Department of Microbiology, Lund University,
Sölvegatan 12, S-223 62 Lund, Sweden
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INTRODUCTION |
The steady-state level of mRNA in a
cell is a function of the rate of mRNA synthesis and the rate of its
decay. For bacteria, there is a wealth of information on the regulation
of mRNA synthesis (see, e.g., reference 27), while
much less is known about mechanisms of mRNA decay (5, 6,
35).
Most of our knowledge about bacterial mRNA decay is based on studies of
Escherichia coli (26, 34). In a simple model, an
initial endoribonucleolytic attack at the 5' end of an mRNA opens up
the molecule for internal downstream cleavages and the fragments
generated are subsequently degraded by exoribonucleases (12). The initial cleavage is performed by one of two
endoribonucleases, RNase E, encoded by the rne gene
(7), or RNase III, encoded by the rnc gene
(3, 8). The endonucleolytic activity of RNase E is localized
to the N-terminal half of the protein, which, unlike the C-terminal
half, is essential for E. coli viability (29,
31). RNase III is primarily involved in maturation of stable RNA
but also in degradation of some mRNA species. The hydrolytic exoribonuclease RNase II and the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase) are important for the final (3'-to-5') degradation of an mRNA to mono- and oligonucleotides. For a
few E. coli mRNA species, binding of specific proteins has been found to have a decisive influence on mRNA half-life (28, 43).
Much less is known about mRNA degradation in Bacillus
subtilis. In several bacterial species, but not B. subtilis, sequence homologues to the N-terminal part of RNase E
have been found (24, 25). E. coli RNase III has a
homologue in B. subtilis called Bs-RNase III which has been
shown to cleave rRNA in an E. coli Rnc mutant
(45). However, E. coli RNase III cannot cleave a B. subtilis phage SP82 mRNA species at a site which is
cleaved by Bs-RNase III (33). RNase III may be an essential
enzyme in B. subtilis (36), but it is not in
E. coli (3). PNPase accounts for more than 90%
of the exoribonuclease activity in B. subtilis cell
extracts. It is unclear if the cells also contain an enzyme related to
RNase II (10). However, the gene for PNPase can be deleted
in B. subtilis with little effect on overall cell physiology or the half-life of bulk mRNA (44).
Transcription of the B. subtilis glpD gene (and other
glp genes) is controlled by termination or antitermination
of transcription at an inverted repeat in the 5' untranslated leader
of glpD mRNA (21, 22, 38). We have isolated
a number of mutants carrying mutations in the glpD leader
which allow increased transcription through the inverted-repeat region.
These mutants have enhanced levels of the glpD gene product
and grow on glycerol as a sole carbon and energy source in the absence
of an activated form of the antiterminator protein GlpP. Some of the
corresponding mutants are temperature sensitive (TS) for growth on
glycerol. This phenotype has been shown to be due to an increased,
temperature-dependent rate of degradation of glpD mRNA. The
TS phenotype is suppressed by the GlpP protein in the presence of
glycerol-3-phosphate (G3P), which is the inducer of the glp
regulon (17). It is possible that the wild-type
glpD transcript is also TS in the absence of GlpP and G3P.
However, we have not yet succeeded in producing sufficient amounts of
wild-type glpD transcript under noninducing conditions to
test this possibility. With this caveat in mind, we will refer to the
class of glpD leader mutations described above as TS.
In the present work we have studied the decay of B. subtilis
wild-type and TS glpD leader transcripts in B. subtilis and E. coli. Fusions were constructed between
B. subtilis wild-type and TS glpD leaders and the
E. coli lacZ gene, and the fusions were integrated into the
chromosomes of B. subtilis and E. coli. In B. subtilis we have found that the stability of the fusion
transcript is determined by the glpD leader sequence, i.e.,
in the absence of GlpP, a TS leader causes rapid and
temperature-dependent degradation of the fusion transcript. In E. coli the TS fusion transcript is much more stable and decays at
the same rate as the wild-type fusion transcript. Additionally, GlpP
does not influence the decay of the fusion transcripts in E. coli although it does function as a specific antiterminator
protein in the species (16). Finally, we show that the
cleavage patterns at the 5' ends of the fusion transcripts are
distinctly different in E. coli and B. subtilis. The most striking difference is that the major cleavage product in
E. coli is barely detectable in B. subtilis. This
cleavage product is missing in an E. coli Rnc mutant.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The bacterial strains and
plasmids used in this work are listed in Table
1.
Growth of bacteria for extraction of RNA.
B. subtilis
was grown in minimal salts (1) with 0.5% casein hydrolysate
and required amino acids (40 mg liter
1) with shaking at
200 rpm. The bacteria were grown at different temperatures to an
optical density at 600 nm (OD600) of 0.5. The cultures were
then induced with glycerol (1.5 g liter1) for 15 min.
E. coli was grown in Luria broth containing 40 mM G3P with
shaking at 200 rpm. The bacteria were grown at different temperatures
to an OD600 of 0.5.
Samples were taken for RNA extraction, or the cells were incubated with
rifampin (B. subtilis, 100 mg liter1; E. coli, 500 mg liter1) and nalidixic acid (E. coli, 20 mg liter1) for various times before samples
were taken. The 0-min samples were taken 2 min after addition of the antibiotics.
Construction of strains.
B. subtilis LUZ1212 was
obtained by transforming B. subtilis LUR252 with pLUM1043
and isolating a kanamycin-resistant, amylase-negative transformant
according to the protocol for the isolation of B. subtilis
LUZ9595 (18). Plasmid pLUM1043 was constructed in the same
way as pLUM1041 with chromosomal DNA from LUR252 as a template for PCR
(18).
E. coli MC4100D2 was constructed in analogy with E. coli MC4100D1 as described by Glatz et al. (16), with
chromosomal DNA from B. subtilis LUR252 as a template for PCR.
DNA and RNA techniques.
PCR and DNA cloning techniques were
applied according to standard protocols (39). Total RNA from
B. subtilis was extracted as described by Resnekov et al.
(37). Total RNA from E. coli was extracted as
described by Emory et al. (13). Electrophoresis of RNA for
Northern blots was done as described by Thomas (42), and the
RNA was then blotted onto Hybond-N filters (Amersham). A
single-stranded (ss) DNA probe for Northern blots was generated by
ssPCR with primer GlpDBamII (18), cold d(A, G, T)TP, and [
32P]dCTP (Amersham). To generate the template for the
ssPCR, a fragment was amplified by PCR from B. subtilis
LUR252 with primers GlpDBamI (18) and GlpDBamII. The PCR
fragment was cleaved with AvaII, and a 215-bp fragment
containing part of the glpD leader together with the first
33 codons of glpD was isolated for use as a template. After
hybridization, the radioactivities of the bands were quantitated with a
PhosphorImager (Molecular Dynamics). Primer extension analysis was
performed according to the method of Ayer and Dynan (2). The
primer used was complementary to positions +111 to +130 of the
glpD leader (5'-ATTGATGATTCATCATTACG-3').
| |
RESULTS |
The glpD leader is a stability determinant of
glpD leader-lacZ fusion mRNA.
We have
previously shown that the 5' untranslated leader of the B. subtilis glpD transcript affects its half-life (17). In
order to determine whether interactions between the leader and other
parts of the glpD transcript are important for stability, the following experiments were done. Gene fusions were made in which a
DNA fragment of about 400 bp containing the 5' part of the
glpD region, including the promoter, the leader sequence, and the first 33 codons, was coupled in frame to E. coli
lacZ. Two fusions were made, one with the wild-type
glpD leader sequence from B. subtilis BR95 and
the other with the mutant glpD leader sequence from B. subtilis LUR252. The mutant leader sequence has an extra GC pair
in the inverted repeat, i.e., the leader RNA has an extra G, which
leads to increased constitutive (GlpP-independent) expression of the
glpD gene. The glpD transcripts produced in the
absence of GlpP are TS. The fusions were inserted in single copies into
the amyE gene of B. subtilis, the wild-type
glpD leader fusion was inserted into BR95, and the mutant
glpD leader fusion was inserted into LUR252. A schematic
description of the resulting strains, LUZ9595 and LUZ1212, is given in
Fig. 1.
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FIG. 1.
Schematic representation of B. subtilis
LUZ9595 and LUZ1212. The glpD promoter and leader from BR95
and LUR252 were amplified by PCR and cloned in frame with
lacZ in pMD433. The glpD leader-lacZ
fusions were integrated into the chromosome at the amyE
locus in the cognate strain.
 ,
promoter;
 ,
glpD leader with inverted repeat; X, glpP12
mutation; wt, wild type; +G, insertion of an extra GC pair in the
inverted repeat;  , intervening
chromosomal DNA.
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The steady-state levels of glpD mRNA and glpD
leader-lacZ fusion mRNA in LUZ9595 and LUZ1212 were measured
under inducing conditions and at different temperatures ranging from 32 to 45°C. The mRNA was analyzed in Northern blots with a probe
specific for the glpD leader. As can be seen in Fig.
2, both a glpD and a
glpD leader-lacZ fusion transcript are detected
at all temperatures in induced LUZ9595, and the amounts are similar at
all temperatures. The smaller band, which increases in intensity with
temperature, represents a truncated fusion transcript that also
hybridizes with a lacZ-specific probe (data not shown). In
LUZ1212, the steady-state levels of glpD and glpD
leader-lacZ mRNA rapidly decrease with increasing growth
temperature and transcripts are not detectable above 40°C. After the
membranes had been probed with the glpD probe, they were
stripped and reprobed with a DNA fragment specific for the
sdhC gene (32). The steady-state levels of
sdhC mRNA were essentially the same at all temperatures in
both strains (data not shown). The 
-galactosidase and G3P
dehydrogenase (GlpD) activities of LUZ9595 and LUZ1212 measured under
inducing conditions at 32 and 45°C correlated well with the
corresponding mRNA levels (15). From these results we
conclude that the glpD leader is a major stability
determinant for both the glpD and the glpD
leader-lacZ fusion transcripts in B. subtilis.
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FIG. 2.
Northern blots showing steady-state levels of
glpD and glpD leader-lacZ mRNA in
B. subtilis LUZ9595 (wild-type glpD leader) (A)
and B. subtilis LUZ1212 (mutant glpD leader) (B).
Total RNA was extracted from cells grown and induced at the
temperatures indicated. The lanes contained 5 µg of LUZ9595 RNA and
20 µg of LUZ1212 RNA.
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A B. subtilis glpD leader transcript is more stable in
E. coli than in B. subtilis.
More is known about
mRNA degradation in E. coli than in any other bacterium, and
mutants affected in different components of the mRNA degradation
machinery are available (26). We next wanted to take
advantage of E. coli to further analyze the decay of
B. subtilis wild-type and TS glpD
leader-lacZ fusion transcripts. It should be emphasized that
GlpP also promotes antitermination of transcription at the
glpD leader in E. coli (16). The
inverted repeat in the glpD leader sequence is, however, a
less efficient stop signal in E. coli than in B. subtilis, as evidenced by a high background of expression of
glpD leader-lacZ fusions in E. coli.
This difference makes possible an analysis of wild-type glpD
leader-lacZ fusion transcripts in E. coli in the
absence of GlpP. In vitro runoff transcriptional analysis has shown
that E. coli sigma-70 RNA polymerase passes through the
inverted repeat unaided, whereas no readthrough was detected with
B. subtilis sigma-A RNA polymerase holoenzyme
(15).
In a previous report (16), the wild-type glpD
leader-lacZ fusion was integrated into the chromosome of
E. coli to give strain MC4100D1, and similarly, the mutant
glpD leader-lacZ fusion was now integrated to
give strain MC4100D2. Plasmids pHP13 and pPHis1 were then introduced
into MC4100D1 and MC4100D2. pHP13 is a B. subtilis-E. coli
shuttle plasmid, and pPHis1 is a derivative which carries a gene coding
for a His-tagged and biologically active derivative of GlpP
(18). The relative steady-state levels of glpD
leader-lacZ mRNA were measured in MC4100D1 and MC4100D2
grown at 32, 37, and 42°C and in the presence (pPHis1) or absence
(pHP13) of GlpP. The resulting Northern blots are shown in Fig.
3. The relative steady-state level of the
fusion transcript at 32°C was assigned an arbitrary value of 1 for
each strain. The steady-state levels at the other temperatures were
then calculated relative to the value at 32°C. By dividing the values
for the MC4100D1 strains with the values for the MC4100D2 strains at
each temperature, we obtained a comparative measure of the temperature
stability of the two fusion transcripts (Table
2). These experiments demonstrate that
there is no temperature-dependent difference between the steady-state
amounts of the glpD leader-lacZ fusion
transcripts in MC4100D1 and MC4100D2. The steady-state amounts of the
transcripts increase in the presence of GlpP. Since it is shown below
that GlpP does not increase the relative stability of the transcripts, this increase should be due to the antitermination effect of GlpP (16). For unknown reasons, this effect is more pronounced at higher temperatures.
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FIG. 3.
Northern blots showing steady-state levels of
glpD leader-lacZ mRNA in E. coli
MC4100D1 (wild-type glpD leader) and MC4100D2 (mutant
glpD leader) carrying no plasmid ( ), pHP13, or pPHis1.
Total RNA was extracted from cells grown at the temperatures indicated.
The lanes contained 20 µg of RNA, except the MC4100D1 plus pPHis
lanes, which contained 10 µg of RNA.
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TABLE 2.
Comparison of steady-state levels of wild-type and mutant
glpD leader-lacZ mRNA in E. coli
MC4100D1 and MC4100D2 at
different temperaturesa
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To confirm the above-mentioned results, the half-lives of the two
fusion transcripts were measured at 42°C in the presence and absence
of GlpP (Fig. 4). Linear regression
analysis gave a half-life of 3 to 4 min in all cases. Importantly, the
experiments show that the mutant glpD leader-lacZ
fusion transcript decays much more slowly in E. coli than in
B. subtilis, where a transcript from the mutant
glpD leader has a half-life of about 1 min at 32°C and
less than 20 s at 45°C (17). Furthermore, the
presence of GlpP does not increase the stability of the transcript as
it does in B. subtilis.
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FIG. 4.
Northern blots showing degradation of wild-type
glpD leader-lacZ mRNA in E. coli
MC4100D1 carrying pHP13 or pPHis1 (A), wild-type glpD
leader-lacZ mRNA in E. coli MC4100D1 carrying
pHP13 (overexposed film) (B), or mutant glpD
leader-lacZ mRNA in E. coli MC4100D2 carrying
pHP13 or pPHis1 (C). (D) Half-life plots. The cells were grown at
42°C, and total RNA was extracted at 0, 4, 8, and 12 min after the
addition of rifampin and nalidixic acid. The lanes contained the
following amounts of RNA: MC4100D1 plus pHP13, 20 µg; MC4100D1 plus
pPHis1, 10 µg; MC4100D2 plus pHP13, 40 µg; MC4100D2 plus pPHis1, 5 µg.
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A glpD leader transcript is differently processed in
B. subtilis and E. coli.
The previous
experiments showed that a glpD leader transcript which is TS
in B. subtilis is much more stable in E. coli.
The following experiments were done to investigate whether this
reflects different processing of the 5' region of the transcript in the two bacteria. RNA was extracted from B. subtilis LUZ1212 and
E. coli MC4100D2, both having in their chromosomes the
mutant glpD leader-lacZ fusion and carrying pHP13
or pPHis1. B. subtilis was grown at 45°C and E. coli at 42°C. RNA samples were taken immediately before the
addition of rifampin (B. subtilis) or rifampin and nalidixic
acid (E. coli) and at various times thereafter. Primer extension products obtained with RNA from each sample were then characterized. The primer used is complementary to a region just downstream of the inverted repeat of the glpD leader. Very
different patterns of primer extension products were obtained from the
two bacteria (Fig. 5A and B). The most
prominent band in E. coli (MC4100D2) is at position +37.
This band is barely detectable in B. subtilis (LUZ1212 plus
pPHis1). It was not possible to identify breakdown products from
B. subtilis in the absence of GlpP (LUZ1212 plus pHP13) due
to the small amounts of fusion mRNA obtained. The bands obtained with
E. coli have higher intensities in the presence of GlpP
(MC4100D2 plus pPHis1), but otherwise the pattern is not different from
that seen in the absence of GlpP (MC4100D2 plus pHP13). Besides the +37
band, many less prominent bands are seen in E. coli, while
only a few are seen in B. subtilis. Two bands, +53 and +57,
are clearly seen in both bacteria. Figure 5C shows the results obtained
with wild-type glpD leader-lacZ mRNA from E. coli MC4100D1 and MC4100 carrying pLUM1041, which
contains a wild-type glpD leader-lacZ fusion. The
patterns of primer extension products, including the +37 band,
are similar to those of mutant glpD leader mRNA from
E. coli MC4100D2. However, the +53 band is not
obtained with wild-type glpD leader mRNA in E. coli. We will return to this in the discussion. It should be noted
that the +56 and +71 bands in the wild-type glpD leader mRNA
correspond to the +57 and +72 bands in the mutant glpD
leader mRNA due to the extra G in the latter. In Fig. 5C, it is also
seen that, similar to what was found with mutant glpD leader
mRNA, the cleavage pattern of wild-type glpD leader mRNA in
E. coli is not affected by GlpP. Figure
6 shows the predicted secondary
structures of wild-type and mutant glpD leader mRNAs, with
cleavage sites +37, +53, and +56-57 indicated.
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FIG. 5.
(A) Primer extension analysis of 5' end points in
glpD leader mRNA in E. coli MC4100D2 and B. subtilis LUZ1212 carrying pHP13 or pPHis1. E. coli was
grown at 42°C, and B. subtilis was grown and induced at
45°C. Total RNA was extracted from samples taken immediately before
the addition of rifampin (B. subtilis) or rifampin and
nalidixic acid (E. coli) (st) and at various times
thereafter. The 0-min (0') samples were taken 2 min after the addition
of the antibiotics. The solid arrowheads indicate possible
endonucleolytic cleavage sites in E. coli, and the open
arrowheads indicate cleavage sites in B. subtilis. F
indicates full-length transcripts, and T indicates fragments caused by
primer extension termination at secondary structures in the base of the
terminator. (B) A longer exposure of the lane containing steady-state
RNA from LUZ1212 plus pPHis1. (C) Primer extension analysis of 5' end
points in glpD leader mRNA in E. coli. Lane 1, MC4100 carrying pLUM1041 (contains a wild-type glpD
leader-lacZ fusion); lane 2, MC4100D1 carrying pHP13; lane
3, MC4100D1 carrying pPHis1. Total RNA was extracted from cells grown
at 42°C.
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FIG. 6.
(A) Computer-predicted folding of the first 84 nucleotides (46) of wild-type glpD leader mRNA.
The arrowheads indicate the +37 and +56 cleavage sites in E. coli. T indicates sites of primer extension termination caused by
secondary structures. (B) Predicted folding of loop III of mutant
glpD leader mRNA. The arrowheads indicate the +53 and +57
cleavage sites, which are seen in both B. subtilis and
E. coli.
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In a control experiment, glpD leader mRNA produced in vitro
was used as a template for primer extension. The major bands found were
a full-length transcript and some shorter products representing a stop
at the 3' end of the stem-loop (data not shown). Thus, we can conclude
that degradation intermediates from the 5' end of the glpD
leader-lacZ fusion transcript are very different in B. subtilis and in E. coli.
To examine the possibility that the +37 fragment in E. coli
is produced from a promoter downstream of the glpD promoter,
we made a deletion starting at the 5' end of the DNA fragment
containing the glpD promoter and leader sequence and ending
at position 6. The deletion caused the fusion transcript to
disappear, indicating that no additional promoter is present downstream
of the glpD promoter.
The +37 cleavage product is missing in an E. coli Rnc
mutant.
Next, we investigated whether either of the two major
endoribonucleases of E. coli is responsible for cleaving at
+37 in glpD leader mRNA. Plasmid pLUM1041 was introduced
into an E. coli TS Rne mutant and an E. coli Rnc
mutant. The Rne mutant was grown at 32°C and shifted to 45°C for 30 min; the Rnc mutant was grown at 37°C. Total RNA was extracted, and
primer extension products were characterized. The result with the Rnc
mutant is shown in Fig. 7, where it is
seen that the +37 band is missing, which implies that this band is
generated by the action of RNase III. A band of much lower intensity at
+71 in the wild type is also missing in the mutant. The RNase
E-deficient mutant gave the same pattern of primer extension products
as the wild type (data not shown).
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FIG. 7.
Primer extension analysis of 5' end points in wild-type
glpD leader mRNA in E. coli carrying pLUM1041
(containing a wild-type glpD leader-lacZ fusion).
Lane 1, BL322 (wild type); lane 2, BL321 (RNase III deficient). Total
RNA was extracted from cells grown at 42°C. The symbols are defined
in the legend to Fig. 5.
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| |
DISCUSSION |
There exists considerable experimental evidence that the 5' end of
an mRNA molecule is an important stability determinant in both B. subtilis and E. coli (4, 11, 13, 20, 30, 32,
41). However, the structures or conditions at the 5' end which
influence mRNA stability may not always be the same in the two
bacteria. For example, ribosome-binding sites, whether coupled to
translation or not, can stabilize a B. subtilis transcript but not an E. coli transcript (23). The present
experiments demonstrate that the glpD leader sequence
determines the steady-state amounts of a TS glpD
leader-lacZ fusion transcript in B. subtilis. Thus, the B. subtilis glpD leader contains the major
stability determinant for the corresponding mRNA. The TS fusion
transcript is about 10-fold more stable in E. coli than in
B. subtilis, indicating different degradation pathways in
the two bacteria.
Different processing of the glpD leader-lacZ
fusion transcripts was reflected in the cleavage patterns obtained from
the 5' ends of the transcripts. Most striking is the fact that the
major cleavage product at +37 in E. coli was barely seen in
B. subtilis. The +37 fragment was absent in an E. coli Rnc mutant, implying that it results from cleavage by RNase
III. When the patterns of primer extension products are further
compared, some additional points can be made. GlpP has no apparent
effect on the patterns in E. coli (Fig. 5A and C). A
comparison of the cleavage pattern of the mutant glpD leader
(Fig. 5A) with that of the wild-type glpD leader in E. coli (Fig. 5C) shows that the +56-57 band is present in both while
the +53 band is missing in the latter. We recall that +53 and +57 bands
in the mutant leader correspond to +52 and +56 bands in the wild-type
leader. Also, in B. subtilis, the mutant leader gives rise
to +53 and +57 bands (Fig. 5A and B) whereas only the +56 band is
obtained with the wild-type leader (data not shown). We suggest that
expansion of loop III due to the G insertion in the mutant leader (Fig.
6) increases the probability for endoribonuclease cleavage between U
and A at +53 in the first part of the loop. Loop III thus seems to be a
target for endoribonucleases of similar specificities in the two
bacteria. It has been shown for a B. subtilis phage SP82
transcript that Bs-RNase III will cleavage in a bulge containing the
sequence CAUG (33). We note that the same sequence is found
at the cleavage site +57 in the loop of glpD leader mRNA.
Our knowledge of mRNA turnover in B. subtilis and of RNases
as well as other proteins involved is quite limited. The fact that
E. coli is often taken as the paradigm for mRNA decay in bacteria mainly reflects a lack of data from other species. We therefore thought that a comparison of the decay of an mRNA in B. subtilis and E. coli should provide valuable
information. Our data on the stability and processing of
glpD leader-lacZ fusion transcripts point to
important differences in the mechanisms of mRNA decay in the two
bacteria. That such differences can exist should be taken into account
in comparative studies of gene control in different bacteria. We find
it particularly interesting that RNase III appears to generate the
major cleavage product in E. coli, a product which can
hardly be detected in B. subtilis. This raises questions
about the roles and substrate specificities of RNase III and its
homologue in B. subtilis.
We thank Lars Rutberg for valuable discussions, Lars Hederstedt,
Charles Kurland, and Lars Rutberg for critically reading the
manuscript, and Bernt Eric Uhlin for sending the RNase-deficient E. coli mutants.
This project was supported by grants from the Swedish Medical Research
Council and the Emil and Wera Cornell Foundation.
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Abstract
Introduction
Present address: Department of Infectious Diseases and Medical
Microbiology, Division of Bacteriology, Lund University,
Sölvegatan 23, S-223 62 Lund, Sweden.
