From the Department of Biochemistry and Molecular Biology,
University of Miami School of Medicine, Miami, Florida 33101-6129
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INTRODUCTION |
Exoribonucleases play an important role in RNA maturation,
turnover, and degradation (for reviews, see Refs. 1 and 2). In
Escherichia coli eight distinct exoribonucleases have been characterized. Most of them display a degree of overlap in their function. For example, six of the eight, including RNases II, D, BN, T,
PH, and polynucleotide phosphorylase
(PNPase),1 participate in the
3'-maturation of tRNA precursors (3). Recently, the maturation of the
small stable RNAs, M1 RNA, 10Sa RNA/tmRNA, 6S RNA and 4.5S RNA, was
examined and found to involve many of the same exoribonucleases (4). It
is also known that strains lacking RNases II, D, BN, T, and PH in
combination are inviable, but the presence of any one of the five
enzymes is sufficient to confer viability, although with varying
degrees of effectiveness (5).
RNase R is one of the eight exoribonucleases. It acts nonspecifically
on poly(A), poly(U), and ribosomal RNAs (rRNA) in vitro (1,
6-8). The enzyme was initially identified 20 years ago in an E. coli strain deficient in RNase II (6). Whereas RNase II accounts
for more than 95% of the activity against poly(A) and poly(U) in crude
cell extracts, the residual activity against these substrates and rRNA
is due primarily to RNase R (1, 5, 7). Based on its gel filtration
properties, RNase R is apparently a protein of ~85 kDa (8). However,
despite all of this biochemical information, essentially nothing was
known about the gene encoding RNase R other than that it mapped to the
last quarter of the E. coli
chromosome.2
In this paper we report the identification and characterization of the
gene that encodes RNase R and show that it is the E. coli
vacB gene. vacB was originally described in
Shigella flexneri as a chromosomal gene required for
expression of the virulence genes carried on the large plasmid of this
organism (9). We were led to consider vacB as a candidate
for the gene encoding RNase R because (a) sequence analysis
revealed that it is homologous to the rnb gene that encodes
another exoribonuclease with similar properties, RNase II (10);
(b) the deduced size of the VacB protein (~92 kDa) agreed
closely with that known for RNase R; and (c) vacB
is located at 95 min on the E. coli chromosome (9), a
position consistent with the earlier mapping studies of the gene
encoding RNase R. Based on these considerations, we cloned and
characterized the E. coli vacB gene. The data obtained from these studies demonstrate that the vacB gene does indeed
encode RNase R, and we propose that it be renamed rnr. The
identification of VacB as an exoribonuclease has important implications
for the understanding of virulence associated with enterobacteria.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Plasmids--
All strains used were
E. coli K-12 derivatives. Strain C600 was used to amplify
phage DD947. Strains UT481 (
(lac-pro)hsdS (r
m
)lacIqlacZ)
(3) and JM109 (Promega) were used for transformation and plasmid
preparations, respectively. Strain CF881 (recB xthA rna) was
used for linear transformation, and wild type strain CA265 and two
RNase II
strains, CA265II
and CAN20-12E
(RNase I
II
D
BN
)
(8), were employed for RNase R assays. Bacteriophage P1vir, used for transduction, was from our laboratory stock.
The E. coli genomic clone,
DD947, which contains a 20-kb
fragment carrying vacB, was a gift from Dr. Frederick R. Blattner (University of Wisconsin-Madison) and was used to prepare DNA for subcloning. Plasmid pBS(+) (Stratagene) was used as the cloning vector. Plasmids pUC4K (Pharmacia) and pBR325 provided the
Kanr and Camr cassettes used to interrupt
vacB.
Plasmid pBSV was constructed by subcloning a 4.58-kb
EcoRI-NheI fragment from
DD947 into the
EcoRI-XbaI sites of pBS(+). To interrupt the
vacB gene on pBSV, a 1.23-kb PstI fragment was deleted from vacB, and the remaining fragment was
self-ligated to generate plasmid pBSVD. The Kanr cassette,
a 1.25-kb PstI fragment from pUC4K, was inserted into the
PstI site of pBSVD to create plasmid pBSVK. Likewise,
plasmid pBSVC was constructed by inserting the 1.23-kb Camr
cassette from pBR325 into the PstI site of pBSVD (see Fig.
1). Plasmid pBSR was constructed by transferring the 2.72-kb
XmnI fragment of pBSV into the HincII site of
pBS(+) such that vacB was placed under the control of the
lac promoter. Because of a stop codon upstream of
vacB, its gene product is not translationally fused to
lacZ. In this plasmid the upstream gene, yjeB,
and most of the downstream gene, yjfH, are deleted (see Fig.
1).
Growth Conditions--
Cells were grown either in YT medium
containing 0, 0.2, or 0.5% glucose in Tryptone broth supplemented with
0.2% lactose or in M9 medium supplemented with 1 mM
thiamine HCl and 0.2% glucose (11). For solid media, 1.5% agar was
added. Antibiotics were present at the following concentrations:
ampicillin, 50 µg/ml; kanamycin, 25 µg/ml; chloramphenicol, 34 µg/ml.
Materials--
Restriction endonucleases, T4 DNA ligase, and
Klenow fragment DNA polymerase I were obtained from New England
Biolabs. Calf intestine alkaline phosphatase,
isopropylthio-
-D-galactoside, and Prime-a-Gene Labeling
System were from Promega. Bacterial alkaline phosphatase was from
Worthington. T4 polynucleotide kinase was a product of Life
Technologies, Inc. [
-32P]ATP (3,000 Ci/mmol) and
[
-32P]ATP (6,000 Ci/mmol) were from NEN Life Science
Products. [3H]Poly(A) was obtained from Amersham
Pharmacia Biotech. Poly(A) was from Sigma. [3H]rRNA was
kindly provided by Zhihua Zhou and was prepared from [3H]uridine-labeled E. coli cells. Ultrogel
AcA44 was from LKB. All other chemicals were reagent grade.
Interruption of vacB on the Chromosome--
Plasmids pBSVK and
pBSVC, which contain deletion-insertion mutations in vacB,
were cleaved with ScaI and FspI, respectively, and introduced into strain CF881 by linear transformation.
Kanr Amps or Camr Amps
transformants were selected, and they served as a source for preparation of bacteriophage P1 lysates. The interrupted
vacB genes were transferred to wild type, CA265, and the
RNase II
strain, CAN20-12E, by P1-mediated transduction.
Extracts of the resulting Kanr or Camr strains
were prepared and assayed against [3H]poly(A) to verify
the decrease in RNase R activity. To ensure that the wild type
vacB gene was replaced by one containing the Kanr or Camr cassettes, chromosomal DNA of
these strains was prepared and analyzed by Southern hybridization.
Preparation of Cell Extracts--
Cells were grown to the
indicated A550 and collected by centrifugation.
Extracts were prepared by sonication of cells suspended in 0.1 volume
of a buffer containing 20 mM Tris-chloride, pH 7.5, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, 300 mM KC1. The protein concentration of the
extract was determined by the Bradford method.
RNase R Assay--
RNase R activity was measured under optimal
conditions by determination of acid-soluble radioactivity released from
the substrates, [3H]rRNA or [3H]poly(A), as
described (8). Assays were carried out in 50-µl reaction mixtures
containing: 20 mM Tris-chloride, pH 8.0, 0.25 mM MgCl2, 300 mM KC1, 5 µg of
[3H]rRNA, or 40 µg of [3H]poly(A). Twenty
microliters of extract were added to each reaction, and incubations
were carried out at 37 °C for 5-30 min.
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RESULTS AND DISCUSSION |
Cloning of the vacB Gene--
To ascertain whether the
vacB gene encodes RNase R, it was subcloned from the
E. coli
genomic clone, DD947, and placed into pBS(+)
(Fig. 1). The resulting plasmid, pBSV,
harbors a 4.58-kb insert containing three genes, yjeB (426 bp), vacB (2,442 bp), and yjfH (732 bp). pBSV was
transformed into an RNase II
strain,
CA265II
, and assays were carried out to determine whether
RNase R activity was elevated. Transformation with the pBS(+) vector
served as a control. Inasmuch as RNase II accounts for >95% of the
nonspecific exoribonucleolytic activity in an E. coli
extract (1, 5, 7), it was necessary to use the RNase II
background to accurately detect changes in RNase R activity. Transformed cells were grown to an A550
1, and sonicated extracts were prepared and assayed using
[3H]poly(A) and [3H]rRNA, substrates of
RNase R (7, 8). Under these conditions, activity against poly(A) was
elevated 2-3-fold and against rRNA, 5-7-fold when extracts from
pBSV-transformed cells were assayed. No elevation of activity was
observed for extracts from pBS(+)-transformed cells (data not shown).
These initial findings supported the conclusion that one of the three
genes present in the insert in pBSV encodes RNase R.

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Fig. 1.
Restriction map of the E. coli
genomic clone DD947 (94.7-95.2 min) and subclones derived
from it. The locations of the restriction sites used in this work
are presented. The three chromosomal genes carried on the fragment
subcloned from DD947 are represented by shaded or
hatched boxes, as indicated. The Kanr
or Camr fragment is shown as a filled
box. The names of the various subclones and the sizes of
their insert DNA are shown on the right. Vector DNA is not
shown. Construction of the various clones is described under
"Experimental Procedures."
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Because the elevation of RNase R activity was lower than expected for
expression from a multicopy plasmid, we examined whether the plasmid
may have been lost during growth. In fact, based on plating on
YT/ampicillin and on analysis of DNA minipreps, only 10-15% of the
cells retained plasmid pBSV; in contrast, all the cells retained
pBS(+). These data suggested that overexpression of at least one of the
genes on the pBSV insert is deleterious to cells. Accordingly, we
repeated the RNase R assays in cells grown only to an
A550
0.3 in an attempt to minimize plasmid loss (Table I). In several experiments,
one of which is presented, activity using poly(A) as substrate was
elevated 3-5-fold, and with rRNA as substrate, 7-12-fold.
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Table I
RNase R activity of strains carrying various cloned vacB genes
Cells were grown to an A550 0.3. Extracts were
prepared and assayed as described under "Experimental Procedures."
All experiments were carried out on the strain CA265II
background.
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To show that vacB was the gene responsible for the elevated
RNase R activity, two additional plasmids, pBSVD, containing a deletion
in the vacB gene, and pBSVK, containing both a deletion and
a Kanr insertion in vacB (Fig. 1), were also
examined for their ability to elevate RNase R activity (Table I). No
increase in activity against poly(A) was observed in the presence of
these plasmids; moreover, these plasmids were stably maintained in
cells. In another experiment to demonstrate that vacB was
responsible for the elevation of RNase R activity, an additional
plasmid, pBSR, in which the two adjacent genes had been removed (Fig.
1), was also examined. In this plasmid vacB is under the
control of the lac promoter. In the presence of 0.2%
lactose and 1 mM
isopropylthio-
-D-galactoside, up to a 100-fold increase
in activity against poly(A) was observed in the CAN20-12E background
(data not shown), indicating that the adjacent genes are not needed for
elevation of RNase R activity.
To confirm that the elevated RNase activity actually corresponds to
RNase R, samples were analyzed by gel filtration on Ultrogel AcA44.
Extracts prepared from CA265II
cells transformed with
either pBSV or pBS(+) were fractionated, and RNase activity was
determined. Assays using [3H]poly(A) as substrate
revealed a peak of activity eluting with a molecular weight of
~86,000 from the pBSV extract; however, this peak of activity was
non-detectable from extracts of cells transformed with pBS(+) (data not
shown). Taken together, these data strongly support the conclusion that
vacB encodes RNase R. Inasmuch as the predicted size of the
VacB protein is ~92,000, these data also indicate that RNase R is a
monomer. Based upon the information presented here, we propose that
vacB be renamed rnr.
Interruption of Chromosomal rnr--
The deletion-interruption
mutations of rnr present in plasmids pBSVK and pBSVC were
introduced into the chromosome of strain CF881 by linear
transformation; Kanr Amps and Camr
Amps transformants, respectively, were selected for further
study. The mutated rnr genes were then transferred to
strains CA265 and CAN20-12E by P1-mediated transduction. RNase
activity assays (Table II) revealed that
the residual activity in strain CAN20-12E, amounting to
2% of wild
type, was decreased even further and rendered undetectable upon
introduction of the interrupted rnr genes.
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Table II
Poly(A) degrading activity in wild type and mutant strains
Cells were grown to an A550 1. Extracts were
prepared and assayed against [3H]poly(A) as described under
"Experimental Procedures."
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To confirm that the chromosomal rnr gene had, in fact, been
substituted with the deletion-interruption mutation, chromosomal DNA from strain CAN20-12Ernr::kan was
subjected to Southern analysis using probes specific for the
rnr gene and the Kanr cassette. DNA from the
parental strain, CAN20-12E, and from CA265II
were used
as controls (Fig. 2). When hybridized
with the probe specific for rnr, EcoRI-digested
chromosomal DNA from all of the strains, including strains
CA265II
and CAN20-12E that carry wild type
rnr and strain
CAN20-12Ernr::kan that contains the
Kanr interruption, gave rise to a 9.6-kb band (Fig.
2A, lanes 2-5). This is as expected because the
interrupted rnr gene is a deletion-insertion mutant and the
fragment deleted is almost identical in size to the Kanr
fragment inserted (see "Experimental Procedures"). When hybridized with the probe specific for the Kanr cassette, no band was
detected with strains CA265II
and CAN20-12E (Fig.
2B, lanes 2 and 3), whereas the same
9.6-kb band was detected with strain
CAN20-12Ernr::kan (Fig. 2B,
lanes 4 and 5). These data demonstrate that the
rnr gene in the latter strain has been interrupted.
Identical results were obtained with the strain containing the
Camr interruption (data not shown).

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Fig. 2.
Southern blot analysis of chromosomal
DNA. Samples were digested with EcoRI and resolved on a
0.7% agarose gel. DNA digested with HindIII and labeled
with 32P at the 5' ends was used as the size marker
(lane 1). The sizes of the marker bands are indicated (in
kilobases). Lanes 2 and 3 were loaded with
chromosomal DNA from strains CA265II and CAN20-12E,
respectively, both of which carry wild type rnr. Lanes
4 and 5 were loaded with DNA from
CAN20-12Ernr::kan. A,
probed with the 1.01-kb StuI-PstI fragment of
rnr; B, probed with the 1.25-kb Kanr
cassette, both prepared by random primer extension using the
Prime-a-Gene Labeling System.
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The isolation of an E. coli strain with a null
mutation in rnr indicates that RNase R is not an
essential enzyme for cells cultured in the laboratory even when several
other exoribonucleases are also absent. To determine whether the
absence of RNase R has any effect on cell growth, strain
CAN20-12Ernr::kan was grown on
rich medium (YT) and on minimal medium (M9/0.2% glucose) plates at 31, 37, and 42 °C. No growth defect was detected compared with the
parental strain, CAN20-12E, under any of these conditions. Moreover,
the doubling time of the mutant strain at 37 °C in YT, 0.2% glucose
was 30 min, the same as that of the parent. Strain CAN20-12Ernr::kan also recovered from
a 24-h carbon source starvation in M9 salts with the same kinetics as
that of the parent, indicating no defect in recovery from starvation.
These data suggest that whatever function is served by RNase R, it can
be rescued completely by the exoribonucleases that are still present in
strain CAN20-12Ernr::kan. Although
this strain lacks RNases II, D, BN, and R, RNases T and PH, PNPase, and
oligoribonuclease still remain.
To test whether any of the remaining, known exoribonucleases can
compensate for the absence of RNase R, the interrupted rnr gene was introduced into mutant strains already lacking either RNase T,
RNase PH, or PNPase using phage P1-mediated transduction. As shown in
Table III, viable transductants can be
isolated when the rnr mutation is combined with mutations
leading to the absence of either RNase T or RNase PH. However, double
mutants lacking RNase R and PNPase do not grow. Inviability of the
rnr,pnp double mutant was also seen when the
pnp mutation was introduced into a RNase R
strain (Table III). Thus, these data indicate that of the known exoribonucleases (except oligoribonuclease), only PNPase overlaps with
RNase R to the extent that at least one of them needs to be retained to
maintain cell viability.
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Table III
Combination of the rnr mutant gene with mutations of other RNase
genes
Phage P1-mediated transduction was carried out as described (12)
using the indicated donor lysates and recipient strains. For
experiment 1, transductants were selected on kanamycin plates; for
experiments 2 and 3, selection was on plates containing kanamycin and
chloramphenicol.
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Sequence Analysis of rnr and RNase R--
Based on sequence
analysis of the E. coli rnr (vacB) gene, its
coding region encompasses 2,442 nt starting with an AUG initiator codon
and ending at a UGA termination codon. This open reading frame would
encode a protein of 813 amino acids with a calculated molecular mass of
92,109 Da, in close agreement with the size of RNase R determined by
gel filtration. The translation start site proposed here is that
predicted in the SWISS-PROT data base (accession number P21499) and
differs from that indicated in the GenBank data base (accession number
G1790622), which begins at a GUG initiation codon 42 nt upstream. We
favor the former assignment because a good Shine-Dalgarno sequence,
GGAGG, is located 7 nt upstream of the proposed AUG codon, whereas no
Shine-Dalgarno sequence is evident upstream of the GUG codon. Also,
plasmid pBSR, which leads to elevated RNase R activity (see above),
lacks the GUG and other upstream codons.
It is likely that rnr is cotranscribed with the adjacent
genes, yjeB and yjfH. First, no promoter sequence
is evident in the short intergenic sequence between yjeB and
rnr or between rnr and yjfH, and
second, no transcription terminator is seen downstream of
rnr. On the other hand, a possible
70
promoter, TAGCGA (18 nt) TATCAT, is present upstream of
yjeB, and a likely rho-independent terminator, a 7-bp stem
followed by 9 U residues, is located 20 nt downstream of the
termination codon of yjfH. If these predictions are
confirmed, it would indicate that rnr is part of an operon
together with the two adjacent genes. Although the identity and
functions of these two genes have not yet been established, we have
found that yjeB is distantly related to a number of
transcriptional repressors and contains a helix-turn-helix motif.
yjfH is homologous to a family of RNA methyltransferase genes, including the E. coli spoU (trmH) gene
encoding a tRNA 2'-O-methyltransferase (13). Computer
analysis also revealed the presence of a REP sequence (14) in the
intergenic region between rnr and yjfH.
Based upon its deduced amino acid sequence, RNase R is a basic protein
with a pI = 8.78. Whereas basic amino acid residues are
distributed throughout the protein, there is a particularly high
positive charge density in the C-terminal region. In fact, 40% of the
73 C-terminal residues of RNase R are basic (5 Arg, 24 Lys). Also
identified in the C-terminal region is one copy of the S1 RNA binding
domain (10). Interestingly, this domain is also present in the
C-terminal region of two other E. coli exoribonucleases,
RNase II and PNPase (10), both of which have substrate specificities
similar to RNase R (1). In addition, as noted earlier, we have now
found that RNase R
, PNPase
double mutant
strains are inviable, and earlier work had shown that RNase
II
, PNPase
strains also do not survive
(15). Moreover, E. coli RNase II and RNase R display a high
degree of sequence similarity, approaching 60% if conservative amino
acid replacements are considered. These observations strongly suggest
that RNase II, RNase R, and PNPase may constitute a subfamily within
the group of eight E. coli exoribonucleases.
Homologues of E. coli rnr are found in essentially all the
sequenced genomes, extending from Mycoplasma to humans (Ref.
16 and this work). Sequence similarities extending over wide regions of
these derived proteins range upward from 30% identity and 40% when
conservative amino acid replacements are included. These data suggest
that the function carried out by RNase R may have been maintained over
a wide range of organisms. On the other hand, we have not found
homologues of rnr in the sequenced Archaeal genomes.
E. coli rnr is clearly orthologous to
the vacB gene of S. flexneri (9). However, upon
comparing the VacB and RNase R protein sequences, we were surprised to
find two interruptions in the near perfect alignment. The first is a
52-amino acid stretch between residues 177 and 228. We found that by
inverting a 150-bp EcoRV fragment (bases 1199 to 1349 of
GenBank D11024) and introducing two single frameshift corrections near
the EcoRV sites, amino acid sequence identity could be
restored. Although the inverted vacB segment could be a
natural variant because it causes a major disruption within a region
conserved among multiple species, we suspect that vacB
sequencing errors are the cause of this difference. The second
discrepancy, which we also attribute to a likely Shigella vacB sequencing error, is caused by a C-terminal frameshift (a missing G after position 2858 of GenBank D11024). We resequenced this
region in E. coli and found perfect agreement with the
published E. coli sequence. The reconstructed
Shigella VacB and the E. coli RNase R sequences
are now 99% identical with only 7 amino acid differences and 29 nucleotide differences. After the C-terminal frameshift reversal, the
Shigella vacB sequence extends to the end of GenBank D11024.
The last 43 amino acids of the reconstructed Shigella VacB
remain unsequenced. The reconstructed partial Shigella VacB
protein sequence will appear in the SWISS-PROT data base as a revised
entry P30851.
Earlier work showed that the vacB gene product is required
for the expression of the virulence phenotype of Shigella
and enteroinvasive E. coli (9). A mutation in
vacB was found to reduce the expression of several
plasmid-encoded virulence antigens, and it was suggested that this
deficiency was because of an effect at the posttranscriptional level
(9). However, the molecular processes affected by the vacB
product were not understood. The new information presented here that
vacB encodes the 3'-5' exoribonuclease, RNase R, narrows the possibilities for VacB action in the expression of virulence and
should aid in clarifying its role in this process. However, it appears
that the function of RNase R extends beyond just affecting virulence.
The fact that mutant E. coli K-12 strains lacking PNPase and
RNase R are inviable suggests that these enzymes carry out an essential
function in RNA metabolism that cannot be taken over by any of the
other cellular exoribonucleases, even the closely related RNase II. It
will be of considerable interest to identify this role as well.