(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 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.
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 ()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.
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.
[P10]corresponds to the nucleotide
5`-CGGATCCCGTAATACGACTCACTATAGG-3`, which is the
10 promoter
sequence of phage T7 (Tabor and Richardson, 1985).
Transcripts were
uniformly labeled with [-
P]ATP and purified
on a tandem gel of 7.5%/10% polyacrylamide, containing 7 M
urea.
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 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.
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, 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.
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 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.
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 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).
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 ( ) and putative (
) RNase E cleavage sites are
indicated.
We dedicate this paper to the memory of the late Dr. David Apirion.