA201
(⌬rnb201::tet) was a kind gift from Dr. C. Arriano (Centro de Tecnologia
Quı́mica e Biológica, University of Lisbon, Lisbon, Portugal) (10). It was
used to prepare the P1vir lysate for conversion of strain BL21(DE3)/
pLys (Novagen) to an RNase II⫺ strain by P1-mediated transduction.
The resulting strain, BL21II⫺(DE3)/pLys, was the host for plasmid
pETR-dependent overexpression of RNase R. The RNase II⫺ strain was
employed to avoid problems in the assay of RNase R during purification
due to the greater activity of RNase II on poly(A). Strain CA244I⫺ was
used to prepare ribosomes and rRNAs.
Plasmid pETR was generated by insertion of a NaeI-BclI fragment
containing the rnr gene and a portion of the upstream yjeB gene at the
NcoI (blunted)-BamHI site of plasmid pET15b (Novagen) (Fig. 1). Dur-
ing the cloning procedure, the His tag coding region of pET15b was
removed, and the recombinant RNase R protein was not tagged.
Overexpression of RNase R—Strain BL21II⫺(DE3)/pLys harboring
pETR was grown to A550 ⬇ 1 with vigorous shaking at 37 °C in 4 liters
of yeast-tryptone medium supplemented with 100 ␮g/ml ampicillin plus
34 ␮g/ml chloramphenicol to maintain the pLys plasmid. Isopropyl-␤-
D-thiogalactopyranoside was then added to a final concentration of 1
mM to induce RNase R expression, and 100 ␮g/ml additional ampicillin
was added to maintain pETR. One h after adding isopropyl-␤-D-thioga-
lactopyranoside, the culture was quickly cooled on ice, and cells were
harvested by centrifugation. Five-ml portions of culture were taken
before and after induction for analysis. The cell pellet was frozen at
⫺80 °C until used.
Preparation of Cell Extracts—To determine the amount of RNase R
expressed from plasmid pETR, cells from the 5-ml portions of culture
taken before and after isopropyl-␤-D-thiogalactopyranoside induction
were resuspended in 1 ml of buffer containing 10 mM Tris-Cl (pH 7.6),
1 mM dithiothreitol, 500 mM KCl, and 0.1 mM phenylmethylsulfonyl
fluoride. Extracts were prepared by sonication using two 15-s pulses.
For RNase R purification, ⬃12 g of wet cells overexpressing RNase R
were suspended in 60 ml of buffer A (10 mM Tris-Cl (pH 7.6), 1 mM
dithiothreitol, 10 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, and
15 units/ml DNase I). Cells were ruptured by two passes through an
Aminco French press at 12,000 p.s.i. The suspension was centrifuged at
30,000 ⫻ g for 2 h. The pellet obtained was resuspended in 30 ml of
buffer B500 (10 mM Tris-Cl (pH 7.6), 1 mM dithiothreitol, 500 mM KCl,
0.5 mM EDTA (potassium salt; pH 7.4), 10% glycerol, and 0.1 mM
phenylmethylsulfonyl fluoride). The sample was centrifuged at
30,000 ⫻ g for 45 min and then at 150,000 ⫻ g for 2 h to obtain an S150
21625
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FIG. 2. SDS-PAGE of RNase R purification fractions. Ten ␮g of
protein from each fraction was denatured and loaded on an 8% SDS-
polyacrylamide gel. After electrophoresis, the gel was stained with
Coomassie Brilliant Blue R-250. Lane 1, sonicated cell extract before
induction; lane 2, sonicated cell extract after induction; lane 3, no-salt
soluble fraction; lane 4, S150; lane 5, pooled Affi-Gel blue (AGB) peak
fractions. The migration positions of molecular mass standards are
shown on the left (in kDa). The position of RNase R is indicated by the
arrow.
supernatant fraction. All steps of the purification were carried out at
4 °C or below.
Affi-Gel Blue Chromatography—The S150 fraction (7.5 ml) was
loaded onto an Affi-Gel blue column (0.8 ⫻ 10 cm) prepared in buffer
B500, washed with 3 bed volumes of the same buffer, and then eluted
with 2 bed volumes of the same buffer containing 1 M KCl. Peak
fractions were combined and dialyzed against buffer B500 to lower the
KCl concentration. The dialyzed sample of purified RNase R was
quickly frozen in an ethanol/dry ice bath and stored at ⫺80 °C.
Gel Filtration—For molecular mass measurement, purified RNase R
was applied to an Ultrogel AcA44 gel filtration column (1.0 ⫻ 56.5 cm)
using either buffer B500 (containing 500 mM KCl) or buffer A (contain-
ing no KCl) as the running buffer. Blue dextran 2000 and bacterial
alkaline phosphatase (86 kDa) were added to the samples and co-
chromatographed as internal molecular mass markers.
Enzyme Assays—RNase R assays were carried out as described (7)
using [3H]poly(A) as substrate, unless indicated. The activity of RNase
R is expressed as nmol of nucleotide released in 1 min at the given
temperature, usually 37 °C. RNase II assays were carried out in 50-␮l
reactions containing 10 mM Tris-Cl (pH 8.2), 100 mM KCl, 10 mM MgCl2,
36 ␮g of [3H]poly(A), and 0.06 ␮g of RNase II. The activity of RNase II
is expressed as described for RNase R. Acid-soluble radioactivity was
measured as described (7). Bacterial alkaline phosphatase assay of the
gel filtration column fractions was carried out by mixing 10 ␮l of each
column fraction with 1 ml of 1 mM p-nitrophenyl phosphate dissolved in
1 M Tris-Cl (pH 8.0) in a cuvette and measuring the ⌬A410/min (11).
Protein Determination—Protein was determined by the method of
Bradford (12) or by absorbance at 280 nm.
SDS-PAGE—Proteins were resolved on 8% SDS-polyacrylamide gels
(13). Gels were stained with Coomassie Brilliant Blue R-250 to visualize
the protein bands.
N-terminal Sequencing—Twenty ␮g of purified RNase R was re-
solved on an 8% SDS-polyacrylamide gel and transferred to a polyvi-
nylidene difluoride membrane by electroblotting. The membrane was
stained with Ponceau S. Protein bands were subjected to N-terminal
sequencing by the Protein Analysis Facility of our department.
Preparation of Ribosomes and rRNAs—Ribosomes were isolated from
E. coli cells (usually in log phase) as described (14). To isolate rRNAs, 10
␮l of 20% SDS was added to 400 ␮l of the ribosome suspension. The
mixture was extracted once with an equal volume of buffered phenol
(buffered with 20 mM sodium acetate (pH 5.3)) and once with an equal
volume of buffered phenol/chloroform/isoamyl alcohol (25:24:1), and
then 2.5 volumes of 95% ethanol was added to the aqueous phase to
precipitate rRNA. The total rRNA pellet was dissolved in 200 ␮l of
diethyl pyrocarbonate-treated H2O and resolved on a 1% agarose gel
(15). 23 S, 16 S, and 5 S RNAs were then isolated using RNaid w/Spin
kit.
21626 Escherichia coli RNase R
TABLE I
Summary of purification of RNase R
Total Total
Step
protein activity
mg nmol/min
Induced extracta 220 346,060
S150 fraction 35 276,850
Affi-Gel blue peak fractions 7.5 147,900
a
Values based on assay of induced extract prepared by sonication.
FIG. 3. Digestion of oligonucleotide substrates by RNase R and
RNase II. Oligonucleotide substrates were labeled at their 5⬘-ends with
32
P. Reactions were carried out at 37 °C in 50-␮l reaction mixtures
containing 20 mM Tris-Cl (pH 8.2), 100 mM KCl, and 10 ␮M labeled
oligomer. For RNase R reactions, MgCl2 was present at 0.25 mM, and
0.15 ␮g of purified RNase R was added; for RNase II reactions, MgCl2
was present at 10 mM, and 0.06 ␮g of purified RNase II was added.
Nine-␮l aliquots were taken at 0, 5, 10, 15, and 20 min as indicated, and
the reactions were stopped with 2 volumes of RNA loading buffer. The
upper panel (marked R) presents the results of RNase R digestion, and
the lower panel (marked II) presents the results of RNase II digestion.
Preparation of a Substrate with a 3⬘-Phosphate—An oligonucleotide
substrate containing a 3⬘-phosphate terminus (A16P) was generated by
periodate oxidation of A17 (16).
RESULTS
Overexpression and Purification of RNase R—Upon isopropyl-
␤-D-thiogalactopyranoside induction of strain BL21II⫺(DE3) /
pLys harboring plasmid pETR, RNase R was overexpressed to an
extent that it was the most abundant protein in the sonicated
extract (Fig. 2, compare lane 2 with lane 1). Quantitatively, with
poly(A) as substrate, an extract of cells collected after induction
had a specific activity of 1,550 nmol/min/mg of protein, which was
⬃100-fold higher than that of an extract from uninduced cells.
RNase R was rapidly purified from the induced cell extract
by taking advantage of its insolubility in low salt (4). Thus,
most of the proteins in the extract were soluble in buffer A (no
KCl), whereas RNase R was not detectable in this fraction (Fig.
2, lane 3). However, resuspension of the pellet in buffer B500
(containing 500 mM KCl) and recentrifugation revealed that
RNase R was the major protein in the S150 supernatant frac-
tion (Fig. 2, lane 4). These precipitation steps resulted in an
⬃5-fold purification of RNase R (Table I). A portion of the S150
fraction was further purified on an Affi-Gel blue column, lead-
ing to an additional 2–3-fold purification (Table I). SDS-PAGE
of the purified RNase R indicated that it was at least 95% pure
(Fig. 2, lane 5). Based on this simple procedure, 7.5 mg of highly
purified RNase R was obtained from the equivalent of 3 g of wet
cells (Table I).
Spectral Analysis—Spectral analysis of purified RNase R in
the range of 200 –700 nm revealed no unusual peaks (data not
shown). The A280/A260 ratio was 1.87, indicating that purified
Specific activity Relative purification Recovery
nmol/min/mg fold %
1,550 1 100
7,910 5 80
19,720 13 40
RNase R does not contain nucleic acid and therefore that
RNase R does not require nucleic acid for activity.
Molecular Mass of RNase R—When gel filtration of purified
RNase R was performed in buffer B500 (containing 500 mM
KCl), RNase R eluted as a globular protein of ⬃95 kDa, in close
agreement with its predicted size of 92 kDa based on the rnr
gene sequence. This observation supports our previous sugges-
tion that RNase R is a monomer (7). In contrast, when gel
Downloaded from http://www.jbc.org/ at Technion-Israel Institute of Technology on March 4, 2014
filtration was carried out in buffer A (lacking KCl), RNase R
eluted in the void volume, indicating that it aggregates at low
ionic strength, as originally observed by Kasai et al. (4) with
partially purified enzyme. The fact that activity could be de-
tected when the protein was aggregated suggests either that
aggregation does not abolish RNase R activity or that the
protein dissociates in the assay mixture.
N-terminal Sequencing of RNase R—To confirm that RNase
R is encoded by the rnr gene and also to conclusively determine
which of the two potential translation start sites is used, puri-
fied RNase R was subjected to N-terminal sequencing. The
enzyme was resolved by 8% SDS-PAGE, followed by electro-
blotting onto a polyvinylidene difluoride membrane. Subse-
quent N-terminal analysis revealed the sequence SQDPFQE,
in agreement with that predicted in the Swiss Protein Data-
base, which is MSQDPFQE. These data indicate that 1) the rnr
gene encodes RNase R; 2) the open reading frame predicted in
the Swiss Protein Database is correct, but that the N-terminal
formyl-Met residue is removed; and 3) the downstream AUG
codon is the initiation site for RNase R synthesis.
Heat Stability of RNase R—To determine the response of
RNase R to heat, its activity was measured at various temper-
atures. RNase II activity was measured at the same tempera-
tures for comparison. Both RNase R and RNase II displayed
similar temperature activity profiles, being most active at
⬃50 °C in a 5-min assay. RNase R stability at 50 °C was fur-
ther examined by preincubating at that temperature for vary-
ing lengths of time and then assaying at 37 °C. The enzyme
was relatively stable, losing ⬃25% of its activity in 15 min and
⬃80% over a 30-min period (data not shown).
Optimal Reaction Conditions for RNase R Activity on
Poly(A)—Purified RNase R was assayed on poly(A) under a
variety of conditions to assess its requirements for optimal
activity; RNase II was assayed in parallel for comparison. Both
RNase R and RNase II displayed a broad optimal pH range
between 7.5 and 9.5. Both enzymes required a divalent cation,
preferably Mg2⫹, for activity. In the presence of 1 mM EDTA,
the activity of each enzyme was abolished. However, the opti-
mal Mg2⫹ concentrations for the enzymes were quite different.
RNase R was most active at 0.1– 0.5 mM Mg2⫹, whereas RNase
II was most active at 10 mM Mg2⫹. Both RNase R and RNase II
were stimulated by the presence of a monovalent cation. RNase
R was stimulated ⬃2-fold by 50 –500 mM K⫹ and 40% more by
Rb⫹. For RNase II, K⫹ was the most stimulatory of the mono-
valent cations tested, increasing activity ⬃5-fold over a simi-
larly wide range of concentrations (data not shown).
Mechanism of Action of RNase R—Earlier work with par-
tially purified enzyme indicated that RNase R is an exoribo-
nuclease releasing 5⬘-nucleoside monophosphates (4). To en-
 Escherichia coli RNase R 21627
TABLE II
Products of RNase R action
In Experiment 1, [3H]poly(A) was digested with RNase R (0.15 ␮g) in a 50-␮l reaction for 30 min as described under “Experimental Procedures.”
The reaction mixture was heated at 70 °C for 10 min to inactivate RNase R, followed by addition of 6 ␮l of 10⫻ alkaline phosphatase buffer and
4 ␮l of 1 unit/␮l calf intestine alkaline phosphatase. After incubation at 37°C for 30 min, the acid-soluble fraction was extracted four times with
ether to remove trichoro acetic acid. The pH of the sample was adjusted to 7.0, and a portion of the sample was loaded onto a 1.2-ml Dowex AG
1-X2 column. Nucleosides were eluted with 6.2 ml of water (in seven fractions), and the nucleotide material (including any charged ol gonucleotides)
was eluted with 6.2 ml of 0.01 M HCl (also seven fractions). In Experiment 2, E. coli tRNA (40 ␮g) was treated with either 0.15 or 3.0 ␮g of RNase
R for 10 min at 37°C in 50-␮l reaction mixtures containing 20 mM glycine/KOH (pH 8.8), 100 mM KCl, and 0.25 mM MgCl2. The reaction mixture
was heated at 70°C for 10 min, followed by addition of 50-␮l tRNA nucleotidyltransferase reaction mixtures containing 80 m glycine/KOH (pH 8.8),
10 mM MgCl2, 1 mM [3H]ATP, and excess tRNA nucleotidyltransferase. After an additional incubation at 37°C for 20 min, acid-precipitable
radioactivity was determined.
After treatment with phosphatase [3H]AMP Amount
incorporation due to
Nucleoside Nucleotide into tRNA RNase R

% pmol pmol
Exp. 1
Acid-soluble products 90 10
Exp. 2
⫺RNase R 653

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⫹RNase R (0.15 ␮g) 693 40
⫹RNase R (3 ␮g) 1232 578

FIG. 5. Digestion of the DNA oligomer dT17 by RNase R and

FIG. 4. Digestion of 16 S and 23 S rRNAs by RNase R and RNase
II. Fifty ␮g of total RNA (purified from isolated ribosomes) was treated
with 0.15 ␮g of RNase R or RNase II in 50-␮l reactions under the
conditions described under “Experimental Procedures,” except that KCl
was present at 100 mM. Nine-␮l aliquots were taken at the times
indicated, and reaction products were resolved on a 1.1% agarose gel,
followed by Northern hybridization using oligomers complementary to
the 5⬘-ends of 16 S and 23 S RNAs. The oligomer used to detect 16 S
RNA was 5⬘-CCTGTTACCGTTCGACTTGC-3⬘, and the oligomer used
to detect 23 S RNA was 5⬘-CCTTCATCGCCTCTGACTGCCA-3⬘. Quan-
titation was done on a PhosphorImager (Molecular Dynamics, Inc.).
sure that the product released was not due to a secondary
reaction by a contaminating activity, we re-examined the
mechanism of action of RNase R using the purified enzyme.
Thus, when 5⬘-32P-labeled RNA substrates (of varying length
from oligomer to polymer) were digested by purified RNase R,
intermediates with sizes between the starting material and
final product were not observed to accumulate (Fig. 3 and data
not shown). This observation is consistent with the conclusion
that RNase R is a processive exoribonuclease. To confirm this
point, the products of the reaction catalyzed by highly purified
RNase R were examined.
If RNase R were an exoribonuclease, the major acid-soluble
product with [3H]poly(A) as substrate would be expected to be
AMP. Upon treatment with alkaline phosphatase, this would
be converted to the uncharged molecule, [3H]adenosine, and
would elute with the nucleoside fraction from an anion-ex-
change column (Dowex AG 1-X2). If, on the other hand, RNase
R were an endoribonuclease, the acid-soluble oligonucleotides
produced would remain charged after phosphatase treatment
RNase II. dT17 was labeled at its 5⬘-end with 32P. Reactions were
carried out at 37 °C for 20 min under the conditions of the T4 polynu-
cleotide kinase reaction: 70 mM Tris-Cl (pH 7.6), 10 mM MgCl2, and 5
mM dithiothreitol with 5 ␮M dT17 and the indicated amounts of RNase
R (R) or RNase II (II).
and would elute with the “nucleotide” fraction. As shown in
Table II, 90% of the acid-soluble radioactivity was eluted from
the anion-exchange column with water, consistent with its
being the nucleoside, adenosine.
A second test of the mechanism of RNase R was its action on
tRNA. Because tRNA is a relatively poor substrate of RNase R,
we reasoned that RNase R might act on it in a distributive
fashion such that tRNA-CC might be generated due to removal
of 1 AMP residue from tRNA-CCA. This product would be a
substrate for the re-incorporation of AMP by tRNA nucleotidyl-
transferase. As shown in Table II, this prediction was fulfilled.
Thus, there was a dramatic increase in AMP incorporation into
tRNA by tRNA nucleotidyltransferase that was dependent on
the amount of RNase R added (40 pmol with 0.15 ␮g of RNase
R and 578 pmol with 3 ␮g of the enzyme). The incorporation of
[3H]AMP in the absence of RNase R was due to the presence of
some tRNA-CC in the tRNA preparation. Nevertheless, these
data show that RNase R can slowly remove a single AMP
residue from tRNA to generate tRNA-CC, supporting the con-
clusion that RNase R is an exoribonuclease.
Substrate Specificity of RNase R—Important clues to the
physiological role of RNase R may come from an analysis of its
substrate specificity. Earlier work suggested that RNase R is a
nonspecific enzyme able to act on homopolymers, rRNA, and
mRNA (4, 7). Inasmuch as the previous studies of RNase R
were carried out with only partially purified preparations, we
felt that it was important to examine the specificity of the
21628 Escherichia coli RNase R
FIG. 6. Digestion of a 17-nucleotide RNA oligomer by RNase R in the presence or absence of a complementary oligomer. An RNA
17-mer was labeled at its 5⬘-end with 32P. Reactions were carried out as described in the legend to Fig. 3. for the times indicated. Complementary
oligomers were present at 20 ␮M, and dT17 (the non-complementary oligomer) was present at 200 ␮M. Twenty-fold as much RNase R was present
when the substrate was the RNA-RNA duplex.
FIG. 7. Action of RNase R and RNase II on an oligomer sub-
strate with a 3ⴕ-phosphate group. A16P was labeled at its 5⬘-end
with 32P. Reactions were carried out as described in the legend to Fig.
3 for the times indicated. R, RNase R; II, RNase II.
purified enzyme. We have used a greatly expanded catalogue of
substrates and also compared its specificity with that of its
homolog, RNase II.
The relative activities of RNase R and RNase II on poly(A)
and the major classes of RNA are presented in Table III. RNase
II was ⬃4-fold more active on poly(A) compared with RNase R,
whereas RNase R was much more active on the natural RNAs.
Both enzymes worked best on poly(A). The Km value for poly(A)
in the RNase R reaction was ⬃30 nM. Among the natural RNAs,
RNase R worked well on 23 S and 16 S RNAs, but relatively
poorly on 5 S RNA and tRNA. Nevertheless, 5 S RNA and tRNA
were potent inhibitors of RNase R action on poly(A) (data not
shown). Fig. 4 shows that the acid-soluble material released
from the 16 S and 23 S rRNA preparations actually led to the
disappearance of the rRNA bands and thus could not be due to
degradation of a contaminating RNA. In separate experiments
using gel analysis rather than acid solubility, degradation of
homopolymers by RNase R was in the order poly(A) ⬎
poly(U) ⬎ poly(C) ⬎⬎ poly(G) (data not shown). The latter
polymer was essentially inactive as a substrate. The limit prod-
ucts of digestion of the poly(A), poly(U), and poly(C) homopoly-
mers were di- and trinucleotides in each instance.
The activities of RNase R and RNase II on A oligonucleotides
6 –17 nucleotides in length were also examined. Both RNase R
and RNase II could hydrolyze RNAs as short as a 6-mer (A6);
however, although RNase R acted processively for RNA oli-
gomers as short as 8-mers, RNase II tended to be more distrib-
utive, especially for the shorter substrates (Fig. 3). The limit
end products of the RNase R reaction were di- and trinucleo-
tides, whereas the major end products of the RNase II reaction
were oligonucleotides ranging from 3 to 5 residues in length
(Fig. 3). Neither RNase R nor RNase II displayed much activity
against DNA oligomers at the enzyme levels usually used.
However, when 30 – 40 times this amount of enzyme was
tested, RNase II could hydrolyze the DNA oligomer dT17, in a
distributive manner; RNase R worked much more poorly, re-
moving only 1–2 residues from a portion of the substrate mol-
TABLE III
Substrate specificity of RNase R and RNase II
Reactions were carried out under the conditions described under
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“Experimental Procedures,” except that KCl was present at 100 mM.
Incubation was at 37 °C for 5 min. The amounts of substrate used were
0.2 ␮M for [32P]rRNAs and [3H]tRNA and 15 ␮M for [3H]poly(A). RNase
(0.15 ␮g) or RNase II (0.06 ␮g) was present. [32P]rRNAs were prepared
from 32P-labeled cells as described under “Experimental Procedures,”
and [3H]tRNA was gel-purified from a [3]tRNA preparation (containing
5 S RNA provided by Dr. P. R. Subbarayan).
Substrate RNase R activity RNase II activity
nmol/min/mg nmol/min/mg
Poly(A) 26,900 109,800
23 S RNA 4,840 160
16 S RNA 2,750 30
5 S RNA 290 20
tRNA 350 ⬍20
ecules (Fig. 5). Moreover, the DNA oligomer dC17 was a potent
inhibitor of RNase II activity with poly(A) as substrate,
whereas it had little effect on RNase R (data not shown).
To examine whether RNase R can hydrolyze double-stranded
molecules, a 5⬘-32P-labeled RNA oligomer (17-mer) was sub-
jected to RNase R action in the presence of a complementary
DNA either 13 or 17 nucleotides in length or a fully comple-
mentary RNA (Fig. 6). In the presence of a 2-fold molar excess
of the complementary 13-mer (to generate a substrate with 13
base pairs and a 4-nucleotide extension at the 3⬘-end) or in the
presence of a complementary DNA or RNA 17-mer (to generate
a completely base-paired substrate), no shortening was evi-
dent. In fact, for the assay of the RNA-RNA duplex, 20 times
the usual amount of RNase R was used. On the other hand,
when even a 20-fold excess of a non-complementary DNA (oli-
gomer dT17) was present, RNase R degraded the RNA sub-
strate at a rate comparable to that when no complementary
oligomer was present (Fig. 6). These data show that the com-
plementary strands inhibit RNase R action and that RNase R
cannot act on double-stranded molecules even when a 4-nucle-
otide 3⬘-RNA overhang is present.
To ascertain whether RNase R can act on an RNA molecule
with a 3⬘-phosphoryl terminus, its ability to degrade A16P was
determined. As shown in Fig. 7, both RNase R and RNase II
could function on a substrate with a 3⬘-phosphate group. The
rate at which A16P was degraded by each enzyme was compa-
rable to that previously observed with A17 (see Fig. 3).
DISCUSSION
Using the rapid purification procedure described here, which
takes advantage of the differential solubility of RNase R in
high and low salt, we were able to purify mg quantities of
essentially homogeneous RNase R in its native untagged form
from 1 liter of culture in just 2 days. Based on activity against
rRNA, the preparation obtained was ⬃20-fold purer than that
 Escherichia coli RNase R
described by Kasai et al. (4). Moreover, the RNase R described
here displayed a native molecular mass of ⬃95 kDa, as ex-
pected based on the sequence of the rnr gene (7). This contrasts
with a mass of ⬃40 –50 kDa reported by Kasai et al. (4) based
on a comparison of the size of RNase R with that of RNase II.
In some preparations of RNase R, we have also found a smaller
active form of RNase R that consisted of the central region and
the C-terminal S1 RNA-binding domain of the protein (1).
Inasmuch as this form of the enzyme could be eliminated by
inclusion of EDTA in the buffers during purification and based
on the fact that the eliminated N-terminal fragment could be
detected by SDS-PAGE (data not shown), we conclude that the
smaller protein arises by proteolytic cleavage due to a metal-
dependent protease. It is possible that this may explain the
form of RNase R purified by Kasai et al. (4). These workers also
reported that RNase R was associated with ribosomes. How-
ever, this has not been observed with the overexpressed
protein.
Both RNase R and RNase II are members of the RNR super-
family of exoribonucleases (1). Structurally, both are large mo-
nomeric proteins. Likewise, RNase R and RNase II share many
catalytic properties. Thus, both enzymes are relatively nonspe-
cific processive 3⬘ 3 5⬘ exoribonucleases that release 5⬘-nucle-
oside monophosphates and leave a residual oligoribonucleotide
core. We have found that this core is somewhat smaller with
RNase R (di- and trinucleotides) than is observed with RNase
II (tri- to pentanucleotides) (this work and Refs. 17 and 18). The
inability of these enzymes as well as polynucleotide phospho-
rylase to act on small oligoribonucleotides is the apparent
reason for the existence of an additional RNase in most cells
(termed oligoribonuclease) that digests such residual molecules
(19). Another similarity in the properties of RNase R and
RNase II is that both enzymes can act on 3⬘-phosphate-termi-
nated RNA molecules, indicating that neither of them requires
a free 3⬘-hydroxyl group to initiate degradation.
On the other hand, there are a number of significant differ-
ences in the actions of RNase R and RNase II. For example, the
latter enzyme works relatively efficiently with DNA, and its
action on RNA is strongly inhibited by DNA oligomers. In
contrast, RNase R acts very poorly on DNA, and its activity is
scarcely affected by the presence of a DNA oligomer. Based on
this information, it is likely that RNase R binds relatively
weakly to DNA. A second major difference between the two
RNases is their substrate specificity. Although both enzymes
work most effectively on synthetic homopolymers such as
poly(A), RNase R also can degrade rRNAs quite well. However,
21629
RNase II is essentially inactive against such natural RNA
substrates. Considering that rRNA molecules contain exten-
sive secondary structure, these observations suggest that
RNase R may be much more effective than RNase II in digest-
ing through such structured regions. It is already known that
RNase II slows down as it approaches within 10 nucleotides of
a double-stranded RNA structure (20). The data presented here
show that RNase R cannot degrade a completely double-
stranded short RNA molecule or one with a 4-nucleotide 3⬘-
overhang. Nevertheless, the fact that RNase R can digest both
16 S and 23 S rRNA molecules indicates that it is able to work
through secondary structure in the context of the rRNA. How
this is accomplished remains to be determined.
The in vivo role of RNase R is not yet known, but the data
presented here indicate that the enzyme would be capable of
acting on a variety of natural RNA molecules. Perhaps, its
ability to act on structured RNAs is the reason why E. coli
Downloaded from http://www.jbc.org/ at Technion-Israel Institute of Technology on March 4, 2014
maintains this enzyme in addition to RNase II.
Acknowledgment—We thank Dr. Cecı́lia Arraiano for providing a
strain.
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