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From the
Received for publication, July 5, 2001, and in revised form, September 6, 2001
Intrinsic transcription terminators are
functionally defined as sites that bring about termination in
vitro with purified RNA polymerase alone. Based on studies in
Escherichia coli, intrinsic termination requires a
palindromic stretch followed by a trail of T (or U) residues in the
coding strand. We have developed a highly efficient algorithm to
identify hairpin potential sequences in bacterial genomes in order to
build a general model for intrinsic transcription termination. The
algorithm was applied to analyze the Mycobacterium
tuberculosis genome. We find that hairpin potential sequences are
concentrated in the immediate downstream of stop codons. However, most
of these structures either lack the U trail entirely or have a mixed
A/U trail reflecting an evolutionarily relaxed requirement for the U
trail in the mycobacterial genome. Predicted atypical structures
were shown to work efficiently as terminators both inside the
mycobacterial cell and in vitro with purified RNA
polymerase. The results are discussed in light of the kinetic
competition models for transcription termination. The algorithm
identifies >90% of experimentally tested terminators in
bacteria and is an invaluable tool in identifying transcription units
in whole genomes.
The interaction of the template DNA, RNA polymerase, and the
nascent RNA chain has evolved so as to minimize the release of the
transcript prematurely (1). At certain sequences, the release occurs at
a rate comparable with that of elongation either spontaneously or in
the presence of assisting factor(s). Based on exhaustive work in
Escherichia coli, terminators are classified into two groups
(2). Functionally, if a sequence can bring about transcript release in
an in vitro system with purified RNA polymerase alone, it is
defined as an intrinsic terminator. These are also referred to as
simple or factor-independent terminators. Terminators that require the
presence of additional factors are classified as complex or
factor-dependent terminators. In E. coli, most
complex terminators depend on the action of the Rho termination factor
(3). These two classes of terminators are not sharply defined as the
efficiency of many intrinsic terminators is enhanced by the presence of
additional factors (4).
Intrinsic terminators are characterized by the presence of a G/C-rich
(interrupted) palindromic region followed by a trail of A residues on
the template strand (5-7). There is evidence from multiple sources
that the palindromic region extrudes out as a hairpin in the nascent
transcript (8-12). Furthermore, there is a strong, although not
absolute, correlation between the predicted stability of the stem-loop
structure and termination efficiency (12). The stem-loop structure is
believed to cause pausing of the polymerase (13-15) and weaken the
interaction of the polymerase with the nascent RNA and template DNA
(16, 17). The release is facilitated by the presence of a U trail (10,
17) probably due to the unusually weak hybrid formed by the dA·rU
base pairing (18). In addition to these two primary determinants,
sequences further downstream have also been shown to affect the
efficiency of termination probably by being an impediment to
transcription elongation (19).
Although intrinsic terminators have been studied extensively in
E. coli, little is known about their orthologs, if any, in other bacteria. Intrinsic terminators from E. coli have been
shown to function in many bacteria. However, recent theoretical
analysis indicates that only a minority of bacteria may employ this
mechanism of transcription termination (20, 21). In an attempt to
formulate a general model for intrinsic transcription termination in
eubacteria, we have developed an algorithm to identify hairpin
potential sequences in bacterial genomes and have applied it to
the Mycobacterium tuberculosis genome. Such sequences appear
to be concentrated in the immediate downstream region of stop codons, a
feature one would expect of intrinsic terminators. Surprisingly, nearly
90% of these structures lack a U trail entirely or possess a mixed A/U
trail. We show experimentally that these atypical structures work
efficiently as terminators both in vivo and in
vitro. Based on our results, the present algorithm represents the
most efficient and accurate software for the identification of
intrinsic terminators in eubacteria.
Algorithm--
The GeSTer (Genome
Scanner for Terminators) algorithm first
segregates the coding, upstream, and downstream regions based on the
feature table entries of the genome sequence. Next it searches for
palindromic sequences downstream of each gene (
The final set of structures used a minimal
With the final parameters, the algorithm identified more than 90% of
all experimentally shown terminators in different bacteria (29-32).
This corresponds to a false negative rate of <10% and a false
positive rate that varies between 5 and 10% in different genomes. All
the putative terminators are classified based on the presence of a U
trail as well as the position of adjacent structures (described below).
The distribution of the structures is also analyzed and represented
graphically. In the case of genes that are followed by multiple
structures, the best candidate is identified again based on the lowest
The whole genome sequences used for the present analysis are as
follows, Bacillus subtilis (AL009126), E. coli K12 (U00096), Mycobacterium leprae (AL450380),
M. tuberculosis H37Rv (AL123456), Neisseria
meningitidis MC58 (AE002098), and Xylella fastidiosa (AE003849). The accession numbers of the GenBankTM entries
are denoted in parentheses.
Bacterial Strains and Plasmids--
E. coli strain
DH10B was used for all cloning experiments, and M. smegmatis
mc2155 was used as the mycobacterial host for all in
vivo assays for termination. The M. tuberculosis strain
H37Ra was used to isolate genomic DNA. The E. coli cells
were grown in Luria-Bertani medium whereas the M. smegmatis
cells were grown as described in Ref. 24. Kanamycin was added at
35 µg/ml where appropriate.
The termination selection series of vectors (pTER) was generated by
cloning various promoter fragments into the E. coli-mycobacterial promoter selection shuttle vector pSD7 (25).
All of them contain different fragments encompassing the M. smegmatis gyr promoter and retain a unique BamHI site
only downstream of the promoter. pTER1 harbors a
257-bp1 fragment whereas
pTER5 harbors a 317-bp fragment. pTER7 harbors a 2.5-kb fragment, which
includes the 5' half of the gyrB gene (26). An internal
BsrGI site was filled to disrupt the reading frame and cause
premature translation termination in pTER7
The putative terminators downstream of tuf and
Rv1324 were PCR-amplified from M. tuberculosis
genomic DNA using primers 5'-ACCAGGATCCTCAAGTAGGTCTAC-3' and
5'-CGGAGGATCCATGTCAGCGTAG-3' and 5'-CGGCGGATCCTCGCCAACGCG-3' and
5'-GAACGGATCCCCCGGGTTGTCGTAG-3', respectively. The putative terminator
downstream of the M. smegmatis gyrA gene was amplified from
the pMN1Bg (26) clone using primers 5'-CCGAGATCTACGCGAGCGAGTTG-3' and
5'-GCGGGATCCCCCGGGGCGCGTCGG-3'. All PCR products were cloned at
the BamHI site in the termination vector after digestion
with BamHI alone or with BglII as required.
Analysis of Termination in Vivo--
M. smegmatis
cells harboring various constructs were grown to midlog phase (1 A600), harvested, washed, and resuspended in 100 mM Tris-HCl (pH 8.0). Cells were disrupted by sonication, and the extracts were recovered by centrifugation. An appropriate dilution was assessed for specific CAT activity as described before (27). All results were normalized to the activity of the equivalent promoter construct.
Analysis of Termination in Vitro--
M. smegmatis
RNA polymerase holoenzyme was purified as described before (28) with
certain modifications. Briefly, the holoenzyme was enriched by
polyethyleneimine and ammonium sulfate precipitation and purified
through Superdex 200 followed by a DNA-cellulose column. Fractions were
assessed for their ability to bind to a fragment encompassing the
M. smegmatis gyr promoter.
Fragments containing the promoter with putative terminator regions were
PCR-amplified using an appropriate primer downstream of the terminator
with a vector-specific forward primer (25). The gel-purified fragments
were used as templates for runoff transcription assays. RNA polymerase
was incubated with 1 µg of template DNA for 10 min at 4 °C in 50 mM Tris-HCl (pH 8.0), 3 mM magnesium acetate,
100 µM EDTA, 100 µM dithiothreitol, 50 mM potassium chloride, 50 mg/ml bovine serum albumin, and
5% glycerol. The reaction was started by adding NTPs (final
concentrations of 100 µM ATP, CTP, and GTP, 0.4 µM UTP, and 1 µCi of [
For single structures, TE = 100 × TP/(RO + TP) where TP is
the amount of terminated product and RO is the amount of runoff transcript. For total termination efficiency of the tandem structures, TE = 100 × (TP1 + TP2)/(RO + TP1 + TP2) where TP1 and
TP2 are the amounts of product terminated downstream of the
first and second structures, respectively.
For the first structure present in tandem, TE = 100 × TP1/(RO + TP1 + TP2). For the
second structure present in tandem, TE = 100 × TP2/(RO + TP1 + TP2).
Algorithm--
To delineate the elements involved in intrinsic
transcription termination conserved in all bacteria, we have developed
the GeSTer algorithm that identifies and classifies structures based on
the trailing nucleotides and the position of adjacent structures. The
algorithm identifies hairpin structures using the following parameters,
a stem length ranging from 4 to 20 nucleotides with a loop of 3 to 10 nucleotides with a maximum of 3 unpaired nucleotides in the form of
gaps or mismatches. These parameters are based on the qualitative
assessment of all known terminators from different bacteria (29-32).
The list of terminators compiled from previous literature has been
provided as Supplemental Material. Whole Genome Analysis--
When structures identified by the
program were compiled, we found that there was a preponderance of
hairpin potential sequences within 50 nucleotides downstream of the
stop codon in bacterial genomes (Table I,
Fig. 1), a characteristic one would
expect of transcription terminators. In a few species either the
L-shaped (B. subtilis and N. meningitidis in
Table I) or I-shaped structures (M. tuberculosis and
M. leprae in Table I) predominate. However, in many cases,
these two classes constitute significant fractions of the structures
identified (E. coli and X. fastidiosa in Table I).
A detailed analysis of M. tuberculosis and E. coli genomes revealed many interesting features. Firstly, the
algorithm detects putative terminators downstream of 20-40% of genes.
This is probably due to the operonic arrangement of many genes. In
addition, some of the other genes may rely on the Rho protein for
transcription termination. In agreement with this, a Rho homologue has
been identified in the M. tuberculosis genome as well.
Secondly, there is dramatic concentration of structures about 21 nucleotides downstream of the stop codon in E. coli with
relatively few structures present in the rest of the downstream region.
In M. tuberculosis, on the other hand, the structures peak
37 nucleotides downstream of the stop codon with a significant fraction
spread throughout the downstream region. Furthermore, as discussed
above, E. coli shows marginal preference in using L-shaped
structures over I-shaped structures whereas M. tuberculosis
almost exclusively uses I-shaped structures. However, it is noteworthy
that irrespective of their frequency of occurrence, the non-L-shaped
structures are concentrated at the same position as the L-shaped
structure (Fig. 1) indicating that they serve a similar purpose,
i.e. of transcription termination. Thirdly, a significant
portion of the genes employ multiple structures, either V- or U-type,
for bringing about termination. Of these the tandem structures are more
frequent. For instance, 10% of the putative terminators in M. tuberculosis and 15% in E. coli are of the U-type.
Lastly, few convergently oriented genes use a single structure present
in the shared downstream region between them.
Structures without a U Trail Are Efficient Terminators in M. smegmatis--
Although L-shaped structures function in many species
including E. coli, the V-shaped structures have been
identified previously at least in Streptomyces (31). In
addition, X-shaped structures function both in E. coli (33)
and Streptococcus (32). Therefore, we decided to test the
ability of structures that lack an obvious U trail to bring about
transcription termination in mycobacteria. Toward this end, we
constructed a mycobacteria-specific termination selection vector
(pTER5; Fig. 2) by cloning the M. smegmatis gyrase promoter (27) upstream of a CAT reporter gene. A
fragment cloned between the promoter and the reporter gene would reduce
transcriptional read-through if it were a terminator, thereby leading
to chloramphenicol sensitivity and a quantitative decrease in specific
CAT activity.
Representative I-shaped terminators were PCR-amplified from the
M. tuberculosis genome and cloned into the termination
vector (see "Experimental Procedures"). The terminator downstream
of tuf gene harbors an AU-rich trail. When present upstream
of the CAT gene, it reduces transcription read-through by ~80% (Fig. 3, A and D)
indicating that a classical U trail as defined in E. coli is
not essential for transcription termination. Surprisingly, the
terminator showed comparable efficiency in the reverse orientation that
lacks an appreciable AU-rich trail. To substantiate this observation,
we tested the terminator present downstream of Rv1324 for
its ability to bring about transcription termination bidirectionally. This structure is flanked on both sides by G/C-rich stretches. In
agreement with the above results, this terminator also functions with
comparable efficiency in both orientations (Fig. 3, B and D). This clearly demonstrates that the U trail is not
essential for the functioning of the terminator in mycobacteria. To
analyze the termination efficiency of U-shaped structures, we used the putative terminator present downstream of the gyrA gene in
M. smegmatis (Fig. 3C). The individual structures
here are weaker than the structures tested so far; however, in tandem
they show a similar termination efficiency in both orientations (Fig.
3, C and D). Thus, both I- and U-shaped
structures function with high efficacy in vivo.
Terminators Work Only in the Untranslated Region--
In the
experiments described so far, the terminators were cloned more than 50 nucleotides downstream of the promoter in the 5'-untranslated region.
When such a structure was moved closer to the promoter (27 nucleotides
downstream), there was no detectable effect on termination efficiency
(compare pTER1 and pTER5 in Fig. 4).
However, when cloned within the coding region (1.1 kb downstream of the
promoter), the structure had no detectable effect on transcription read-through in either orientation (compare pTER7 and pTER5 in Fig. 4).
On the other hand, in the same construct, when translation was moved
out of frame, leading to a premature stop codon, the structure brings
about termination with efficiency comparable with the 5'-untranslated
region context (compare pTER7 Terminators Work Efficiently in Vitro--
To ensure that the
structures were genuine intrinsic transcription terminators, we
analyzed their ability to bring about termination in vitro
using purified RNA polymerase from M. smegmatis. The templates containing the promoter and the various terminators in either
orientation were generated as described under "Experimental Procedures." Fig. 5 shows results of a
representative in vitro transcription termination assay. The
majority of the transcript appears to terminate a few nucleotides
downstream of the structure in every case. The termination efficiency
of these structures is comparable with those obtained in
vivo (Table II). Furthermore, in
agreement with the results obtained in the in vivo
experiments, all three structures work bidirectionally (compare
"Forward" and "Reverse" in Table II). Of particular interest is
the tandem terminator in which transcription terminates downstream of
each structure. Notably, the first structure encountered by the
polymerase (t1 in the forward orientation and
t2 in the reverse orientation) works at low
efficiency (~45%, Table II). On the other hand, the same structure
when encountered second (t2 in the forward
orientation and t1 in the reverse orientation)
shows appreciably higher termination efficiency (~70%, Table II),
probably due to a slowing down of the polymerase at the first
structure. Together the two structures in tandem show an efficiency
comparable with the other individual structures that have a longer
stem. Thus, in conclusion, structures with long stems (>27 bp)
function alone efficiently as terminators whereas structures with
shorter stems (~8 bp) individually form inefficient terminators.
However, the cell recruits these shorter structures in tandem where two
of them together now constitute a single efficient terminator. This is
especially important because the U-shaped structures constitute 10% of
the structures in M. tuberculosis.
Intrinsic terminators represent an extremely economical mechanism
of transcription termination. Earlier attempts to identify intrinsic
terminators have, in general, had limited success in bacterial species
other than E. coli (21, 30, 34, 35). This is probably
because they fail to take into account the possibility that secondary
structure alone could work as a terminator. As a result, they identify
only the L (and possibly the X) subsets of the terminators identified
by the present algorithm. The only other theoretical analysis of the
distribution of secondary structures in the non-coding region similarly
failed to detect a concentration of structures downstream of the
stop codon in the majority of genomes (20). This is probably
because of the 60-base window (moved in steps of 10 bases) employed in
the study. Such a rigid window and large step size would lead to
blunting of peaks, which reduces the resolution of their analysis.
Therefore, in many organisms, including M. tuberculosis, the
modest concentration of structures is no longer statistically
distinguishable from the background We used the M. tuberculosis genome as a test for our
algorithm. Surprisingly, although there were many secondary structure potential sequences present downstream of genes, most of these were
devoid of a trail of U (Table I). Notably, irrespective of whether the
structures are followed by a U trail or not, they are concentrated
approximately within 50 nucleotides downstream of the stop codon (Fig.
1). Thus, both classes of structures appear to have evolved for a
conserved function in transcription termination. Interestingly, even in
E. coli, a significant fraction of the structures lack a
discernible U trail. In agreement with this, we have experimentally
shown that the U trail does not play a primary role in transcription
termination both inside the mycobacterial cell (Fig. 3) and in
vitro with purified mycobacterial RNA polymerase (Fig. 4). On the
other hand, the significance of the trail sequence in E. coli is not completely clear. In different systems, the U trail
has been shown to be either essential (10, 17), unnecessary (36), or
necessary only in the absence of appropriate elements downstream of the
termination site (19).
The efficiency of termination is believed to be determined by the
opposing influences of the rates of elongation and release (1, 37).
Recently, a paused form of the polymerase that reacts slowly with the
nucleotides has been proposed as an intermediate prior to the actual
step of release (38, 39). Revised models based on single molecule
experiments evoke kinetic competition between elongation rates and the
largely irreversible formation of the paused complex rather than the
actual step of release (39). Most bacterial coding sequences have
evolved to favor the former rather than latter reaction.
Terminators represent sequences, which specifically alter one or both
of these reactions leading to transcription termination.
Support for the above models came from the analysis of mutationally
altered polymerases that have a lower elongation rate and show a
concomitant increase in termination (40). Similar results are obtained
using a wild-type polymerase in the presence of limiting concentrations
of nucleotides (7, 40). Furthermore, recent work shows that the primary
role of the U trail may be to decrease the rate of elongation (38) and
thereby allow the hairpin to extrude and dislodge the nascent chain
from the catalytic site. In M. tuberculosis, where the rate
of RNA chain elongation is about 10-fold slower than E. coli
(41), such a role for the U trail would be redundant. Therefore, an
I-shaped structure, even without the stalling effect of the U trail,
could work as efficiently as an L-shaped structure. Thus, in the
framework of the kinetic competition model, a lower elongation rate
would mean that the enhancement required in the rate of pausing/release
to bring about termination would be correspondingly lower. An
alternative explanation for the low representation of the L-shaped
structures in M. tuberculosis could be the high G/C content
of the organism. However, M. leprae, an organism closely
related to M. tuberculosis, shows a similar preference for
I-shaped structures although it has a considerably lower G/C content.
In addition, we find no simple relationship between the G/C content of
an organism and its preference for one or the other type of structure.
On the other hand, our hypothesis predicts a correlation between the prevalence of the I-shaped structure with a lower rate of RNA chain
elongation. In agreement with this prediction, when the E. coli RNA polymerase itself is made to move slowly in the presence of limiting amounts of nucleotides, it terminates efficiently even in
the absence of a U-trail (7). Thus, our results substantiate the model
of kinetic competition between the rates of elongation and termination
(1, 39).
Another point of interest is that we found that the cells are protected
against premature termination at structures within the coding region by
the translating ribosomes (Fig. 4). This mechanism would not be
operational in tRNA and rRNA genes. These two classes of highly
transcribed genes are known to have extensive secondary structure in
their RNA without the protective influence of translation. Therefore, a
terminator structure within the coding region of such genes would be
disastrous to the cell. Significantly, the algorithm does not identify
putative terminators in the coding regions of these genes, implying
that the identified structures are genuine terminators.
We thank Anil K. Tyagi for pSD7, Narasimha
Prakash for suggestions and discussion, and M. Chatterji for technical
assistance, discussion, and critical reading of the manuscript.
*
The research was supported by grants from the Indian Council
of Medical Research, Government of India.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of an Indian Academy of Sciences fellowship.
Published, JBC Papers in Press, September 10, 2001, DOI 10.1074/jbc.M106252200
The abbreviations used are:
bp, base pair(s);
kb, kilobase(s);
PCR, polymerase chain reaction;
CAT, chloramphenicol
acetyltransferase;
TE, termination efficiency.
Alternate Paradigm for Intrinsic Transcription Termination in
Eubacteria*,
,§, and¶
Department of Microbiology and Cell Biology,
Indian Institute of Science, Bangalore 560012, India and
¶ Jawaharlal Nehru Centre for Advanced Scientific Research,
Bangalore 560064, India
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 to +270 nucleotides
of the stop codon) without entering adjacent coding regions. The search
is initiated at a G/C-rich (>50%) tetranucleotide, and a reverse
complementary match is sought within the next 70 nucleotides. This
defines the base of the stem, and the match is extended inward. Once a
mismatch is encountered, all possible structures are computed allowing
for different combinations of mismatches and gaps. The 
G
of formation of each of these structures was computed using the
parameters from Turner et al. (22) and Jaeger et
al. (23). Among all these structures, the one with the lowest
G was retained. Then the program moved to the next G/C-rich tetranucleotide and reinitiated the search.
G
filter based on the G/C content of the bacteria. The species-specific
Gcutoff was set at 0.230 × (%GC) + 3.44 based on two premises. Firstly, the basal G of the downstream region (20) is strongly
correlated with the G/C content of the genome. Secondly, the algorithm
should identify preferentially structures in the downstream rather than in the upstream region. The optimized cutoff value for G
was derived by iteratively weighing down
Gdownstream so as to maximize the likelihood
of identifying only downstream and not upstream structures. Under these
constraints, the algorithm detects 10-fold or more structures in the
downstream region compared with the upstream region. Furthermore, the
distribution with respect to the stop codon shows a characteristic peak
indicating a non-randomness in the distribution of structures. To
further substantiate the statistical significance of this peak, a
t test was performed to compare the average around the peak
and that of a region that shows a background level of occurrence of
structures 190-200 nucleotides downstream of the stop codon. The
p value of the t tests for each genome is listed
in Table I.
G value. The program is available on request from the authors.
.
-32P]UTP) and
shifting to 37 °C. After 1 min, the reaction was supplemented with
UTP (final concentration of 100 µM) and heparin (final
concentration of 150 µg/ml). Reactions were stopped by the addition
of equal volumes of formamide containing 0.025% bromphenol blue and
0.025% xylene cyanol and resolved on an 8% denaturing polyacrylamide gel. The reactions were visualized and quantitated by phosphorimaging (Fujifilm). The termination efficiency (TE) was calculated as follows.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 to +270 nucleotides around the
stop codon for each gene were searched, without entering adjacent
coding regions. In the case of overlapping structures, the one with the
lower G was retained. Finally, structures were filtered
using a minimal G requirement based on the G/C content of
the genome. With these parameters, the algorithm identified more than
90% of all experimentally shown terminators in different bacteria
(29-32, Supplemental Material). Structures were classified as follows:
(a) E. coli type/L-shaped, those with >3 U
residues present in the 10 nucleotides trailing the structure; (b)
Mycobacterium type/I-shaped, those with 3 or fewer U
residues in the trail; (c) V-shaped, structures that are
immediately followed (or preceded) by another structure; (d)
Tandem/U-shaped, multiple structures that are present downstream of a
single gene; and (e) Convergent/X-shaped, structures present
between adjacent convergently oriented genes. It should be noted that
all structures, other than the L-shaped ones, are symmetric and could
potentially work in either orientation.
Representative whole genome analysis of bacterial sequences
View larger version (27K):
[in a new window]
Fig. 1.
Distribution of terminators in bacterial
genomes. The distribution of all classes of terminators with the
stop codon in E. coli (A) and M. tuberculosis (B). The distribution of L- and I-shaped
terminators is also shown.
View larger version (25K):
[in a new window]
Fig. 2.
The termination selection vector series
(pTER). These vectors are E. coli-mycobacteria shuttle
vectors and have a unique BamHI site between the promoter
and the CAT reporter gene. The origins of replication for mycobacteria
(oriM) and E. coli (oriE) are indicated. The M. smegmatis gyr promoter is shown as an arrow. Upstream
of the reporter system, there are stop codons in all frames.
View larger version (31K):
[in a new window]
Fig. 3.
Different classes of terminators in
mycobacteria. Representative terminators with (A) or
without (B) an A/U trail or present in tandem (C)
identified by the algorithm are shown along with a representative CAT
activity indicating a decrease in read-through transcription. All
terminators were tested in pTER5 (see "Experimental Procedures" and
Fig. 2). D, summary of the analysis of read-through
transcription. Each value is an average of at least three independent
experiments.
and pTER7 in Fig. 4). Thus,
terminators appear to be effective only in the non-coding region. The
close coupling of transcription and translation in bacteria probably
prevents the extrusion of these structures in the RNA in the coding
region.
View larger version (24K):
[in a new window]
Fig. 4.
Effect of distance from the promoter and
translation on termination efficiency. The terminator from Fig.
2A was placed at different distances from the promoter, 27
bp (pTER1), 77 bp (pTER5), and 1.1 kb (pTER7) downstream of the
transcription start site. Representative CAT assays and the means
obtained from at least three independent experiments is shown. The
promoter (arrow), terminator (filled box), and
translated regions (hatched box) are indicated.
View larger version (24K):
[in a new window]
Fig. 5.
In vitro termination assay.
A, schematic representation for the assay. Runoff
transcription assays were performed with constructs harboring the
terminators shown in Fig. 2, in either orientation. The terminators
used in the assay are as follows, tuf terminator
(B), Rv1324 terminator (C), and the
gyrA terminator (D). The positions of full-length
runoff and terminated (Term. Prod.) products are indicated.
A sequencing ladder was used as molecular mass marker.
Termination efficiency of various structures in vitro
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G. In contrast, the
present algorithm varies the window size dynamically to specifically
identify individual stem-loop structures, thereby improving the
sensitivity and accuracy of the prediction.
ACKNOWLEDGEMENTS
FOOTNOTES
The on-line version of this article (available at
http://www.jbc.org) contains a list of terminator
sequences used to optimize the algorithm.
To whom correspondence should be addressed. Tel.:
91-80-360-0668; Fax: 91-80-360-2697; E-mail:
vraj@mcbl.iisc.ernet.in.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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