Department of Protein Biosynthesis, Institute
of Molecular Genetics, Academy of Sciences of the Czech Republic,
166 37 Prague 6, Czech Republic
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INTRODUCTION |
The streptomycin operon
(str) belongs among the most-conserved operons in
prokaryotic evolution (20, 27). The str operon of
Escherichia coli, from which most of our knowledge about
this operon is derived, is composed of four genes: rpsL
(coding for ribosomal protein S12), rpsG (ribosomal protein
S7), fus (elongation factor G [EF-G]), and tufA
(elongation factor Tu [EF-Tu]). It is transcribed from its main
promoter situated upstream of the rpsL gene (43).
Besides the main promoter, two additional promoters, located within the
fus gene, direct the transcription of the tufA gene (1, 57, 59). Zengel and Lindahl estimated the combined activity of these additional promoters to be about 30% of the activity
of the main promoter (58, 59). These promoters, as well as
the second copy of the tuf gene (tufB) present in
the E. coli chromosome (21), contribute to the
increased expression of EF-Tu relative to the expression of other genes
of the str operon.
Approximately 50 str operons have already been sequenced.
However, only a few of them were also characterized functionally by
their transcriptional products and transcriptional starts. In our
previous work, we cloned and characterized the tuf gene of
Bacillus stearothermophilus. A tuf-specific
promoter (tufp) was detected in the fus-tuf
intergenic region. Based on Northern blot experiments, the ratio
between the tuf-specific and the polycistronic transcript
was estimated to be at least 10:1 (28).
In the present work, we extended our studies to the rest of the
str operon of B. stearothermophilus with a
special focus on the characterization of the promoters of this operon.
As a prerequisite, the complete primary structure of the str
operon of B. stearothermophilus was determined, and the main
operon promoter (strp) was mapped. By using the
chloramphenicol acetyltransferase (CAT) assay technique, the strength
of the strp promoter was compared with the strength of the
previously identified tufp promoter. The proposed
stimulatory function of an A/T-rich block preceding tufp was
experimentally confirmed and an inhibitory cis element
acting on strp was discovered. Based on the comparison of
EF-G and EF-Tu expression, we suggest that the promoter for the
tuf gene plus the cis elements acting on
tufp and on strp significantly contribute to the
differential expression of the str operon genes in
gram-positive B. stearothermophilus. An "additional"
gene, one preceding the rpsL gene and designated ybxF, was found to belong to the str operon in
B. stearothermophilus and B. subtilis. The
implications derived from the presence of the ybxF gene in
this highly conserved operon are outlined.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
B. stearothermophilus
(CCM 2184) and B. subtilis 168 were used as the sources of
DNA and RNA, and B. subtilis MI 115 (leu rm
mm
recA4) as a host in CAT assay experiments. E. coli DH5
served as a host in cloning and subcloning
experiments. Plasmids pUC18 and pUC19 (54) were used in
cloning and subcloning experiments. Promoter probe E. coli-B.
subtilis shuttle vector pCPP-3, obtained upon request from Band et
al. (3), was modified by insertion of a multiple cloning
site (MCS). The resulting constructs were named pCPP-31 and pCPP-32,
depending on the orientation of the MCS (see Fig. 6). Plasmids are
available upon request. Shuttle vector pCPP-31 was used as a promoter
probe vector in CAT assay experiments. Plasmids pCPP-31.X1-7 and
pCPP-31.Y1-3, derivatives of pCPP-31, were constructed to evaluate the
activity of strp and tufp (see Fig. 8). pCPP-31
was digested with EcoRI and PstI, and
appropriate, PCR-amplified, inserts were ligated into it. Precise
endpoints of cloned fragments are indicated in Fig. 2 and 7. Primer
sequences used to amplify the promoter regions that were cloned into
these plasmids are available on request. The plasmid copy number of the
constructs (in B. subtilis) was assessed regularly,
according to the method of Labes et al. (29). The plasmid
copy number was approximately 20, and it varied <10% among the
individual constructs. Sequence inspection of the region upstream of
the MCS did not reveal any known motif (secondary structures; presence
of core promoter-like sequences in both the plasmid and the inserted
DNA that could upon ligation create an artificial promoter; and UP
element-like sequence adjacent to the MCS) that could, in the context
of the inserted DNA, influence the results of the CAT assays.
Media and transformation.
B. stearothermophilus and
B. subtilis 168 were grown as described by
Krásný et al. (28). E. coli competent
cells were prepared, and consecutive transformations were carried out
as described by Hanahan (17). Transformants were selected by
plating onto RMK (composition per liter: Bacto-tryptone, 20 g;
Bacto-yeast extract, 5 g; 1 M KCl, 10 ml; 20% glucose, 16.7 ml;
80% [wt/vol] MgSO4 · 7H2O, 10 ml)
agar plates supplemented either with ampicillin (80 µg/ml) or
neomycin (20 µg/ml). B. subtilis MI 115 cells were made
competent, and transformation experiments were carried out according to
the method of Dubnau and Davidoff-Abelson (12). Transformants were selected by plating them onto agar plates prepared from the enriched Spizizen medium (composition per liter:
(NH4)2SO4, 2 g;
KH2PO4, 6 g; sodium citrate · 2H2O, 1 g; MgSO4 · 7H2O, 0.2 g; glucose, 0.5%;
K2HPO4 · 3H2O, 18.3 g;
tryptone, 20 g; yeast extract, 5 g) supplemented with
neomycin (20 µg/ml).
Enzymes and chemicals.
Restriction enzymes, T4 DNA ligase,
shrimp alkaline phosphatase, DNA Megaprime Labeling Kit, T7 Sequenase
DNA Sequencing Kit, QUAN-T-CAT kit, [-32P]dCTP, and
[
-32P]ATP were purchased from Amersham (Germany);
RNase A, T4 polynucleotide kinase, RNasin, and avian myeloblastosis
virus reverse transcriptase were purchased from Promega. The Expand
High-Fidelity PCR System was purchased from Boehringer Mannheim. Deep
Vent DNA polymerase was from New England BioLabs. All enzymes and kits
were used according to the manufacturers' recommendations.
Oligonucleotide primers were purchased from GeneriBiotech (Czech Republic).
Nucleic acids preparation and manipulation.
B.
stearothermophilus and B. subtilis genomic DNAs were
extracted and purified as described earlier (33) with some
modifications. Plasmid DNAs were prepared with the Wizard System
purchased from Promega. Gel extractions were carried out with the
QiaexII kit from Qiagen. Restriction mapping, Southern hybridization
analysis, agarose gel electrophoresis, and subcloning of DNA fragments
were performed by standard procedures (47). B. stearothermophilus and B. subtilis RNA was prepared
from cells harvested in the mid-log-growth phase with the RNAeasy kit
purchased from Qiagen. Formaldehyde agarose electrophoresis, blotting,
and Northern blot hybridization were carried out as described
previously (47). RNA marker from Gibco-BRL was used as a
molecular weight marker in Northern blot hybridization experiments.
Cloning of the fus gene.
A pair of primers was
designed to amplify the region containing the entire fus
gene: PIII from residues 1053 to 1073 (25) (5'-GCGTGCGCGATCATTTCTCTG-3') and PIV from residues 16 to 36 (28) (5'-GAGCGCACGAAACCGCACGTC-3'). The Expand
High-Fidelity PCR System was used to ensure the fidelity of the PCR.
The 2.3-kb product was cloned into pUC18. Three recombinant clones were
selected for further analysis.
Cloning of the region upstream of rpsL.
Ligation-mediated PCR (LM-PCR) (44) was used to clone the
region upstream of the rpsL gene with the following
modifications. Primers PV (5'-TCTCCGTTGACCAGTTTGCC-3') and
PVI (5'-GTTTTTTCGGCGTCATG-3') were used to amplify a part of
the rpsL gene, which was used as a probe in Southern
hybridization experiments. A BclI digest of chromosomal DNA
was established to contain a 1.4-kb fragment containing at its 3' end
the 5'-end region of the rpsL gene. Primers PVII (5'-TAGCTTATTCCTCAAGGCACGACG-3') and PVIII
(5'-GATCCGTCGTGC-3') were assembled into a linker. The
linker was ligated to the BclI digest of chromosomal DNA.
Subsequently, the ligation mixture was digested with BclI.
Primers PVII and PIX (5'-CAAACTGGTCAACGGAGAATCGCC-3'; from
positions 
105 to 83 [25]) from the region adjacent
to the rpsL gene were used to amplify the 1.4-kb fragment.
The Expand High-Fidelity PCR System was used to ensure the fidelity of
the PCR. The 1.4-kb fragment was cloned into pUC18, and three clones were selected for further analysis.
DNA sequence determination.
DNA sequencing was done in both
directions with either T7 Sequenase DNA Sequencing Kit or fluorescent
AutoRead Sequencing Kit purchased from Pharmacia. At least three clones
from independent PCR reactions of each DNA fragment were sequenced. The
fus gene sequence was obtained by sequencing of subcloned
DNA fragments. The 1.4-kb fragment from the region upstream of the
rpsL gene was first sequenced from both sides to verify its
identity. It contained the 3' end of the rpoC gene at its 5'
end, and it ended upstream of the rpsL gene. The 3'-end half
of the fragment reaching far into the rpoC gene was then
separately cloned and sequenced.
PCR: Northern blot hybridization probes.
The following pairs
of primers were used to amplify the respective regions of the
str operon: for B. stearothermophilus,
fus probe, PAN (5'-GCACAATGGAAAGGCCACCGC-3') and
VOK (5'-GCCCAGCCGGAGAAGACCGGG-3'), and ybxF probe
(containing the entire ybxF gene), B. subtilis, fus probe, nucleotides (nt) 3772 to 3791 (forward primer)
and nt 4019 to 4038 (reverse primer), and ybxF probe, nt
2429 to 2428 (forward primer) and nt 2661 to 2680 (reverse primer)
(numbering is according to Yasumoto and coworkers
[55]).
Primer extension experiments.
The primer extension
experiments were carried out as described previously (28)
with the following modifications. A total of 25 µg of B. stearothermophilus total RNA was hybridized to the primer SMICH
(5'-CGATCACTTCCGTTGCTTCC-3'), complementary to the 5' end of
the ybxF gene. The hybridization temperature was set at
30°C.
2D electrophoresis.
B. stearothermophilus EF-Tu and
EF-G were isolated essentially as described earlier (23,
28). B. stearothermophilus S30 fractions from the
mid-exponential and stationary (overnight culture) phases were prepared
from bacterial cells resuspended in the following buffer: 20 mM
cacodylic acid sodium salt (pH 6.8), 60 mM KCl, 5 mM MgCl2,
5 mM 
-mercaptoethanol, 10 µM phenylmethylsulfonyl fluoride, and
15% glycerol. Estimation of the protein concentration was performed by
the method of Bradford (6). A total of 0.5 µg of the S30
fraction was resolved on Mini 2D electrophoresis gels (Bio-Rad)
according to the manufacturer's recommendations. The density of the
second-dimension gel was 12%. EF-Tu and EF-G were analyzed in a
parallel experiment to allow the identification of EF-Tu and EF-G spots
in the B. stearothermophilus proteome map. Proteins were
visualized either by staining with Coomassie blue R-250 or by silver
staining (4). The relative staining efficiencies of
identical amounts of isolated EF-Tu and EF-G were about equal. The
relative amounts of EF-Tu and EF-G in the two-dimensional (2D) map were
determined by densitometric measurements, normalized to the molecular
weights of these proteins.
CAT assay experiments.
B. subtilis MI 115 transformed
with respective plasmids (see Fig. 8) was grown in enriched Spizizen
medium at 37°C. The cells were harvested at an optical density at 600 nm of ~0.6. Then, 1 ml of the cell suspension was centrifuged at
12,000 × g at 4°C. The supernatant was discarded,
and the pellet was resuspended in 1 ml of ice-cold 0.1 M Tris-Cl (pH
7.8). Next, 100 µl of this suspension was transferred into a fresh
tube, and the cells were disrupted by sonication at 0°C (twice, for
10 s each time; amplitude, 0.6 µm; Soniprep 150). The sonicated
cells were pelleted, and the supernatant (S30) was used in subsequent
steps. Then, 0.04 µl of the supernatant, diluted in 40 µl of 0.1 M
Tris-Cl (pH 7.8), was used to conduct CAT assays with the QUAN-T-CAT
kit (Amersham). The CAT assays were conducted according to the
manufacturer's recommendations. Estimation of protein concentration
was performed by the method of Bradford (6).
FCA.
Factorial correspondence analysis (FCA) was performed
with the assistance of the database Indigo, accessible through the
World-Wide Web at http: //indigo.genetique.uvsq.fr).
Accession numbers.
The fus gene sequence and the
sequence of the region upstream of the rpsL gene have been
submitted to the EMBL database under accession numbers AJ249559 and
AJ249558, respectively. The MCSs of pCPP-31 and pCPP-32 have been
submitted to the EMBL database under accession numbers AJ133759 and
AJ133760, respectively.
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RESULTS |
Structure and genomic organization of the str
operon.
Sequences of two fragments of the str operon of
B. stearothermophilus have already been reported: (i) the
rpsL (S12) and rpsG (S7) genes (25)
and (ii) the tuf gene (28). To determine the primary structure of the remaining parts of the str operon,
the fus gene located between the rpsG and
tuf genes was cloned and sequenced (Fig.
1). The fus gene consists of
2,079 nt. It codes for EF-G, a protein of 692 amino acid (aa) residues
with a calculated 77,038-Da molecular mass (excluding possible
posttranslational modification) and a pI of 5.10. EF-G is a monomeric
GTP-binding protein involved in translocation of peptidyl-tRNA from the
A to the P site on the ribosome. Starting from the aa 46 (Gly), the
amino acid sequence of the N terminus of EF-G deduced here differs from
that proposed by Kimura (25). This difference is due to a
missing G nucleotide at this position in his published DNA sequence.
The nucleotide identity of the B. stearothermophilus fus
gene to the fus gene of B. subtilis is 72%.
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FIG. 1.
str operon of B. stearothermophilus. Horizontal arrows represent PCR primers used
in cloning experiments (for details, see Materials and Methods). The
EMBL accession numbers assigned to nucleotide sequences are indicated
below the scheme in combination with the sequences of Kimura
(25) and Krásný et al. (28). The
positions of the functional promoters strp and
tufp and their transcriptional products are shown.
Transcripts: i, ybxF transcript terminated in the
ybxF-rpsL intergenic region; ii, full-length transcript of
the str operon; and iii, tuf gene transcript
transcribed from the tuf-specific promoter (see reference
28). The A/T-rich block and the IR are indicated.
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Subsequently, the LM-PCR cloning strategy (44) was used to
clone the unknown region upstream of the rpsL gene (Fig.
2; see also Materials and Methods).
Directly upstream of the rpsL gene is an open reading frame
(ORF) of 249 nt separated from the 5' end of the rpsL gene
by 90 nt. It shares 61% nucleotide identity with an analogous ORF of
B. subtilis. It was therefore assigned to ybxF.
The ybxF gene codes for a protein of 82-aa residues with a
calculated molecular mass of 8,591 Da and a pI of 8.91. The region
upstream of the ybxF gene is occupied by the 3' end of another ORF. Since it is 72% identical to the rpoC gene of
B. subtilis that codes for the ' subunit of RNA
polymerase (55), it was assigned to rpoC. The
intergenic region between rpoC and ybxF is 136 nt
long and contains a palindromic structure that most likely serves as a
transcription termination signal for the rpoC gene (Fig. 2).
At the 3' end, the str operon is followed by the S10 operon
(28).
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FIG. 2.
Nucleotide sequence of the region upstream of the
rpsL gene, and deduced amino acid sequences of the
ybxF gene and the 3' end of the rpoC gene of
B. stearothermophilus. The Shine-Dalgarno sequences are in
underlined italics. The 35 and 10 regions of the strp
are boxed. The +1 nucleotide of the str operon transcript is
in boldface, indicated by an asterisk. The A-tract upstream of
strp is underlined. The transcription termination signal of
the rpoC gene is underlined with shaded arrows. The top of
the palindrome is indicated by a triangle. The stability of this
structure was calculated to be 49.3 kJ mol1 at 65°C
(RNAdraw V1.0 [34]). The IR extends from the 3' end of
the rpoC gene (from 159) to the 5' end of the A tract. The
circled amino acids denote the RNA-binding motif (26). The
underlined boldface letters denote the 5' and 3' ends of the cloned DNA
fragments (see also Fig. 8).
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The str operon is a five-gene transcriptional
unit.
Based on the cloning and sequencing experiments, a question
emerged: does the ybxF gene of B. stearothermophilus belong to the str operon? Even in
B. subtilis the answer to this question was unknown. So far,
it has been assumed that the str operon of B. subtilis is a four-gene transcriptional unit (20). To
address this question experimentally, Northern blot hybridization
experiments were carried out. In Fig. 3A,
lane 1, total RNA of B. stearothermophilus was hybridized
with a DNA fragment from the fus gene. A single band
corresponding in size to 4.9 kb was detected. In parallel, the
ybxF was used as a probe. A signal appeared in precisely the same position as in the experiment with the fus gene
fragment as a probe (Fig. 3A, lane 2). This experiment was repeated
with B. subtilis RNA and corresponding probes. The result
was identical: the ybxF probe hybridized in the same
position as the fus probe (Fig. 3B). The conclusion drawn
from these experiments is that the ybxF gene is a part of
the str operon in both B. stearothermophilus and
B. subtilis.
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FIG. 3.
Northern blot hybridization analysis of B. stearothermophilus (A) and B. subtilis (B) RNA. Each
lane contains 5 µg of total RNA. Hybridization was carried out with
fus probes (lanes 1 and 3) or ybxF probes (lanes
2 and 4). The polycistronic (str) and ybxF
(ybxF) transcripts are indicated. The RNA molecular weight
marker was from Gibco-BRL.
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Besides the polycistronic transcripts of 4.9 kb (B. stearothermophilus) and 4.7 kb (B. subtilis), the
ybxF probes of both bacteria hybridized to a small
transcript of approximately 0.3 kb (Fig. 3, lanes 2 and 4). Its length
suggests that it comprises the entire ybxF gene and
terminates in the ybxF-rpsL intergenic region. This
transcript is most likely not a processing product because both the
ybxF and fus probes detected no other transcripts indicative of processing of the str mRNA.
Mapping of the str promoter.
S1 nuclease
experiments mapped the 5' end of the str mRNA transcript
upstream of the ybxF gene (data not shown). The +1
nucleotide, determined by primer extension experiments (Fig.
4), was mapped 6 bp downstream of the
TAGTCT sequence that has 5 nt identical to the B. stearothermophilus 10 promoter consensus sequence (TATTA/CT [52]; Fig. 2). Further upstream, separated by a
spacer of 17 nt, is a hexanucleotide TTGACA sequence that in
5 out of 6 nt matches the B. stearothermophilus 35
promoter consensus sequence (TTGACT/C). Inspection of the
corresponding region of B. subtilis revealed a similar
promoter-like structure. The 10 region (GATAAT, consensus
TATAAT) and the 35 region (TTGACA, same as
consensus) are separated by 16 nt. The promoter sequences of both
organisms comply with all requirements on Bacillus promoter
sequences necessary for efficient recognition by the 
A
subunit of the RNA polymerase involved in transcription of housekeeping genes (16).
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FIG. 4.
Primer extension analysis of the polycistronic
transcript of B. stearothermophilus str operon. The 5' end
of the transcript, initiated at strp, is indicated by an
arrow. Lanes A, C, G, and T contain sequencing ladder of the respective
genomic region generated with the same primer. The 10 region is
indicated by a bracket.
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Intracellular level of EF-G and EF-Tu and quantitative evaluation
of the tufp and strp promoters.
Prior to
quantitative evaluation of strp and tufp, the
relative intracellular concentrations of EF-G and EF-Tu were
determined. Direct densitometric measurements within the proteome, the
2D profile map of B. stearothermophilus proteins, indicated
that the ratio of molar concentrations of EF-Tu and EF-G is
approximately 9:1 (Fig. 5). The
experiments were performed with cells from the exponential and
stationary phases (see Materials and Methods) with identical results.
This experiment was also carried out with B. subtilis. The
EF-Tu/EF-G ratio was almost the same as in B. stearothermophilus: 10:1 (data not shown).
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FIG. 5.
2D electrophoresis of B. stearothermophilus
S30 fraction from the mid-exponential phase. The first dimension was
run from left to right, with the more acidic proteins migrating further
to the right. The second dimension was run from the top down. The
positions of EF-Tu and EF-G are indicated.
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Since both our previous experiments (28) and the experiments
presented here suggested that transcription of the str
operon of B. stearothermophilus is controlled from two
promoters, various portions of the putative promoter regions were
cloned into a newly constructed promoter probe E. coli-B.
subtilis shuttle vector pCPP-31 (Fig.
6) to assess promoter strengths of the
cloned DNA fragments. The cloning experiments were conducted in
gram-negative E. coli, and the constructs were used to
transform gram-positive B. subtilis to provide for a
homologous environment in subsequent CAT assay experiments. The
nucleotide sequences of the cloned DNA fragments are shown in Fig. 2
(the strp region) and 7 (the tufp region).
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FIG. 6.
Map of pCPP-31 and pCPP-32. The ribosome binding site
(RBS) is underlined with a rectangle shaded in gray. The cat
gene accession number (GenBank) is K00544 (18). The MCS
sequences and their flanking regions are deposited in the EMBL database
under accession numbers AJ133759 (pCPP-31) and AJ133760 (pCPP-32). For
details, see Materials and Methods.
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FIG. 7.
Sequence alignment of B. stearothermophilus
and B. subtilis tuf promoter regions. The 3' ends of the
sequences end with start codons of tuf genes. The black
rectangle below the sequence denotes the stop codon of the
fus gene. The 35 and 10 hexamers are indicated by thick
lines above the sequence. The asterisk denotes the
transcription-initiation site as determined previously (28).
The ribosome binding site (RBS) is italicized. The shaded box contains
the A/T-rich block. The thick underlined letters mark the endpoints of
cloned DNA fragments as described in Fig. 8.
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Figure 8
schematically represents all of the recombinant constructs used in this
study and summarizes the results of the CAT assay experiments obtained
for bacteria from the mid-exponential phase. (i) The region upstream of
the rpsL gene, where the main promoter of str
operons is regularly found, e.g., in E. coli
(43), did not possess any promoter activity (pCPP-31.X1).
Its effect on the expression of CAT remained at a level comparable to
the background expression from the insertless pCPP-31. (ii) In
contrast, insertion of strp and tufp core
promoters (fragments containing 35 and 10 hexamers) upstream of the
otherwise promoterless cat gene of pCPP-31 induced a strong
expression of CAT (constructs pCPP-31.X2 and pCPP-31.Y1, respectively).
Both core promoters were equally active in vivo. (iii) The previously
identified A/T-rich block preceding the 35 region of tufp
(Fig. 7) (28) stimulated transcription from the
tufp core promoter (10 and 35) by a factor of about
three (pCPP-31.Y2). (iv) The DNA sequence upstream of the A/T-rich
block was not found to significantly influence the transcriptional
activity of tufp. The DNA fragment containing tufp and the A/T-rich block, extended by about 300 bp
further upstream of the A/T-rich block (pCPP-31.Y3), was approximately as active in CAT assays as the nonextended fragment (pCPP-31.Y2). The
~10% difference in activities between pCPP-31.Y2 and pCPP-31.Y3 may
rather be attributed to the overall nucleotide composition of the
region upstream of the A/T-rich block than to a particular structural
element. (v) In contrast to tufp, the activity of the strp promoter was not influenced by an A-tract (construct
pCPP-31.X3) situated directly upstream of the 35 hexamer, i.e., in a
position analogous to the A/T-rich block of tufp (Fig. 2).
(vi) However, further upstream extension of the strp region
for approximately 70 bp (pCPP-31.X5) resulted in a sharp, ~10-fold
drop in the promoter activity. This unexpected finding indicated the
presence of an inhibitory element in the intergenic region partially
suppressing the core promoter activity. (vii) Further upstream
extension of the promoter region for 180 bp (pCPP-31.X6) slightly
relieved the suppression brought about by the 70-bp inhibitory region
(IR). Nevertheless, ~7-fold inhibition of the core promoter activity was retained. (viii) The IR contains the termination palindrome for the
transcription of the rpoC gene. To test whether the
palindromic structure could be responsible for the inhibition by
extruding into a cruciform, pCPP-31.X4 was constructed. The insert
comprised only the 3'-end half of the palindrome. Despite this
substantial modification, which abolishes the formation of any
cruciform based on the palindrome, the inhibitory activity of the
insert was retained, although 2.4-fold decreased with respect to
pCPP-31.X6, indicating that the palindrome is not essential for the
inhibition. (ix) Since the IR is in vivo most likely transcribed, the
pCPP-31.X7 was constructed to check whether the simultaneous
transcription of the rpoC gene influences transcription
initiation from strp. The simultaneous transcription of the
3'-terminal part of the rpoC gene fragment, controlled by
the inserted tufp region including the A/T-rich block
(pCPP-31.X7), was not found to significantly modify the promoter
suppression imposed by the IR.
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FIG. 8.
Schematic structure of pCPP-31 derivatives (A) and their
transcriptional activity in CAT assays (B). (A) All DNA inserts were
cloned into the EcoRI-PstI site of the polylinker
upstream of the cat gene and its ribosome binding site (RBS)
of pCPP-31. The picture is not drawn to scale, and the pCPP-31 part of
the plasmid name is omitted. The 'X2-'X7 plasmids carry the
strp core promoter sequence extended with its upstream DNA
regions of increasing size as indicated. The 'Y1-'Y3 plasmids carry the
tufp core promoter extended in the same way. * and **,
see Fig. 2 and 7. #, the DNA insert upstream of the strp
core promoter is the same as in X6 extended for the tuf
promoter region specified in 'Y2; $, the 5' end of the insert is 499 bp
upstream of the tuf gene translational start (for nucleotide
sequence see EMBL database entry AJ249559). (B) CAT assay results. The
background value of the negative control (plasmidless B. subtilis) was subtracted. The
results are averages of at least two experiments done in duplicates,
normalized to the same amounts of protein. a, the activity of the most
active promoter region (pCPP-31.Y3) was taken as 100% (underlined); b,
relative strengths of promoter regions compared to the activity of the
strp promoter (pCPP-31.X6). Its activity was taken as 1 (underlined).
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The deletion mapping experiments provided evidence that the two
cis-acting regulatory elements, one enhancing (the A/T-rich block) and the other silencing (the rpoC-ybxF intergenic
region preceding the strp core promoter), set the in vivo
ratio between strp and tufp transcriptional
strength to approximately 1:20.
Codon usage.
Our experiments have established a direct
positional and transcriptional link between the rpsL,
rpsG, fus, and tuf genes and the
ybxF gene within the B. stearothermophilus and
B. subtilis str operons. Since most proteins of the
translational apparatus have a highly biased codon usage in a
particular organism (35), FCA was carried out to address the
functional relatedness of ybxF with other genes of the
str operon. This method relates genes according to their
usage of synonymous codons (reference 40 and
references therein). Genes highly expressed under specific physiological conditions should display a similar codon usage bias
(36). Figure 9 shows that the
rpsL, rpsG, fus, and tuf genes of B. subtilis group together, while the
ybxF gene is positioned at a distance from this cluster (for
further interpretation, see Discussion).
View larger version (37K):
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|
FIG. 9.
Factorial correspondence analysis of codon usage in the
B. subtilis ORFs. The large black crosses represent
rpsL, rpsG, fus, and tuf.
The position of ybxF is marked by the large white cross (for
details, see the text).
|
|
| |
DISCUSSION |
Transcription of the str operon.
Our experiments
have established that the str operons of both B. stearothermophilus and B. subtilis are transcriptional
units composed of five genes: 5'-ybxF-rpsL
(S12)-rpsG (S7)-fus (EF-G)-tuf (EF-Tu)-3' (Fig. 1). Besides the highly conserved rpsL,
rpsG, fus, and tuf genes, the
ybxF gene was found to extend the 5' end of the operon in
both organisms. This is a rare observation because until now all genes
found to precede the rpsL gene of the str operon,
with the exception of the archaebacterium Methanococcus vannielii (30), have been considered to be genes
adjacent to the str operon but never as members of the
operon (5, 20).
The measurements of the strength of strp and tufp
of B. stearothermophilus revealed that the transcription
proceeds about 20 times more efficiently from tufp than from
strp. The experiments demonstrated that the observed
concentration difference between the polycistronic and the
tuf-specific transcript is based on two differently active
promoters. The difference in transcription from these two promoters is
brought about by a combined effect of two cis elements
acting on equally active core promoters (Fig. 1 and 8). The main operon
core promoter is downregulated (by the IR), while the
tuf-specific core promoter is upregulated (by the A/T-rich block).
Sequence inspection of the IR revealed the presence of a palindrome
representing the transcription termination signal for the
rpoC gene. If the palindrome created an unusual DNA
structure, such as a cruciform, it might interfere with formation of
the transcription-initiation complex (37, 49). We tested
this alternative on a plasmid containing only the 3'-end half of the palindrome (pCPP-31.X4). This modification excluded the formation of
any palindrome-based cruciform. However, the inhibitory effect of the
region was not abolished, only decreased. The promoter activity of the
construct was still 2.9-fold lower than that of the strp
core promoter. Also, in vitro S1 nuclease mapping experiments (41) failed to detect any cruciform in pCPP-31.X6 (data not shown). These results suggested an additive, stepwise-like inhibitory effect within the IR sequence. We further tested the strp
region for the presence of DNA curvature, which could be responsible for the suppression (10, 42, 45). DNA bending manifests itself by anomalous electrophoretic mobility in polyacrylamide gels.
DNA fragments containing bends migrate more slowly than unbent DNA
fragments (11). The K value (the ratio of the
apparent length of a fragment to its actual length) of the fragment
cloned into pCPP-31.X5 was 1.2, strongly suggesting the presence of a DNA bend (data not shown). Further experiments are required to characterize the detected DNA bend, its position and distribution. Furthermore, the suppression can be aided by protein factors, and we
intend to study the topology of the IR to determine its mode of function.
The A/T-rich block preceding the 35 region was demonstrated to
stimulate the tufp activity by a factor of three. Generally, A/T-rich blocks, also referred to as upstream (UP) elements, are sequence elements that increase promoter strength (39, 53). The UP elements contribute to promoter recognition via their
interaction with the C-terminal domain of the subunit (CTD) of
RNA polymerase (9, 22, 46). Therefore, a contact between the
A/T-rich block and the CTD RNAP may be the mechanism that
contributes to the efficient transcription of the tuf gene
in B. stearothermophilus and B. subtilis.
It should be emphasized that the experimental design of the CAT assay
experiments described here does not rule out the possibility that some
unknown regulatory factor may be neutralized by the elevated gene
dosage (the plasmid copy number is approximately 20; see Materials and
Methods). However, the ratios of EF-Tu to EF-G (this work) and
str mRNA to tuf mRNA (28) are based on measurements of the products of the chromosome-borne genes. The relative strength of the two tested promoters correlates with the
relative concentrations of these products. Therefore, we feel it is
reasonable to suggest that the results of our experiments reflect the
promoter activities in the chromosome.
Our findings are consistent with the idea that the differential
transcription of the fus and tuf genes
significantly contributes to the observed 1:9 EF-G/EF-Tu ratio in
B. stearothermophilus, provided the two classes of the
tuf mRNAs are not translated with significantly different
efficiencies. In E. coli, neither the two additional, weak
tufA-specific promoters nor the second copy of the
tuf gene, which is transcribed from a promoter approximately as active as the additional tufA promoters (1),
provide a sufficient excess of the tuf-containing mRNAs over
the fus-containing mRNAs that would explain the 1:10
EF-G/EF-Tu ratio in this organism (56). Thus, while in
E. coli other mechanisms, such as translational efficiency
or mRNA or protein stability, must adjust the final EF-G/EF-Tu ratio by
increasing the concentration difference between these two proteins, in
B. stearothermophilus the difference is already created at
the transcriptional level. In fact, the difference is more pronounced
at the transcriptional than at the translational level, most likely
also further accentuated by the partially readthrough termination
downstream of the ybxF gene, and additional mechanisms must
be required to adjust the final EF-G/EF-Tu ratio by reducing the
concentration difference created at the transcriptional level. Thus,
E. coli and B. stearothermophilus represent two
solutions of the same problem.
According to DNA sequence data, it is likely that elements similar to
those described here are also active in the B. subtilis (55) and B. halodurans str operon
(50). Therefore, it is tempting to hypothesize that
mechanisms of control of the EF-G/EF-Tu ratio have common features
among bacilli. The work on streptomycetes conducted by Tieleman and
coworkers (51) implies that the presence of a
tuf-specific promoter may also be characteristic for other gram-positive bacteria. The relative activities of tufp and
strp in streptomycetes, however, were not assessed.
Implications derived from the presence of the ybxF gene
in the str operon.
The ybxF gene codes for
a protein of unknown function, homologous to eukaryotic ribosomal
protein L30. The ybxF gene product shares 41% amino acid
identity and 82% amino acid similarity with a 30-aa conserved region
of human-rat liver L30 (38). L30 proteins bind to 28S rRNA
(31, 32). A conserved, tentative RNA binding motif (Fig. 2)
was identified in these proteins by Koonin and coworkers
(26). The deduced amino acid sequences of the
ybxF genes of both B. stearothermophilus and
B. subtilis contain the same RNA binding motif, and their
cotranscription with ribosomal proteins and basic pI suggest that they
are ribosomal proteins. However, none of the known primary structures
of B. stearothermophilus ribosomal proteins (2,
19) matches the deduced aa sequence of the ybxF gene.
To resolve whether the product of the ybxF gene is a part of
the ribosome or possesses a different function, or both, will be the
subject of further studies.
The factorial correspondence analysis of B. subtilis ybxF
gene revealed its codon usage as being significantly different from the
remaining four genes of the operon. This result suggests that the
rpsL, rpsG, fus, and tuf
genes have evolved under similar selective constraints, whereas the
ybxF gene was linked to the rest of the str
operon at a point in evolution when the aforementioned four genes had
already coexisted and participated in translation. This result supports
the view that the ybxF translation product has a
nonribosomal destination.
The ybxF gene has a paralogous counterpart on the B. stearothermophilus and B. subtilis chromosomes, the
ymxC gene. The ymxC gene is organized in the
nusA-infB region and codes for a protein of unknown function
(7, 48). In most of the known archaebacterial genomes, the
nusA gene and the ybxF orthologue are organized
upstream of the rpsL gene (see, for example, references
8, 24, and 30). FCA of B. subtilis genes positions nusA and ybxF close to each other (data not shown). Thus, the ybxF gene in
bacilli may have originated by duplication of the ymxC gene
or vice versa. It is also interesting to note that the gene adjacent to
ymxC, infB, codes for a GTP-binding protein,
translation initiation factor IF2, that is partially homologous to
EF-Tu and EF-G.
The presence of the ybxF gene in the str operon
bears further phylogenetic implications. A database search revealed
that also some other gram-positive bacteria have the ybxF
gene organized in the same genomic context (e.g., Clostridium
acetobutylicum and Staphylococcus aureus), but nothing
is known about their transcriptional organization. In contrast,
gram-negative bacteria do not have a homologue of the ybxF
gene. Thus, our results support the hypothesis that archaebacteria are
more closely related to gram-positive bacteria, especially to the
low-G+C gram-positive bacteria (e.g., bacilli), than to gram-negative
bacteria (13, 14, 15).
This work was supported by grant 75195-540305 from the Howard Hughes
Medical Institute (to J.J.) and by grant 204/98/0863 from the Grant
Agency of the Czech Republic (to J.J.).
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Vacík,
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