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(Received for publication, July 6, 1995; and in revised form, August 23, 1995) From the
The processing endoribonuclease RNase E (Rne), which is encoded
by the rne gene, is involved in the maturation process of
messenger RNAs and a ribosomal RNA. A number of deletions were
constructed in order to assess functional domains of the rne gene product. The expression of the deletion constructs using a T7
promoter/RNA polymerase overproduction system led to the synthesis of
truncated Rne polypeptides. The smallest gene fragment in this
collection that was able to complement a temperature sensitive rne
Out of over 20 ribonucleases identified so far in Escherichia coli (Deutscher, 1993) there are only three
enzymes known to possess processing endonucleolytic activity. They
catalyze very specific reactions and act only on a subpopulation of RNA
molecules. The enzymes are RNase III, RNase P, and RNase E (Deutscher,
1985, 1993; Apirion et al., 1992; Apirion and Miczak, 1993).
They cleave RNA in such a manner as to generate 3`-hydroxy termini and
require a divalent cation for catalysis. Each of these enzymes seems to
have limited substrate specificity and defined cleavage sites. Genetic
evidence suggests that the contribution of these enzymes (RNase III,
RNase P, and RNase E) is unique, since in each case it was found that
when the enzyme activity was abolished by mutation, the cleavage
ascribed to it could not be accomplished by any other enzyme in the E. coli cells. The rnc gene encoding RNase III is not
essential for bacterial survival (Takiff et al., 1989),
whereas the genes encoding RNase P (rnp) and RNase E (rne/ams/hmp1) are. RNase P cleaves all of the tRNA precursors
to generate the 5`-phosphoryl terminus of mature tRNA (Altman, 1989).
Both RNase E and RNase III are involved in processing of the precursor
molecules of ribosomal and messenger RNAs (Apirion et al.,
1992; Apirion and Miczak, 1993; Court, 1993). RNase III is specific for
double-stranded RNA. It introduces a double cleavage in each of two
stems that produce 16 S and 23 S rRNA (Ginsburg and Steitz, 1975;
Talkad et al., 1978; Gegenheimer and Apirion, 1980) and
contributes to the decay and stability of some mRNAs (Gitelman and
Apirion, 1980; Regnier and Grunberg-Manago, 1989; Bardwell et
al., 1989; Faubladier et al., 1990; Regnier and
Grunberg-Manago, 1990; Robert-Le Meur and Portier, 1992; Hajnsdorf et al., 1994). RNase K has been defined as the enzyme
implicated in the growth rate-dependent regulation of the expression of ompA mRNA by introducing a cleavage in the 5` leader of the ompA message (Lundberg et al., 1990). Recent data
suggest that RNase K is a proteolytic product of RNase E. ( RNase E was initially defined as a processing
ribonuclease that catalyzes the maturation of 5 S rRNA (Apirion, 1978;
Ghora and Apirion, 1978). This enzymatic activity also cleaves RNA I, a
small RNA that controls the replication of ColE1 plasmid DNA (Tomcsanyi
and Apirion, 1985) and is involved in the maturation and turnover of
many bacterial and bacteriophage T4 mRNAs (Mudd et al., 1988;
Lundberg et al., 1990; Nilsson and Uhlin, 1991; Regnier and
Hajnsdorf, 1991; Mackie, 1991, 1992; Klug et al., 1992; Gamper
and Haas, 1993; Deutscher, 1993). Inactivation of RNase E has a
stabilizing effect on the bulk of mRNA (Ono and Kuwano, 1979; Babitzke
and Kushner, 1991; Mudd et al., 1990). The entire rne gene, suggested to be the RNase E structural gene, was cloned and
sequenced (Casaregola et al., 1992), but the exact size of its
open reading frame remained unclear. Recently RNase E has been purified
and shown to be the rne gene product. It is now established
that the E. coli rne/ams/hmp locus encodes the RNA processing
endonuclease RNase E (Cormack et al., 1993; Carpousis et
al., 1994; Taraseviciene et al., 1994). There were some
discrepancies in the molecular size of the gene product in different
reports. According to the latest corrections ( Here we describe a deletion analysis of the rne gene and its product, endoribonuclease E (Rne). Our observations
suggest that (i) the endonuclease catalytic domain most probably is
located at the N terminus of the protein molecule, (ii) the Rne protein
has an RNA binding region that correlates with the endonucleolytic
cleavage, since the truncated Rne polypeptides lacking the RNA binding
region did not exhibit endonucleolytic activity (our deletion analysis
data and the computer-predicted Rne structure analysis suggest that the
RNA binding region is located between amino acids 580 and 700), and
(iii) the rne-3071 and ams1 mutations, located at the
N terminus and known to abolish the endonucleolytic activity, did not
eliminate the RNA binding activity. RNAs that were known not to be
substrates for the enzyme did not bind to the RNase E polypeptides.
Furthermore, the truncated Rne polypeptides lacking the RNA binding
region did not exhibit endonucleolytic activity.
Figure 1:
Physical map of the rne gene
locus of the E. coli chromosome and the restriction map of the rne gene and different deletion constructs. Locations of genes orfX-30K-orfY-rpmF(L32) are from Oh and Larson(1992).
Locations of flg genes are from Casaregola et
al.(1993). A-AvaII, B-BanI, BHI-BamHI,
C-ClaI, H-HindIII, M-MluI, N-NruI,
P-PstI, S-SphI, X-XmnI. The direction of
transcription of the genes and the translation start of the Rne protein
are indicated.
[P Transcripts were
uniformly labeled with [
The expression of the cloned
fragments in vivo led to the synthesis of truncated proteins (Table 2). The yields of the synthesized proteins amounted to as
much as 10-20% of the total cell proteins (Fig. 2a). The smaller polypeptides seemed to be
present in relatively higher amounts than the larger ones (data not
shown). The antibodies raised against the truncated protein expressed
from the plasmid pRE141, containing the N-terminal two-thirds of the
intact Rne sequence, cross-reacted with all the polypeptides expressed
from the deletion constructs (Fig. 2b). Even the
smallest (184 amino acids encoded from the rne cistron)
polypeptide expressed from pRE160 was recognized by the antibodies (Fig. 2, lane 6), suggesting that the N-terminal part
of the protein contains strong immunological epitopes. The expressed
polypeptides migrated with some discrepancy on the SDS-polyacrylamide
gel in comparison with the molecular weight calculated from the cloned
sequence. The largest inconsistency was exhibited by the protein
expressed from the plasmid pRE171 containing the intact rne gene sequence (Table 1).
Figure 2:
Expression of the rne gene and
different deletion constructs. In each case the cells contained two
plasmids pGP1-2 and pT7-5 (Tabor and Richardson, 1985) with
different DNA fragments from the rne gene (Table 2). A, Coomasie Brilliant Blue G-250 stained SDS-polyacrylamide
gel; B, Western blot using antibodies against 110-kDa
polypeptide. Lane 0, pT7-5 (vector plasmid, no insert); lane 1, pRE171; lane 2, pRE141; lane 3,
pRE154; lane 4, pRE155; lane 5, pRE156; lane
6, pRE160.
Figure 3:
RNase E activity of different truncated
Rne polypeptides (see Table 2). A, substrate, 9 S RNA; B, substrate, 7 S RNA. Reaction conditions are described under
``Materials and Methods.'' RNA fragments were separated on
5%/12% polyacrylamide gels containing 7 M urea. Lane
0, control RNA; lanes 1-7, substrate RNA treated
with enzyme preparations from cells carrying plasmids: 1,
pRE171; 2, pRE141; 3, pRE153; 4, pRE154; 5, pRE155; 6, pRE156; 7, pRE160. C,
enzymatic activity of refolded polypeptides expressed from 1,
pRE171; 2, pRE141; 3, pRE154; 4, pRE155; 5, pRE160; 0, untreated substrate, 9 S
RNA.
The overexpressed truncated
polypeptides were purified by eluting the proteins from the
SDS-polyacrylamide gel. The eluted polypeptides were at least 95% pure,
as only traces of other polypeptides were visible when the samples were
overloaded on the gel (data not shown). The proteins were subjected to
a denaturation-renaturation procedure as described under
``Materials and Methods.'' Enzymatic activity was exhibited
by the renatured polypeptides expressed from the plasmids pRE171 (the
entire Rne protein), pRE 141, and pRE154 (Fig. 3C). No
enzymatic activity was detected with the renatured proteins expressed
from the plasmids pRE155 and pRE160 (Fig. 3C). This is in good
agreement with the activity test of the protein extracts. Taken
together, our data from the enzymatic activity tests of deletion
mutants suggest that the endonucleolytic activity is encoded by the
N-terminal part of the RNase E protein molecule.
Figure 4:
A,
hydrophilicity plot of RNase E using the MacVector program. The
deletion sites are indicated by arrows. The lengths of the
truncated polypeptides (number of amino acids) are: 1, 842; 2, 794; 3, 652; 4, 635; 5, 410. B, secondary structure predictions of an N-terminal region of
wild-type and temperature-sensitive mutant Rne
proteins.
Figure 5:
9
S RNA binding to RNase E. Proteins were separated on SDS-polyacrylamide
gel, transferred onto nitrocellulose using electroblotting, and
hybridized with
The overexpressed proteins from the deletion
mutants (Table 1) were tested for their activity to bind
different RNAs: 9 S RNA, 7 S RNA, RNA E1 (untranslated region of Rne
mRNA, see Fig. 1), tRNA
Figure 6:
Protein blots hybridized with different
RNA probes. A and Ga, nitrocellulose membrane stained
with Amido Black. B-I, protein blots probed with
different
Figure 7:
Substrate
specificity of RNase E. Cell extracts were passed through a gel
filtration column and precipitated with 40% saturated ammonium sulfate
as described earlier (Taraseviciene et al., 1994). Reaction
conditions are described under ``Materials and Methods.'' A, E1 RNA; B, PAP5 RNA; C, 9 S RNA; D, PAN5 RNA; E, aPAN5 RNA treated with enzyme
preparations from cells carrying the plasmids: 1, pRE155; 2, untreated RNA; 3, pRE171; 4, pRE141; 5, pRE154. Note that only RNAs that are able to bind to the
protein are cleaved by RNase E and that the truncated polypeptide that
has RNA binding activity exhibits nucleolytic
activity.
The experiments presented here provide evidence that the
catalytic domain of Escherichia coli processing
endoribonuclease RNase E is located within the N-terminal half of the
protein and that it includes an RNA binding region, which is likely to
play a crucial role in the recognition and cleavage of specific
substrate RNAs. The deletion analysis and RNA-protein blotting
technique used in this study demonstrated that RNase E encodes an RNA
binding region. A putative RNA binding motif, rich in arginines, was
earlier predicted by computer analysis from the sequence similarity
with the human U1 RNA-associated 70-kDa protein (Casaregola et
al., 1992). Out of the 104 arginines present in the Rne protein,
31 (29%) are clustered between amino acid residues 601 and 731 of the
polypeptide (Fig. 4A). The present results ( Fig. 5and Fig. 6) suggest that the RNA binding motif is
located in the same region, i.e. in the central part of the
protein molecule. RNase E is the largest ribonuclease identified so
far in E. coli. The molecular mass of the protein calculated
from the updated sequence It has been established that 9 S RNA
contains two cleavage sites at which RNase E acts. Our deletion
analysis is consistent with the suggestion that both cleavages are
caused by the same enzymatic activity since extracts from all of the
deletion mutants that allowed the synthesis of a peptide with
endonucleolytic activity could carry out both types of cleavage, while
extracts from all the deletion mutants that failed to produce
functional enzyme did not perform either cleavage (Fig. 3). The
interesting question still remains if it is a sequential two-step
reaction such that the substrate must be released (and subsequently be
bound by the same or a different enzyme molecule) for the second
cleavage to occur, or if both cleavages can occur while the RNA remains
in the same active site but is repositioned for cleavage at the
alternative sites. The Rne protein seemed to bind RNA in a highly
specific manner. Only those RNAs that were substrates for RNase E were
bound by Rne protein ( Fig. 6and Fig. 7). Therefore,
quite particular features of the RNA secondary structures presumably
play a critical role in the specificity of the protein-RNA interaction.
The importance of the stem-loop structure in the processing of 9 S RNA
has been assessed earlier by in vitro experiments (Cormack and
Mackie, 1992). Mutations affecting the 5` domain of 9 S RNA, which are
likely to affect the secondary structure upstream of the first cleavage
site, were tested for their effect on processing. Removal of the
stem-loop region significantly slowed the processing efficiency,
suggesting that secondary structural features adjacent to the cleavage
site play a direct and critical role in RNase E recognition of its
substrate (Cormack and Mackie, 1992). On the other hand, it has been
shown that the addition of a 5`-terminal stem-loop structure (probably
not in a correct/native context) can slow down RNase E cleavage of RNA
I (Bouvet and Belasco, 1992). Recently it has been shown that the
unusual longevity of the E. coli ompA transcript is determined
by its untranslated region and that in some context the stem-loop
structure might stabilize mRNA by impeding access of RNase E to
downstream cleavage sites (Hansen et al., 1994). Plots of the
predicted folded structures of different RNAs used as in vitro test substrates in the present study are shown in Fig. 8.
In all cases there is potential for stem-loop formation, and the
location and extent of the stem-loop near the cleavage site may be
hypothesized to carry the feature(s) recognized by the RNase E protein.
The RNase E polypeptides described here and our findings regarding
protein-RNA binding specificity provide a basis for further
localization and characterization of the specific functional domains in
the structure of this important enzyme.
Figure 8:
Secondary structure models of different
RNAs used in this study. The RNA was folded using the FOLD program of
Zuker and Stiegler(1981) run in the GCG package on a VAX computer.
Established (
We dedicate
this paper to the memory of the late Dr. David Apirion.
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26391-26398
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
mutation and to restore the
processing of 9 S RNA was a 2.3-kilobase pair fragment with a
1.9-kilobase pair N-terminal coding sequence that mediated synthesis of
a 70.8-kDa polypeptide. Antibodies raised against a truncated 110-kDa
polypeptide cross-reacted with the intact rne gene product and
with all of the shorter C-terminal truncated polypeptides, indicating
that the N-terminal part of the molecule contained strong antigenic
determinants. Furthermore, by analyzing the Rne protein and the
truncated polypeptides for their ability to bind substrate RNAs, we
were able to demonstrate that the central part of the Rne molecule
encodes an RNA binding region. Binding to substrate RNAs correlated
with the endonucleolytic activity. RNAs that are not substrates for
RNase E did not bind to the protein. The two mutated Rne polypeptides
expressed from the cloned gene containing either the rne-3071 or ams1 mutation also had the ability to bind 9 S RNA,
while their enzymatic function was completely abolished. The data
presented here suggest that the endonucleolytic activity is encoded by
the N-terminal part of the Rne protein molecule and that the central
part of it possesses RNA binding activity.
)
)intact Rne
protein consists of 1061 amino acids. The molecular mass calculated
from the DNA sequence amounts to 118 kDa, while the protein in
SDS-polyacrylamide gel electrophoresis migrates as a 180-kDa
polypeptide.
Bacterial Strains and Plasmids
The bacterial
strains and plasmids used in this study are listed in Table 1.
DNA Manipulations
All DNA manipulations
(restriction enzyme digestion, fill-in reactions, transformation,
ligation, and DNA preparations) were performed as described by Sambrook et al. (1989) unless otherwise stated.Preparation of Cell Extracts
Cells containing the
coupled T7 polymerase/promoter system (Tabor and Richardson, 1985) were
grown at 30 °C in LB medium. At a cell density of about 2 
10
cells/ml, the culture was shifted to 42 °C for 25
min to induce the transcription of T7 RNA polymerase and then shifted
back again to 30 °C for 1 h. The whole cell extract for protein
analysis was prepared by boiling the cells in a lysis buffer (60 mM Tris-HCl, pH 6.8, 1% SDS, 1% 
-mercaptoethanol, 10% glycerol,
and 0.01% bromphenol blue) for 3 min prior to loading onto the
SDS-polyacrylamide gel. For testing the enzymatic activity, cells were
lysed in a buffer containing 0.01 M Tris-HCl, pH 8.0, 0.01
MgCl, 0.01 M KCl, and 7 mM -mercaptoethanol by six ultrasonic bursts of 20 s with
intervals of 30 s in a XL2020 Sonicator ultrasonic processor using the
smaller probe. Cell debris and intact cells were removed by
centrifugation at 5000 g for 10 min followed by
centrifugation at 20,000 g for 30 min. The supernatant
was removed, and the P-20 pellet was resuspended in the same buffer and
used to test the enzymatic activity.Protein Refolding Assay
Whole cell extracts
containing overexpressed polypeptide were separated on 5%/8%
SDS-polyacrylamide gel. Proteins were visualized with ice-cold 0.25 M KCl containing 1 mM DTT. (
)The gel was
rinsed several times with deionized water containing 1 mM DTT.
The bands containing overexpressed polypeptides were excised from the
gel. Proteins were eluted in an elution buffer (0.05 M NaHCO, 0.1% SDS) using the Bio-Rad electroeluter
(model 422). The eluted polypeptides were precipitated with 4 volumes
of acetone and recovered by centrifugation. Proteins were denatured
with 6 M guanidine hydrochloride in buffer D (0.05 M Tris-HCl, pH 8.0, 0.1 mg/ml bovine serum albumin, 0.15 M NaCl, 1 mM DTT, and 0.1 mM ETDA) (Hager and
Burgess, 1980). Renaturing of the proteins was performed by 50-fold
dilution of the guanidine hydrochloride in buffer D for 1-2 h at
room temperature. After the refolding, guanidine hydrochloride was
removed by dialyses against buffer containing 0.05 M Tris-HCl,
pH 8.0, 0.15 M NaCl, 1 mM DTT and 0.005 mM
EDTA. The protein solution was concentrated against 50% glycerol in the
same buffer and used for the enzymatic activity test as described
below.Assay for RNase E Activity
P-Labeled
RNA transcripts (1-2
10 cpm) were incubated
in 20 µl of a mixture containing 10 mM Tris-HCl, pH 8.0,
100 mM NH
Cl, 0.1 mM NaEDTA, 5
mM magnesium acetate, 50 µg/ml yeast tRNA, and cell
extract (10-15 µg). To inactivate the enzyme encoded by the
chromosomal gene copy, protein extracts from the rne(Ts) were
preincubated for 20 min at 43 °C. The reaction mixtures were
incubated at 30 °C for 30 min, and reactions were terminated by the
addition of 5 µl of loading buffer (50 mM
NaEDTA, 1% SDS, 50% glycerol, 0.01% bromphenol blue).
Samples were heated at 95 °C for 3 min and subjected to
electrophoresis in a 5%/10% tandem polyacrylamide gel containing 7 M urea. The bands were visualized and quantitated on the
PhosphorImager (Molecular Dynamics).Western Blot Analyses
Rabbit anti-Rne antibodies
were raised against the 110-kDa protein overexpressed from the plasmid
pRE141 using a T7 polymerase/promoter system (Tabor and Richardson,
1985). Affinity purification of the antibodies and the Western blot
procedure were performed as described earlier (Taraseviciene et
al., 1994).Preparation of Substrates for RNase E Activity
Test
As templates for RNA synthesis in vitro, we have
polymerase chain reaction-derived DNA fragments corresponding to 9 S
RNA and intercistronic region of papBA with a sequence
corresponding to the T7 promoter 10 at the 5`-end (see Fig. 1B). The following primers were used: Pr1,
5`-[P
10]-GAAGCUGUUUUGGCGGAUGAG-3`, and
Pr2, 5`-ACGAAAGGCCCCAGTCTTTC-3`, for 9 S RNA; Pr3,
5`-[P
10]-TCCGTGTCATCCTTGTTAAAACAA-3`,
and Pr4, 5`-AGTTGCGTTGATTAACATTCTTTTCAT-3`, for the untranslated region
of the rne gene (Fig. 1); Pr5, 5`-[P
10]-TTATGGCATTCCGGAGTTTCTGGAAG-3`, and Pr6,
5`-ATAGCTACCGCACCGGCA, for the intercistronic region of papBA (sense); Pr7, 5`-[P
10]-AAATAACAACCTCTTTTTCATTACTCAAC-3`, and Pr8,
5`-ATTATGGCATTCCGGAGTTTCTGGAAG-3`, for the intercistronic region of papBA antisense RNA.
10]corresponds to the nucleotide
5`-CGGATCCCGTAATACGACTCACTATAGG-3`, which is the
10 promoter
sequence of phage T7 (Tabor and Richardson, 1985).
-P]ATP and purified
on a tandem gel of 7.5%/10% polyacrylamide, containing 7 M
urea.
RNA-Protein Binding
Proteins from the whole cell
extracts were separated on the 5%/10% SDS-polyacrylamide gel. After
electrophoresis, the gel was soaked in a TGS (48 mM Trizma
base, 39 mM glycine, 10 mM SDS) buffer for 30 min.
Proteins were transferred to a nitrocellulose membrane in the same
buffer using a Bio-Rad semi-dry transblot apparatus for 45 min at a
constant current of 0.2 mA/cm. The membrane was placed in TEN50 buffer
(10 mM Tris-HCl, pH 8.0 containing 1 mM EDTA, 50
mM NaCl, and 1 mM DTT) and stored at 4 °C
overnight. Prehybridization was performed for 90 min at 44 °C in 5
ml of RNA binding buffer RBB (TEN50, containing 0.02% Ficoll 400, 0.02%
polyvinylpyrrolidone, 0.02% bovine serum albumin, and 150 µg/ml
single-stranded DNA) (Cormack et al., 1993). P-Labeled RNA probes (1
10
cpm)
generated from in vitro transcription were added, and the
hybridization was performed for 2 h at 44 °C. The membranes were
washed 5 times for 10 min in TEN50 buffer. To visualize the proteins
the membranes were stained with 0.1% Amido Black in 25% isopropanol,
10% acetic acid for about 3 min and destained in 25% isopropanol, 10%
acetic acid. The blots were dried, and radioactive bands were detected
by PhosphorImager (Molecular Dynamics).
Construction and Analysis of Deletions in the rne
Gene
Plasmid pRE171 contains a 6-kilobase pair PstI-PstI chromosomal DNA fragment encoding the
entire rne gene (Taraseviciene et al., 1994). The rne gene deletions were constructed by recloning DNA fragments
from a smaller derivative pRE141 (Taraseviciene et al., 1991)
using different restriction enzymes. Deletion constructs were expressed
in the coupled T7 polymerase/promoter system (plasmid vector
pT7-5) described by Tabor and Richardson(1985). The deletion
constructs are shown in Fig. 1. E. coli conditionally
lethal (temperature sensitive) mutant strains N3438 (rne-3071,
recA), N3431 (rne-3071,
recA
), and CH1828 (ams-1,
recA
) were transformed with the different
deletion constructs and the ability of the plasmids to complement the
Ts mutations was tested. As shown in Table 1, only the plasmids
pRE171, pRE141, pRE152, pRE153, and pRE154 were able to complement the
Ts mutation in a recA
background. In recA
strains N3431 and CH1828, even plasmid
pRE160 carrying a 1.2-kilobase pair insert with the coding sequence of
about 600 nucleotides reversed the temperature sensitivity, since
recombination events could occur. The plasmids pRE184 and pRE185
containing a 858-nucleotide deletion at the 5` end of the gene
(starting at codon 29) did not overcome the temperature sensitivity and
did not restore enzymatic activity. The results are fully consistent
with the recent findings that the rne-3071 and ams-1 mutations in the rne gene are located near the 5`
terminus within the first 600 nucleotides (the ams-1 mutation
is a G
A transition in codon 66 and the rne-3071 mutation
is a C
T transition in codon 68 of the rne gene (McDowall et al., 1993)). Plasmid pRE158 contains a deletion in the
promoter region, and it suppressed the Ts phenotype only in recA
strains but not in recA
strains, since it fails to express the
protein (data presented below).
Enzymatic Activity of the Truncated Products
The
protein extracts prepared from the deletion constructs expressed in E. coli strain N3438 (rne-3071) were tested for
enzymatic activity using as substrates 9 and 7 S RNAs transcribed in vitro. As is shown in Fig. 3and Table 2,
extracts from the strains containing plasmids pRE171, pRE141, pRE153,
and pRE154 exhibited endonucleolytic activity. Extracts prepared from
the strains containing the plasmids with smaller inserts (less than 2.5
kilobase pairs), pRE155-160, and with the deletion at the N
terminus (pRE184 and pRE185), were not able to process 9 or 7 S RNA (Fig. 3, A and B, and data not shown).
Extracts from the cells containing the plasmid pRE158 also were lacking
ribonucleolytic activity, since no protein was expressed from this
plasmid (see Table 2).
RNase E Contains an RNA Binding Region
The
hydrophilicity plot of RNase E (Fig. 4A) from the
computer structural analysis using the MacVector program revealed a
highly charged area between amino acids 580 and 720, which has been
suggested to bind RNA (Casaregola et al., 1992). In order to
test RNA binding by RNase E and deletion constructs, proteins expressed
from the plasmids carrying the full-length wild-type, rne-3072, or ams-1 gene (pRE171, pRE181, pRE182) and
truncated ams-1 gene (pRE183) were tested by Western-Northern
blots. The proteins were separated by SDS-polyacrylamide
electrophoresis and blotted onto a nitrocellulose membrane. The
membrane was then hybridized with P-labeled 9 S RNA and
RNA-protein binding was visualized by autoradiography. The data
presented in Fig. 5(lanes 1-4) clearly
demonstrate that the full-length wild-type and mutated polypeptides
were able to bind the classical Rnase E substrate 9 S RNA with high
efficiency and specificity. Moreover, the 286-amino acid deletion
(amino acids 29-314) at the N-terminal part of the protein
(plasmids pRE184 and pRE185) did not affect the binding ability,
suggesting that the binding occurs downstream of codon 315 and that the
lack of catalytic activity of the mutated proteins does not abolish the
RNA binding activity.
P-labeled 9 S RNA. Left panel,
Coomasie Brilliant Blue-stained SDS-polyacrylamide gel. Right
panel, protein-RNA blot. Whole cell extract prepared from the
cells carrying plasmids: 1, pRE171; 2, pRE181, 3, pRE182; 4, pRE184; 5, pRE183; and 6,pRE185.
, PAP5 RNA (papBA intercistronic region), PAN5 RNA (deletion in the papBA intercistronic region), aPAP5 RNA (antisense), and aPAN5
RNA (antisense). Data in Fig. 6clearly demonstrate that only
the proteins expressed from the plasmids pRE171, pRE141, and pRE154
(plasmids pRE152 and pRE153 were not tested) have the ability to bind
RNA. The smaller truncated proteins (expressed from the plasmids pRE155
or pRE160) were not able to bind RNA. Therefore, the Rne molecule
contains RNA binding activity, and the region for that activity is in
the central part of the protein. Furthermore, the RNA-protein
hybridization experiments revealed another very interesting finding:
only those RNAs that were substrates for the RNase E endonucleolytic
activity were bound by the proteins. As is shown in Fig. 6, the
RNase E polypeptides exhibited an apparent high substrate specificity
toward 9 S RNA, 7 S RNA, PAP5 RNA and RNA E1, while PAN5 RNA,
tRNA
, antisense PAP5 and antisense PAN5 RNAs
did not bind either to the intact Rne protein or to its truncated
polypeptides. The data suggest that the substrate RNAs for RNase E also
encode the determinant for RNA-protein recognition.
P-labeled RNA probes. Whole cell extracts were
prepared from the cells carrying plasmids: 1, pRE171; 2, pRE141; 3, pRE154; 4, pRE155 and
hybridized with
P-labeled RNAs. B, 9 S RNA; C, E1 RNA; D, PAP5 RNA; E, aPAP5 RNA; F, tRNA
; Gb, 7 S RNA; H, PAN5 RNA; I, aPAN5 RNA. aPAP5,
tRNA
, PAN5n, and aPAN5 RNA did not give any
signal, suggesting that they do not bind to RNase
E.
The RNA Binding Region Is Required for Expression of
Endonucleolytic Activity
The ability of the active truncated
polypeptides to cleave the RNA substrates was also tested. The
experiments showed that all of the RNAs that were able to bind intact
Rne protein were cleaved by RNase E (Fig. 7). On the other hand,
mutations or deletions at the N terminus of the Rne polypeptide totally
eliminated the catalytic activity, while the RNA binding activity was
not affected (Fig. 5). Furthermore, only the polypeptides
containing the intact N terminus, which were found to bind RNA, were
able to perform the cleavage reactions. It was clear that the substrate
specificity of the endoribonucleolytic activity and of the RNA binding
were similar. Thus, it seems that both protein and RNA contain
important determinants for recognition and for performing the enzymatic
reaction.
is 118 kDa. The protein migrates
in the SDS-polyacrylamide gel as a 180-kDa polypeptide. The deletion
analyses used in this study allowed us to demonstrate that the Rne
polypeptide lacking much of the molecule from the C terminus still
maintained the enzymatic activity (Fig. 3). The calculated
molecular mass of the smallest of our polypeptides still exhibiting
endoribonucleolytic activity was 70.8 kDa (Table 2; plasmid
pRE154). Recently it was demonstrated that a degraded RNase E
preparation may exhibit enzymatic activity (Carpousis et al.,
1994). Sedimentation analysis of a proteolized Rne polypeptide on a
glycerol gradient revealed that it contained 73- and 69-kDa
polypeptides that correlated with the enzymatic activity. Presumably
such partially degraded polypeptides retained the N terminus; this
would be consistent with our present finding that the RNase E catalytic
site is located in the N-terminal region of the Rne protein. Nucleotide
sequencing analysis of the two conditionally lethal
temperature-sensitive mutations of the rne gene, rne-3071 and ams-1 (McDowall et al., 1993), revealed that
these two mutations are located in a region near the 5` end of the gene
and separated by only six nucleotides. The ams-1 mutation
causes the change of glycine to serine at position of 66 in the
predicted Rne amino acid sequence, while rne-3071 causes a
conservative change of phenylalanine for leucine at position 68. Thus,
one might suspect that glycine 66 and phenylalanine 68 are part of the
active center located at the N-terminal part of the Rne molecule. The
predicted two-dimensional structures of the mutant proteins suggested
that mutation rne-3071 caused an enhancement of helix in the
region of the mutation, whereas mutation ams-1 did not affect
helix, but introduced a new predicted -turn in this region (Fig. 4B).
) and putative ( 
) RNase E cleavage sites are
indicated.
)))
We thank Dr. John M. Stewart for valuable discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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