Department of Biochemistry and Molecular
Biology, Mount Sinai School of Medicine of New York University, New
York, New York 10029
 |
INTRODUCTION |
One goal of studies on RNA
processing in Bacillus subtilis is to determine the function
of various RNases. To date, the B. subtilis RNases shown to
be involved in mRNA processing are Bs-RNase III, a narrow-specificity
endonuclease (15, 19), and polynucleotide phosphorylase
(PNPase), a 3'-to-5' exonuclease (2, 11). Previously, we
constructed a strain in which part of the PNPase gene
(pnpA) is deleted and that contains no PNPase activity
(18). Decay of mRNA encoded by ermC, a
plasmid-borne erythromycin resistance gene, was studied in the
pnpA deletion strain. We showed that 5'-proximal
ermC RNA fragments were easily detectable in the
pnpA strain but barely detectable in the wild-type strain
(2). Since mRNA turnover is thought to be essential for
viability (6), the lack of a strong growth phenotype in the
strain deficient in PNPase indicated the presence of at least one other
3'-to-5' exoribonuclease that can compensate for the missing PNPase
activity. We proposed that the 5'-proximal ermC fragments
were mRNA decay intermediates that are normally degraded rapidly by
PNPase. The 3'-to-5' exonuclease that is responsible for mRNA turnover
in the pnpA deletion strain is less efficient at degrading
these fragments, possibly due to particular structures at their 3'
ends. In addition to its function in mRNA turnover, PNPase processing may be required for the expression of particular genes. The absence of
such processing events in the pnpA strain may be the cause of the phenotypes displayed by this strain: competence deficiency, cold
sensitivity, tetracycline sensitivity, and filamentous growth (11,
18).
Although PNPase is apparently able to degrade ermC mRNA
fragments efficiently, we have found that PNPase processivity can be
impeded. In the course of assaying for purified Bs-RNase III, we
discovered an RNA-processing product that was the result of PNPase
stalling (14). A similar PNPase stall site was detected in
vivo (2).
In this study, we examined in more detail the previously observed
PNPase block by using a model RNA that contained 5'-proximal SP82
sequences and 3'-proximal ermC sequences. Experiments show that PNPase activity behaves similarly in vitro and in vivo, suggesting that the block to processivity is an inherent property of the RNA.
Decay intermediates of the model RNA that were detected only in the
pnpA strain suggest that other sequences, which are not a
barrier to PNPase activity, function to block a different 3'-to-5' exonuclease.
| |
MATERIALS AND METHODS |
Bacterial strains.
The wild-type B. subtilis host
was BG1, which is trpC2 thr-5. The pnpA mutant
host was BG119, a derivative of BG1 in which an internal portion of the
pnpA gene was replaced with a kanamycin resistance cassette
(18). B. subtilis growth media and competent B. subtilis cultures were prepared as described previously
(7). E. coli DH5
(9) was the host
for plasmid constructions.
Plasmids.
Plasmid pYH199 was a derivative of pYH188
(2). Plasmid pYH188 contains an SP82-ermC
transcriptional unit which is similar to the one described here but has
an altered transcription terminator sequence. It also contains a second
(promoterless) copy of ermC downstream. We found that
strains carrying pYH188 were erythromycin resistant, indicating
substantial readthrough transcription from the SP82-ermC
sequence into the downstream ermC sequence. This would give
additional RNA products upon probing with an ermC-specific probe. To avoid this, we constructed plasmid pYH199, an HpaI
deletion derivative of pYH188 which does not contain any
ermC sequences other than that of the SP82-ermC
RNA transcriptional unit and which has the native ermC
transcription terminator.
Mutations in the SP82 sequence were constructed either by the method of
Kunkel et al. (10) on M13 single-stranded phage DNA
templates or by the QuikChange site-directed mutagenesis protocol (Stratagene) on plasmid DNA templates.
In vivo RNA analysis.
Growth of strains and isolation of RNA
were as described previously (2). Northern blot analysis,
using 6% denaturing polyacrylamide gels, was performed as described
previously (19). Electroblotting from a high-resolution
(sequencing) gel was performed in the same manner as for low-resolution
gels, except that the blotting was done at 10 V overnight followed by
30 V for 30 min. Radioactivity in the bands on Northern blots was
quantitated with a PhosphorImager instrument (Molecular Dynamics).
Half-lives were determined by linear regression analysis on
semilogarithmic plots of percent RNA remaining versus time. Reverse
transcriptase products were generated with reagents from the Gibco-BRL
Superscript Preamplification System. Riboprobes were synthesized by T7
RNA polymerase transcription, in the presence of
[-32P]UTP, with isolated PCR fragments as templates.
For the PCR amplifications, the downstream primer contained at its 5'
end the T7 RNA polymerase promoter sequence (TAATACGACTCACTATA),
allowing transcription of the PCR product.
In vitro RNA analysis.
Replicative-form preparations of M13
DNA containing the SP82 sequence and mutant derivatives thereof were
linearized by HindIII digestion for use as transcription
templates. The M13 clone containing the SP82 sequence carries a T7 RNA
polymerase promoter upstream of the SP82 sequence such that
transcription begins as shown in Fig. 2 (see below). Transcription was
performed in the presence of [-32P]UTP, and the
labeled RNA products were isolated from a 6% denaturing polyacrylamide
gel as described previously (1). Labeled RNA was incubated
in the presence of 10 mM Tris (pH 7.8)-100 mM NaCl-3 mM
Mg2+, either with or without 0.5 mM
Na2HPO4, and with approximately 4 µg of a
B. subtilis protein extract. The extract was prepared by
sonication of lysozyme-treated cells that were harvested from a
late-logarithmic-phase B. subtilis culture grown in rich
medium. After centrifugation at 15,000 × g for 20 min,
the supernatant was dialyzed overnight against 20 mM Na-Tricine (pH
8.0)-100 mM KCl-10% glycerol-0.2 mM EDTA-0.1 mM
phenylmethylsulfonyl fluoride, and 1.0 mM dithiothreitol. Aliquots were
stored at 
70°C. After incubation at 37°C with the extract, the
reaction mixture was extracted with phenol-chloroform (1:1) and
precipitated in 2 M ammonium acetate with 2.5 volumes of ethanol. The
products were analyzed on a 6% denaturing polyacrylamide gel.
| |
RESULTS |
Properties of the model RNA.
To study RNA processing in
B. subtilis, a plasmid (pYH199) was constructed that
contained a transcriptional unit with the following characteristics
(Fig. 1): (i) transcription from the constitutive ermC promoter to give a 320-nucleotide (nt)
RNA; (ii) 150 nt of bacteriophage SP82 sequence at the 5' end and 170 nt of ermC sequence, including the transcription terminator,
at the 3' end; (iii) a Bs-RNase III cleavage site between nt 108 and
109 of the SP82 moiety; and (iv) a strong block to PNPase processivity,
which was characterized in this study, located in the SP82 moiety.
Thus, the expressed RNA contained elements (Bs-RNase III cleavage site,
PNPase stall site) that had been found previously in vitro (13,
14), as well as a classical transcription terminator consisting
of a strong stem-loop followed by a run of U residues. The proposed
secondary structure for the SP82 moiety of this
"SP82-ermC" RNA is shown in Fig.
2 and is based on a previous analysis by using structure-specific RNases (14). SP82 sequences are up to the HpaI site shown, followed by the 3'-terminal 170 nt
beginning at the ermC HpaI site.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 1.
Diagram of SP82-ermC RNA. Straight lines,
SP82 sequences; wavy lines, ermC sequences. (A) Sites of
PNPase stalling and Bs-RNase III cleavage are indicated. Stem
structures in the SP82 moiety are numbered 1, 2, and 3. The
ermC transcription terminator is indicated by the
3'-terminal stem-loop. (B) Location of riboprobes used in Northern blot
analyses and oligonucleotide primers used for reverse transcriptase
mapping. (C) Schematic diagram of major RNAs detected in Northern blot
analyses. BSR, Bs-RNase III; PNP, PNPase.
|
|
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 2.
Secondary structure of the SP82 moiety of
SP82-ermC RNA. The sequence shown is the one transcribed in
vitro by T7 RNA polymerase. In vivo RNA contains an additional 2 nt
(GU) at the 5' end. Numbering is from the in vitro transcriptional
start site. The 4-nt deletions in stems 2 and 3 are shown in boldface.
The CAGC sequence that was deleted in stem 3 was replaced with an A to
create an AflII site. The boxed UAG sequence in the loop
portion of stem 2 is the stop codon for the putative SP82 gene
60 coding sequence.
|
|
Block to PNPase processivity.
The presence of a
PNPase-specific block in SP82-ermC RNA encoded by pYH199 was
demonstrated by the Northern blot analysis in Fig.
3 (pYH199 lanes). The blot was probed
with a riboprobe complementary to the SP82 moiety (Fig. 1B). Little of
the full-length RNA was observed due to rapid Bs-RNase III cleavage. In
addition to the 5' Bs-RNase III cleavage product, a group of bands in
the range of 60 to 80 nt, with a concentration of bands of
approximately 65 to 70 nt, was observed in the wild-type strain but not
in the pnpA deletion strain. Previous in vitro experiments,
using 5'-end-labeled RNA, demonstrated that these products started at
the native 5' end and terminated with 3' ends located upstream of the
Bs-RNase III cleavage site (14). Since PNPase is a 3'-to-5'
exonuclease, we hypothesized that this set of bands was the result of
an encounter by PNPase with a structural element in the SP82 moiety
that stalled PNPase processivity. It was also observed that the 5'
Bs-RNase III cleavage product was present in somewhat greater abundance in the pnpA strain, indicating that PNPase is capable of
degrading this RNA fragment.
View larger version (86K):
[in this window]
[in a new window]
|
FIG. 3.
Northern blot analysis of SP82-ermC RNA,
using the SP82 5' riboprobe. RNA was isolated from wild-type (+) and
pnpA () strains containing the wild-type SP82 sequence
(pYH199), the 4-nt deletion in stem 2 (pYH204), and the 4-nt deletion
in stem 3 (pYH205). The marker lane (M) contained 5'-end-labeled DNA
fragments of TaqI-digested plasmid pSE420 (3).
Values to the left are molecular sizes in nucleotides. The location of
the 220-nt RNA detected in the strain carrying pYH205, which encodes
the SP82-ermC RNA that is not cleaved by Bs-RNase III, is
indicated by the open arrow to the right of the blot. FL, full-length
RNA.
|
|
Stem 2 is the strongest predicted structure in the SP82
moiety; it is located upstream of the 3'
end of the PNPase-specific products detected in the wild-type strain. A
mutation was made that deleted four nucleotides (CAGU) in stem 2 (Fig.
2), severely disrupting the potential to form secondary structure at
this site. Northern blot analysis of RNA encoded by a pYH199 derivative
containing this mutation (pYH204) showed an absence of PNPase-specific
products, even in the wild type (Fig. 3, pYH204 lanes). Thus, stem 2 appeared to be important for formation of the PNPase block.
We wished to test whether the particular nucleotides at which PNPase
stalls were important for the stalling effect. In addition, we wished
to test whether prior Bs-RNase III cleavage was necessary for the
attack and subsequent stalling of PNPase. In other words, is the 3' end
generated by Bs-RNase III cleavage necessary for attack by PNPase and
subsequent stalling, or can PNPase initiate degradation at a distal 3'
end and stall at the same site? For this, a 4-nt deletion (CAGC) was
made in the left side of stem 3 (Fig. 2), in the middle of the region
at which PNPase processivity is stalled. Deletion of these nucleotides
was expected to alter the secondary structure of stem 3 such that it
may not serve as a target for Bs-RNase III cleavage. Previous in vitro
experiments have shown that perturbations in the lower stem portion of
the SP82 Bs-RNase III cleavage site affect Bs-RNase III activity
(13). The plasmid carrying the stem 3 mutation was
designated pYH205. Northern blot analysis of RNA encoded by pYH205
showed that, indeed, Bs-RNase III cleavage was not detectable (Fig. 3,
pYH205 lanes). The stall to PNPase processivity was apparent in the
wild-type strain, although the distribution of bands was more
restricted than for pYH199 RNA. (The identity of the strong band [ca.
220 nt] below the full-length RNA, which is seen in the
pnpA deletion strain carrying pYH205, is discussed below;
the identity of the other bands [ca. 150 nt] detected in this strain
has not been studied.) We concluded that PNPase degradation and
subsequent stalling can initiate downstream of the Bs-RNase III
cleavage site and that the specific nucleotides at the stall site are
not necessary for stalling.
Single-nucleotide changes in stem 2.
The 4-nt deletion in stem
2, contained on plasmid pYH204, was predicted to severely affect the
stem structure. To determine the effect of less drastic structural
alterations, we made single-nucleotide changes in stem 2. Plasmid
pYH214 contained an SP82-ermC RNA with an A-to-T change at
nt 60, while plasmid pYH221 had a G-to-C change at nt 61 (Fig. 2).
Northern blot analysis of RNA isolated from strains carrying these
plasmids showed that the PNPase-stalled products were detected but in
sharply decreased amounts relative to the wild type (Fig.
4). Quantitation of the group of bands representing PNPase stalling revealed that the amount of these products
in strains with the single-nucleotide mutations in stem 2 was 7- to
7.5-fold smaller than in the wild type.
View larger version (88K):
[in this window]
[in a new window]
|
FIG. 4.
Northern blot analysis of SP82-ermC RNA with
single-nucleotide mutations. RNA was isolated from wild-type (+) and
pnpA () strains carrying the A-to-T change in nt 60 (pYH214) and the G-to-C change in nt 61 (pYH221).
|
|
In vitro analysis of PNPase stalling.
Since the RNA products
ending at the SP82 stall site were observed only in the wild-type
strain and not in the pnpA deletion mutant, it could be
assumed that these products were generated by PNPase activity that was
stalled at this site. However, it could also be that the absence of
PNPase disrupts the function of other RNA cleavage/recognition proteins
that are involved in the formation of these RNA products. Furthermore,
the evidence that stem 2 creates a block to PNPase processivity could
be explained differently. For example, the observed products could be
the result of endonucleolytic cleavage, followed by limited degradation
or by 3' polyadenylation. Perturbation of the stem 2 sequence might interfere with such endonucleolytic cleavage. We therefore used a
B. subtilis protein extract to examine PNPase-dependent
stalling in vitro. The substrates used were transcribed by T7 RNA
polymerase from a template that contained the same 150 nt of SP82
sequence to the HpaI site as in pYH199 (except for the first
2 nt at the 5' end), followed by an additional 145 nt of SP82 sequence.
Mutant templates were prepared which contained the same changes as
those present on the plasmids used for the in vivo analyses described above.
PNPase-specific activity was tested by the presence or absence of
inorganic phosphate (Pi). To avoid the complication of
Bs-RNase III activity, which is present in an extract from a wild-type strain, an extract from strain BG218 was used. BG218 contains a partial
disruption of the rncS gene such that Bs-RNase III activity is virtually undetectable in an extract from this strain
(19). The results with the wild-type substrate are shown in
Fig. 5A, lanes 1 to 3. Incubation in the
presence of Mg2+ alone (lane 2) resulted in little
degradation of the full-length substrate. This is consistent with our
previous findings of low-level Mg2+-dependent decay in a
B. subtilis extract (18). When Pi was added (lane 3), the PNPase activity resulted in degradation of about
85% of the full-length substrate, with the appearance of an RNA
product representing a stalling of PNPase processivity. The size of
this RNA product was similar to that observed in vivo, but there was a
tighter distribution of 3' ends. Thus, the observed products were
phosphate dependent, indicating strongly that they are generated by
PNPase activity. Reducing the substrate concentration 10-fold did not
affect the results (data not shown), suggesting that PNPase activity
was in excess.
View larger version (68K):
[in this window]
[in a new window]
|
FIG. 5.
In vitro analysis of PNPase processing. (A)
Low-resolution denaturing gel showing processing of labeled SP82 RNA
incubated in the presence of a BG218 protein extract. The RNA substrate
is indicated at the top of each set ( , deletion mutant). The three
lanes in each set contained a control in which RNA processing was
inactivated by the immediate addition of phenol-chloroform (lanes 1, 4, 7, 10, and 13), the mixture without Pi (lanes 2, 5, 8, 11, and 14), and the mixture with Pi (lanes 3, 6, 9, 12, and
15). (B) High-resolution denaturing gel showing the migration of the
PNPase processing product from the wild-type (lane 1) and stem 3 deletion mutant (lane 3) substrates. The leftmost lanes show a
sequencing reaction used as a molecular size marker (sizes in
nucleotides are indicated at the left). Lane 2 contains RNA that was
incubated with the extract in the presence of Pi and 3 mM
ATP. The products of this incubation migrated near the top of the gel
(not shown), suggesting the presence of polyadenylating activity in the
extract. This result is not relevant to the subject of this paper.
|
|
RNA substrates containing the mutations described above were tested in
the in vitro assay. The amount of degradation of the full-length
substrate in the presence of Pi was approximately 85% in
all cases. The stem 2 deletion mutant (Fig. 5A, lanes 4 to 6) showed no
RNA resulting from PNPase stalling, as was observed in vivo. The stem 3 deletion mutant showed the same pattern as the wild-type substrate
(lanes 7 to 9). On closer examination, using a higher resolution
(sequencing) gel, the PNPase stalling products for the wild type and
stem 3 deletion mutant were very similar (Fig. 5B). The amount of RNA
resulting from PNPase stalling in the substrates that contained
single-nucleotide changes in stem 2 was dramatically affected, as it
was in vivo. The A-to-T change at nt 60 resulted in a weak PNPase stall
(Fig. 5A, lanes 10 to 12), while the G-to-C change at nt 61 resulted in
a barely detectable PNPase stall (lanes 13 to 15).
Since the in vitro system appeared to mirror the in vivo results, the
in vitro assay was used to determine whether PNPase was completely
blocked by stem 2 or whether PNPase was able to proceed past the block
at some frequency. For this, uniformly labeled SP82 RNA that terminated
at the HpaI site was incubated for 15 min at 37°C in the
presence of extract with Mg2+ only or with Mg2+
plus Pi. The products were run on a 6% denaturing
polyacrylamide gel, and the counts in the full-length and the
PNPase-processed RNA bands were measured. It was assumed that the RNA
substrate was uniformly labeled; thus, based on the number of U
residues in each of the RNA species, quantitation of the full-length
input RNA and the small RNA resulting from the PNPase block would give the percentage of RNA molecules on which PNPase was blocked and could
not be further degraded. The percentage of RNA remaining in the
PNPase-blocked form was 97.8 ± 10.8 (mean of five experiments; data not shown). Thus, the block to PNPase processivity on RNA molecules that had been attacked by PNPase was virtually complete. We
tentatively concluded that the nature of the stem 2 structure provides
an absolute block to PNPase processivity.
Alternatives to this conclusion were considered and were tested (data
not shown). For example, it could be that the PNPase activity in the
extract dies after a few minutes of incubation at 37°C. To test this,
we analyzed the PNPase activity after preincubation of the extract in
buffer containing Pi but without labeled RNA substrate (the
extract contains cellular RNAs, including substantial amounts of rRNA).
Preincubation for as long as 30 min showed no effect on the ability of
PNPase to degrade the substrate up to the block. It was also possible
that upon incubation with the extract, the substrate undergoes some
change (e.g., protein binding), which would interfere with PNPase
activity. To test this, the RNA was preincubated in the extract prior
to addition of Pi. This treatment also did not affect the
pattern of PNPase-dependent degradation. It appeared, therefore, that
the block to PNPase processivity was solely due to the presence of stem 2.
Block to decay in the pnpA deletion strain.
The
data presented so far focused on the block to PNPase processivity,
which occurs in the SP82 moiety of SP82-ermC RNA. We next
examined the ermC moiety of SP82-ermC RNA by
using probes directed at the upstream and downstream portions of the
ermC sequence, as shown in Fig. 1B (probes A and B). RNA
isolated from wild-type and pnpA mutant strains carrying
pYH199 was examined by Northern blot analysis (Fig.
6). The A probe, which is complementary
to 3'-proximal sequences, detected the 3' Bs-RNase III cleavage product as well as a faint signal for full-length SP82-ermC RNA.
(The A probe consistently gave weaker signals than other probes.) By using the A probe, only the 3' Bs-RNase III cleavage product was detected, with the pnpA strain containing about twice the
amount of this product as the wild-type strain. This suggested that
PNPase is not required for decay of the 3' Bs-RNase III cleavage
product. On the other hand, the B probe, which also detected the 3'
Bs-RNase III cleavage product, gave a strong signal for a band of about 100 nt in the pnpA strain only (Fig. 1C). This band was also
detected in the strain carrying pYH204, which contained the stem 2 deletion mutant.
View larger version (63K):
[in this window]
[in a new window]
|
FIG. 6.
Northern blot analysis of SP82-ermC RNA,
using the A and B riboprobes. RNA was isolated from wild-type (+) and
pnpA () strains containing the wild-type SP82 sequence
(pYH199), the 4-nt deletion in stem 2 (pYH204), and the 4-nt deletion
in stem 3 (pYH205). Values to the left are molecular sizes in
nucleotides. The location of the 220-nt RNA detected in the strain
carrying pYH205, which encodes the SP82-ermC RNA that is not
cleaved by Bs-RNase III, is indicated by the open arrow to the right of
the blot probed with the B riboprobe.
|
|
The nature of the band that was detected only in the pnpA
strain was examined further by blotting from a sequencing gel and by
reverse transcriptase analysis. The sequencing gel blot was used to
more accurately determine the size of this RNA or group of RNAs. The
data in Fig. 7A show a group of bands
detected in the pnpA strain ranging from 108 to 120 nt, with
the strongest signal at about 110 nt. Below, we call this group of RNAs
the 110-nt RNA. To map the 5' end of this RNA, reverse transcriptase analysis was performed (Fig. 7B). For this analysis, two
oligonucleotide primers (primers 1 and 2) were used (Fig. 1B). Primer 1 was complementary to ermC sequences, with its 5' end located
145 nt downstream of the Bs-RNase III cleavage site. A single 5' end
was mapped for the SP82-ermC RNA in the wild-type and the
pnpA strains, and this corresponded precisely to the
Bs-RNase III cleavage site. The amount of reverse transcriptase product
in the pnpA strain was about three times that in the
wild-type strain, which reflects the relative abundance of the 3'
Bs-RNase III cleavage product detected by the A probe (Fig. 6). Primer
2 was complementary to SP82 sequences, with its 5' end located 39 nt
downstream of the Bs-RNase III cleavage site. Again, a single 5' end
was mapped, which was at the Bs-RNase III cleavage site. The amount of
reverse transcriptase product from the pnpA strain (in which
there was abundant 110-nt RNA) was approximately 13-fold more than in
the wild-type strain (which had no detectable 110-nt RNA). From these mapping data, we conclude that there is a block to 3'-to-5' degradation in the ermC sequence, which is located about 110 nt
downstream of the Bs-RNase III cleavage site.
View larger version (70K):
[in this window]
[in a new window]
|
FIG. 7.
Analysis of 110-nt RNA. (A) Northern blot analysis of
RNA from wild-type and pnpA strains carrying pYH199, which
were separated on a sequencing gel. The probe was the B riboprobe (Fig.
1B). The leftmost lanes show a sequencing reaction used as a molecular
size marker (sizes in nucleotides are indicated at the left). The
figure is a composite of two exposures of the same filter. (B) Reverse
transcriptase analysis of RNA isolated from wild-type and
pnpA strains carrying pYH199, using primer 1 (left) or
primer 2 (right). The four leftmost lanes in each panel show sequencing
reactions used as molecular size markers (sizes in nucleotides are
indicated at the left). Lanes: 1, wild-type strain with no plasmid; 2, wild-type strain with pYH199; 3, pnpA strain with pYH199.
|
|
We would expect that in the strain carrying pYH205, which contains the
stem 3 deletion mutant for which no Bs-RNase III cleavage occurs, the
block to decay in the ermC sequence would produce an RNA
that was 110 nt larger than the 110-nt RNA (since the Bs-RNase III
cleavage site is at nt 110 of the in vivo-transcribed RNA [Fig. 2
legend]). In the Northern blot analysis probed with the B riboprobe, a
band running at about 220 nt was detected in the lane for the
pnpA strain carrying pYH205 (Fig. 6). The same result was
observed for the pnpA strain carrying pYH205 when the SP82 5' riboprobe was used (Fig. 3). This band was not detected in the lanes
for the wild-type strain. These results indicate that blockage of
3'-to-5' decay at the site in the ermC moiety is not dependent on Bs-RNase III cleavage.
The stability of the 110-nt RNA detected in the pnpA strain
carrying pYH199 was measured relative to the stability of the 3'
Bs-RNase III cleavage product (Fig. 8).
The stability of the 3' Bs-RNase III cleavage product in the wild-type
and pnpA strains was determined to be 5.7 and 6.5 min,
respectively. However, the 110-nt RNA was about three times as stable,
with a half-life longer than 15 min (and projected to be 19.5 min).
Since it is likely that the 110-nt RNA is generated by processing of
the 3' Bs-RNase III cleavage product, the half-life measurement of the
110-nt RNA probably reflects both the production and degradation of the 110-nt RNA.
View larger version (58K):
[in this window]
[in a new window]
|
FIG. 8.
Northern blot analysis of processed RNAs after the
addition of rifampin. Total RNA from wild-type and pnpA
strains was probed with the B riboprobe. Above each lane is the time
(minutes) after rifampin addition.
|
|
| |
DISCUSSION |
That stem 2 of the SP82 moiety afforded the block to PNPase
processivity was confirmed by showing an absence of this block when 4 nt of stem 2 was deleted and a strong reduction in PNPase stalling when
single-nucleotide changes were introduced. The site of stalling was
downstream of stem 2, but the distribution of RNA bands detected in
vitro was narrower than that detected by Northern blotting of in vivo
RNA (compare Fig. 5A to Fig. 3). This may reflect further processing
events in vivo that occur after the block to PNPase; e.g., it is
possible that the 3' ends generated by PNPase degradation at this site
are polyadenylated by a poly(A) polymerase, thus generating a more
extensive ladder of bands in vivo.
The block to PNPase activity was independent of the 3' end at which
PNPase initiated degradation. The in vitro results shown here were
obtained with a protein extract from BG218, which does not have
detectable Bs-RNase III activity. Thus, PNPase degradation initiated at
the 3' end of the substrate located approximately 190 nt downstream of
the Bs-RNase III cleavage site. Similar results were obtained with RNA
substrates whose 3' end was at the HpaI site (Fig. 2) or
whose 3' end coincided with the site of Bs-RNase III cleavage (data not
shown). The in vivo results with the stem 3 deletion mutant, which was
not recognized as a substrate for Bs-RNase III cleavage, demonstrated
that the same PNPase block was observed even though degradation must be
initiating at a site downstream of the Bs-RNase III cleavage site.
Further experiments are required to determine whether PNPase can
initiate degradation in vivo at the native 3' terminus, which is
immediately downstream of a strong secondary structure, or whether
degradation of SP82-ermC RNA by PNPase requires a prior
endonucleolytic cleavage upstream of the terminator.
We found that the block to PNPase processivity in vitro could not be
overcome by extended incubation. In earlier studies of 3'-to-5'
exonuclease processivity (4, 12), blocks to E. coli PNPase or RNase II were found to be transient. It is likely
that the transient nature of a block is the result of alternative
conformations that can be assumed by the blocking secondary structure.
If an alternate structure, which does not block exonuclease
processivity, can be assumed, then at some frequency during the
incubation period this structure will form and will allow passage of
the exonuclease. The complete block to PNPase activity offered by
stem 2 may be due to the assumption of an extraordinarily stable
structure or may be a special feature of B. subtilis PNPase.
In terms of the biological significance of the SP82 sequence, we note
that in the native SP82 sequence, stem 2 is located at the end of an
open reading frame that for most of its coding sequence is identical to
gene 60 of phage SPO1 (16). The stop codon for
the SP82 gene 60 is located in the loop region of stem 2 (Fig. 2). Since gene 60 is undoubtedly expressed as an early SP82 phage gene, processing by PNPase may be an important step in gene expression.
Although stem 2 provided a strong block to PNPase processivity, RNA
bands that had 3' ends at the site of PNPase stalling were not observed
in the pnpA strain. Thus, although we assume that there are
other 3'-to-5' exonuclease activities present in the pnpA
strain that can accomplish mRNA turnover, such activities are not
blocked at the site of PNPase stalling. This may indicate a difference
in the processivity mechanism of PNPase and other exonucleases, such
that the stem 2 block is specific for PNPase.
An interesting parallel to the case of the stem 2 block was the
finding, in the pnpA strain, of a strong RNA band with its 3' end located in the ermC moiety
the 110-nt RNA. It has
not been possible, so far, to recreate this processing event in vitro
(unpublished results). We have not resolved whether the 110-nt RNA is
formed by a block to exonuclease degradation or by endonuclease
cleavage. The evidence arguing against endonuclease cleavage is that we have been unable to detect the 3' RNA product that would result from
endonucleolytic cleavage at this site. Neither the A riboprobe nor
several different oligonucleotide probes directed at ermC sequences downstream of the putative cleavage site could detect such an
RNA fragment (unpublished results). It is possible, however, that the
3' cleavage product is extremely unstable and therefore undetectable
even in the pnpA strain. An argument in favor of the 110-nt
RNA representing an endonuclease cleavage site and not a block to
exonuclease processivity is that there is no apparent upstream RNA
structure in the vicinity of the 110-nt RNA 3' end that would be
predicted to block processivity. Tests for computer-predicted secondary
structure show little potential for even moderately strong stem-loops
in this region of the ermC sequence.
Strikingly, the sequence at the site of the 3' end of the 110-nt RNA is
GAUUU, which conforms precisely to the consensus sequence originally
described for RNase E endonuclease cleavage in E. coli (8). RNase E is thought to be the major RNase involved in
initiation of mRNA decay in E. coli, and evidence for a
B. subtilis RNase E activity has been obtained by Condon et
al. (5); however, a homologue to RNase E is not identifiable
from the B. subtilis genome sequence. It is tempting to
speculate that the 3' end of the 110-nt RNA represents a B. subtilis "RNase E" cleavage site. Experiments involving
site-directed mutagenesis of this site in the ermC sequence
may reveal the requirements for formation of the 110-nt RNA and may
resolve the issue of exonucleolytic versus endonucleolytic processing.
The fact that the 110-nt band was observed only in the pnpA
strain and not in the wild type suggests that PNPase is capable of
degrading this portion of SP82-ermC RNA, while the
exonuclease(s) that is active in the pnpA strain cannot
proceed easily through this region. Thus, we have mapped two sites on
SP82-ermC RNA that appear to represent blocks to 3'-to-5'
processivity: one which is specific to PNPase (stem 2) and one (in the
ermC moiety) which is specific to exonucleases other than
PNPase. These results indicate that the RNA products that form as a
result of exonuclease activity may depend on particular degradative
activities. Different RNA sites may act as blocks to different
exonucleases, thus increasing the potential number of processed
products. A study on the decay of the 3.7-kb cry1Aa mRNA in
B. subtilis came to the conclusion that decay occurs through
multiple endonuclease cleavages, whose sites are clustered near the 5'
and 3' ends of the mRNA (17). It was pointed out, however,
that the same mapped decay intermediates could result from limited
exonuclease digestion. Our results with a much smaller RNA demonstrate
that such limited exonuclease digestion does in fact occur. Further
experiments to define the processing pathway of model RNA substrates,
such as SP82-ermC RNA, are needed before we can better
understand the role that RNA processing plays in mRNA decay and gene expression.
This work was supported by Public Health Service grant GM-48804
from the National Institutes of Health.
| 1.
|
Alberta, J. A.,
K. Rundell, and C. D. Stiles.
1994.
Identification of an activity that interacts with the 3' untranslated region of c-myc mRNA and the role of its target sequence in mediating rapid mRNA degradation.
J. Biol. Chem.
269:4532-4538[Abstract/Free Full Text].
|
| 2.
|
Bechhofer, D. H., and W. Wang.
1998.
Decay of ermC messenger RNA in a polynucleotide phosphorylase mutant of Bacillus subtilis.
J. Bacteriol.
180:5968-5977[Abstract/Free Full Text].
|
| 3.
|
Brosius, J.
1992.
Compilation of superlinker vectors.
Methods Enzymol.
216:469-483[Medline].
|
| 4.
|
Coburn, G. A., and G. A. Mackie.
1996.
Differential sensitivities of portions of the mRNA for ribosomal protein S20 to 3'-exonucleases dependent on oligoadenylation and RNA secondary structure.
J. Biol. Chem.
271:15776-15781[Abstract/Free Full Text].
|
| 5.
|
Condon, C.,
H. Putzer,
D. Luo, and M. Grunberg-Manago.
1997.
Processing of the Bacillus subtilis thrS leader mRNA is RNase E-dependent in Escherichia coli.
J. Mol. Biol.
268:235-242[Medline].
|
| 6.
|
Donovan, W. P., and S. R. Kushner.
1986.
Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12.
Proc. Natl. Acad. Sci. USA
83:120-124[Abstract/Free Full Text].
|
| 7.
|
Dubnau, D., and R. Davidoff-Abelson.
1971.
Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex.
J. Mol. Biol.
56:209-221[Medline].
|
| 8.
|
Ehretsmann, C. P.,
A. J. Carpousis, and H. M. Krisch.
1992.
Specificity of Escherichia coli endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site.
Genes Dev.
6:149-159[Abstract/Free Full Text].
|
| 9.
|
Grant, S. G. N.,
J. Jessee,
F. R. Bloom, and D. Hanahan.
1990.
Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants.
Proc. Natl. Acad. Sci. USA
87:4645-4649[Abstract/Free Full Text].
|
| 10.
|
Kunkel, T. A.,
K. Bebenek, and J. McClary.
1991.
Efficient site-directed mutagenesis using uracil-containing DNA.
Methods Enzymol.
204:125-139[Medline].
|
| 11.
|
Luttinger, A.,
J. Hahn, and D. Dubnau.
1996.
Polynucleotide phosphorylase is necessary for competence development in Bacillus subtilis.
Mol. Microbiol.
19:343-356[Medline].
|
| 12.
|
McLaren, R. S.,
S. F. Newbury,
G. S. C. Dance,
H. C. Causton, and C. F. Higgins.
1991.
mRNA degradation by processive 3'-to-5' exoribonucleases in vitro and the implications for prokaryotic mRNA decay in vivo.
J. Mol. Biol.
221:81-95[Medline].
|
| 13.
|
Mitra, S., and D. H. Bechhofer.
1994.
Substrate specificity of an RNase III-like activity from Bacillus subtilis.
J. Biol. Chem.
269:31450-31456[Abstract/Free Full Text].
|
| 14.
|
Mitra, S., and D. H. Bechhofer.
1996.
In vitro processing activity of Bacillus subtilis polynucleotide phosphorylase.
Mol. Microbiol.
19:329-342[Medline].
|
| 15.
|
Panganiban, A. T., and H. R. Whiteley.
1983.
Bacillus subtilis RNase III cleavage sites in phage SP82 early mRNA.
Cell
33:907-913[Medline].
|
| 16.
|
Stewart, C. R.,
I. Gaslightwala,
K. Hinata,
K. A. Krolikowski,
D. S. Needleman,
A. S.-Y. Peng,
M. A. Peterman,
A. Tobias, and P. Wei.
1998.
Genes and regulatory sites of the "host-takeover module" in the terminal redundancy of Bacillus subtilis bacteriophage SPO1.
Virology
246:329-340[Medline].
|
| 17.
|
Vázquez-Cruz, C., and G. Olmedo-Alvarez.
1997.
Mechanism of decay of the cry1Aa mRNA in Bacillus subtilis.
J. Bacteriol.
179:6341-6348[Abstract/Free Full Text].
|
| 18.
|
Wang, W., and D. H. Bechhofer.
1996.
Properties of a Bacillus subtilis polynucleotide phosphorylase deletion strain.
J. Bacteriol.
178:2375-2382[Abstract/Free Full Text].
|
| 19.
|
Wang, W., and D. H. Bechhofer.
1997.
Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci.
J. Bacteriol.
179:7379-7385[Abstract/Free Full Text].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
Present address: Section of Microbial Pathogenesis, Yale University
School of Medicine, New Haven, CT 06536.
