(Received for publication, May 11, 1995)
From the
In vitro transcription systems are a classic means to
dissect mechanisms of gene expression at the molecular level. To begin
an analysis of the biochemistry of gene expression in trypanosomes, we
established an in vitro transcription system from cultured
insect forms of Trypanosoma brucei. As a model we used the U2
snRNA gene which in vivo is transcribed by an RNA polymerase
with characteristics of animal RNA polymerase III. To obtain maximum
sensitivity in our assay, we adapted the so-called G-less cassette
approach to the U2 snRNA gene promoter. Since an intragenic control
region is required for accurate expression in vivo, we
generated a series of mutations to substitute all guanosine residues in
the intragenic control region. These mutants were shown to retain full
transcriptional activity in vivo after transient expression in
insect-form trypanosomes. In a cell-free extract, synthesis of the U2
G-less cassette RNA is correctly initiated, is mediated by RNA
polymerase III as determined by RNA polymerase inhibitor studies, and
is dependent on the integrity of the upstream B box element.
Studies of transcription in the protozoan family
Trypanosomatidae have revealed unusual features such as polycistronic
transcription units and RNA polymerase I-mediated transcription of
protein-encoding genes. However, compared to what we know about the
transcriptional machinery in a number of different organisms, including
yeast and higher eukaryotes, our understanding of promoter structures
and trans-acting factors in trypanosomatid protozoa is
extremely limited. This is in large part due to the fact that only a
few functional promoters have been identified and that no in vitro transcription system is available. To date, RNA polymerase I and
III promoters have been characterized in detail in Trypanosoma
brucei. RNA polymerase I transcription units include the genes
encoding the large ribosomal rRNAs(1, 2) , the variant
surface glycoprotein gene expression sites(3, 4) , and
the procyclic acidic repetitive protein
genes(1, 5, 6) .
The analysis of RNA
polymerase III-mediated transcription in T. brucei has led to
a number of surprising findings. Based on RNA polymerase inhibitor
studies with the drugs
The
characterization of cis-acting elements required for
expression of the U2 and U6 snRNA genes in T. brucei has been
a major focus of our research. In vivo transcription of both
genes is dependent upon the integrity of three regulatory elements (7, 8) .
Although the above studies
have unraveled a novel promoter architecture for the U2 and U6 snRNA
genes, the experiments reported thus far do not provide insight into
the underlying mechanism. Therefore, as a first step toward a detailed
biochemical understanding of the transcriptional apparatus assembling
on these U-snRNA genes, we developed a T. brucei DNA-dependent in vitro transcription system. Using the G-less cassette
approach (11) and a modified Dignam extract(12) , RNA
polymerase III-mediated transcription was shown to initiate at position
+1 of the U2 snRNA gene, and transcription activity was dependent
on the upstream B box element.
The U2 G-less
cassette has the following sequence (sequence of the nontranscribed
strand, +1 indicates the U2 transcription start site):
The cassette was constructed by using two complementary
oligonucleotides. Oligonucleotide U2-GL2 is 111 nt long and
complementary to the G-less sequence listed above. It spans the whole
cassette and has additional CG and CT dinucleotides at its 5` and 3`
end, respectively. Oligonucleotide U2_GL3, 5`-CCACTACATATCTTCTCAAC-3`,
was designed to generate an overhang characteristic for the restriction
enzyme SalI at the 5` end of the sequence shown above. The
oligonucleotides were hybridized to each other, and double-stranded DNA
was prepared by the Klenow-mediated fill-in reaction. The G-less
cassette was cloned into the SalI and NruI
restriction sites of plasmid pTbU2_BS-12/-2b thereby replacing the U2
snRNA coding region. pTbU2_BS-12/-2b is a derivative from mutant
BS-12/-2 (7) and was generated by removing a SalI site
in the vector. Two U2 G-less cassette mutants were obtained. TbU2_GLa
contains the G-less cassette sequence as shown above, whereas TbU2_GLb
lacks three of the five 3`-terminal thymidine residues. Therefore,
transcription of TbU2_GLb is expected to terminate 10 nt downstream at
the putative endogenous termination signal. Standard transcription
reactions were carried out with TbU2_GLb, because this template
reproducibly gave rise to stronger transcription signals. The extra
sequence in TbU2_GLb contains a guanosine residue. Thus, RNase T1
cleavage at this site will trim TBU2_GLb RNA to the same size as RNA
obtained from TbU2_GLa (see Fig. 2B).
Figure 2:
In vivo expression of the U2
G-less cassette gene. A, schematic outline of the U2 G-less
cassette gene. Filled in and open boxes represent
wild-type and G-less sequences, respectively. Transcripts expected from
correct initiation (97 base pairs) and nonspecific readthrough
transcription (106 base pairs) are indicated. GLESS_PE denotes
the oligonucleotide complementary to the tag at the 3` end of the
G-less cassette. B, 3`-terminal sequences of mutants TbU2_GLa
and TbU2_GLb. C and D, total RNA was isolated from
transfected T. brucei cells and subjected to Northern blot (C) and primer extension (D) analyses with
oligonucleotide GLESS_PE. Cells transfected with vector DNA served as a
control (lane 1). Lane 2, pTBU2_GLa; lane 3,
pTbU2_GLb; M, MspI-digested pBR322
marker.
In transcription reactions without radioactive
labeled UTP, the unlabeled UTP concentration was raised to 0.8
mM; in reactions without 3`-OMeGTP, GTP was added to a final
concentration of 0.8 mM. For the time course experiments, the
standard reaction was scaled up 10 times, and 25-µl aliquots were
removed at specified time intervals.
Figure 1:
In vivo expression of mutant
U2 genes containing G replacements in their intragenic control region. A, schematic outline of the tagged U2 snRNA gene. The upstream
A and B box promoter elements and their orientation are
indicated(7) . The mutated guanosine residues in the sequence
of the intragenic control region (ICR) are printed in bold and marked by arrows (+1 marks the transcription
start site). B, wild-type (WT) and mutated U2 genes
were transiently transfected into T. brucei cells, and total
RNA was analyzed by primer extension with oligonucleotide U2k which is
complementary to the 3` end of U2 snRNA. The letters above each
lane indicate the G replacements within the intragenic control
region, i.e. TCA stands for the mutant in which the guanosine
residues at positions +11, +12, and +20 are replaced by
a thymidine, a cytidine, and an adenosine residue, respectively
(- marks a deletion).
After varying extract preparation procedures, testing different
extract fractions, and trying out different reaction conditions, we
eventually were able to detect a weak signal of the correct size. In
order to improve the transcription efficiency, we varied a number of
components in the incubation mixture. Most dramatically was the
addition of polyethylene glycol, a compound which reduces the reaction
volume and, therefore, can increase transcription efficiencies. We
found that polyethylene glycol at a final concentration of 3% resulted
in an approximately 20-fold increase of the specific transcription
signal (data not shown). In vitro transcription was sensitive
to small changes of the magnesium chloride concentration, and maximal
synthesis occurred at 2.5 mM MgCl
Figure 3:
Magnesium chloride titration of the in
vitro transcription reaction. A, in vitro transcription
reactions were carried out in the presence of 1.0, 2.0, 2.5, 3.0, or
4.0 mM MgCl
Figure 4:
Effects of template DNA, RNase T1,
3`-O-methyl-GTP, and MgCl
Figure 5:
The
5` end of the U2 G-less cassette RNA coincides with the mapped
initiation site of the U2 snRNA gene. In vitro transcription
reactions were carried out in the absence of radiolabeled nucleotides,
and RNA was analyzed by primer extension with 5`-end-labeled
oligonucleotide GLESS_PE (lane pTbU2_GL). In a control
reaction, the vector without the U2 gene was used as template (lane
vector). For comparison, the U2 G-less cassette gene was sequenced
by chain-terminating cycle sequencing with the same end-labeled
oligonucleotide (lanes marked pcr seq.). On the right
side, the double-stranded sequence surrounding the U2
transcription start site is shown. The sequence on the left is
from the transcribed strand and corresponds to the sequencing ladder.
The adenosine residue at position +1 (nontranscribed strand) is
aligned with the sequencing ladder as indicated by the arrow. Marker, MspI-digested
pBR322.
Figure 6:
Kinetic analysis of U2 G-less cassette
gene transcription in vitro. A, after incubation
times of 0, 5, 10, 20, 30, 40, 60, 90, and 120 min, aliquots were taken
from a 250-µl in vitro transcription reaction, and the RNA
was analyzed on a 6% polyacrylamide-50% urea gel. B,
quantitation of the transcription signal. The strongest signal at 90
min was set to 100%.
Figure 7:
In vitro transcription of the U2
G-less cassette gene is mediated by RNA polymerase III and depends on
the upstream B box promoter element. A, in vitro transcription
reactions with radiolabeled UTP were carried out in the presence of 0,
2, or 100 µg/ml
In this study we report the development of the first in
vitro transcription system for the protozoan family
Trypanosomatidae. This goal was achieved by adapting the G-less
cassette approach to the RNA polymerase III-transcribed U2 snRNA gene
of T. brucei. With this in vitro transcription
system we are now in a position to identify and characterize
transcription factors essential for U2 snRNA gene transcription.
We thank Tim Nilsen for valuable suggestions and
Philippe Male for photography.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amanitin and tagetitoxin and on the
architecture of the promoters, the U2, U4, and U-snRNA
(
)B (the U3 snRNA homolog) genes are transcribed by
RNA polymerase III(7, 8) . This is in contrast to most
other eukaryotic organisms studied to date, where these RNAs are
synthesized by RNA polymerase II(9) . The only exception being
the plant U3 snRNA gene which is also transcribed by RNA polymerase III (10) . In particular, studies with tagetitoxin, a specific
inhibitor for RNA polymerase III, showed that transcription inhibition
of the T. brucei U2, U4, and U-snRNA B genes closely followed
that of typical RNA polymerase III genes, namely 5S, 7SL, U6 snRNA, and
tRNAs, but not that of RNA polymerase I or II genes(7) .
(
)Two upstream
elements coincide with divergently oriented A and B boxes, which in the
case of the U6 snRNA gene locus, are part of a functional threonine
tRNA gene. In addition, expression of the U2 and U6 snRNA genes
requires intragenic elements close to the 5` end of the coding region,
which are most likely responsible for positioning the RNA polymerase at
the correct transcription start site.
Plasmid Construction
Plasmids pTbU2_GL-AAA,
pTbU2_GL-AT-, pTbU2_GL-CAA, pTbU2_GL-CAT, pTbU2_GL-TAT, and
pTbU2_GL-TCA were derived from construct pTU283 (7) by
oligonucleotide-directed mutagenesis of single-stranded
template(13) . The oligonucleotide used in this mutagenesis was
U2-GL1, 5`-GCATATCTTCTC(ATC)(ATC)CTATTTA(ATC)CTAAGATC-3` (parentheses
indicate degenerate positions), containing the sequences of the U2
snRNA gene from nucleotides -2 to +28.
Mutant
pTbU2_GL-B52 is a derivative of pTbU2_GLb carrying six point mutations
in the upstream B box element(7) .
DNA Transfection and RNA Analysis
Transfections,
RNA isolation, primer extension analysis, and Northern blot
hybridizations were carried out as described(7, 8) .
The following oligonucleotides were used in primer extension analysis
and/or Northern blot hybridizations: U2k(14) , complementary to
nucleotides 128 to 148 of the U2 snRNA; GLESS_PE,
5`-GAGTGAATGATGATAGATTTG-3`, complementary to nucleotides +75 to
+94 of the U2 G-less cassette gene.
Preparation of Nuclear Extracts
A 2-liter culture
of the procyclic forms of T. brucei rhodesiense strain YTaT
1.1 was grown as described previously (15) to a density of 1
10
cells per ml. Cells were harvested at room
temperature, yielding a packed cell volume of 2 to 2.5 ml. The pellet
was rinsed twice with 10 ml of wash solution (20 mM Tris-HCl,
pH 7.4, 100 mM NaCl, 3 mM MgCl
) and once
with 10 ml of ice-cold transcription buffer (150 mM sucrose,
20 mM potassium L-glutamate (Sigma), 10 mM Hepes-KOH, pH 7.9, 2.5 mM MgCl
, 1 mM
dithiothreitol, 10 µg/ml leupeptin). The following steps were
carried out at 4° C. Cells were finally resuspended in 1.5 times
packed cell volume of transcription buffer, preincubated for 5 minutes,
and broken in a 7-ml dounce with a type A pestle by applying rapid
strokes continuously for about 10 min, until the vast majority of cells
were broken. The resulting cell extract was spun in an Eppendorf
centrifuge at 4,000
g for 10 min, the supernatant was
removed, and the pellet was subjected to another spin for 2 min at
16,000
g. The final pellet was resuspended in an equal
volume of transcription buffer. KCl was added to a final concentration
of 400 mM, and the nuclear fraction was extracted by rotating
the tube at 4° C for 30 min. The extract was spun in an Eppendorf
centrifuge for 10 min at 16,000
g, and the supernatant
was desalted using a Centricon-10 concentrator (Amicon) until the KCl
concentration was around 40 mM. Final extracts ranged in
protein concentration from 3 to 6 mg/ml and were stored at
-70° C. No correlation was detected between protein
concentrations within this range and extract activity.
In Vitro Transcription Reactions
A standard
transcription reaction was carried out in a volume of 25 µl
containing 10 µl of nuclear extract. The reaction further contained
1 µg of circular plasmid DNA (40 µg/ml final concentration), 20
mM potassium L-glutamate, 2.5 mM MgCl, 10 mM Hepes-KOH, pH 7.9, 2 mM
ATP, 0.8 mM CTP, 0.8 mM 3`-O-methyl-GTP
(3`-OMeGTP), 5 µM UTP, 0.26 µM [
-
P]UTP (20 µCi), 20 mM creatine phosphate, 0.48 mg/ml creatine kinase, 3% polyethylene
glycol, 10 units of RNase T1 (Calbiochem), 1 mM dithiothreitol, and 10 µg/ml leupeptin. Unless otherwise
stated, the reaction was conducted at 28° C for 90 min and stopped
by the addition of 400 µl of HES buffer (5 mM Hepes-KOH,
pH 7.9, 5 mM EDTA, 0.5% SDS, 0.3 M NaAc, 5 µg/ml
tRNA). The RNA products were extracted with an equal volume of buffered
phenol/chloroform (1:1), precipitated from the aqueous phase with
ethanol, and analyzed on 6% polyacrylamide/50% urea gels. Following
electrophoresis, the gels were dried and transcription signals were
visualized by autoradiography and quantitated by phosphorimaging
(Molecular Dynamics).
Determination of the Transcription Start Site
A
standard transcription reaction in the absence of radiolabeled
nucleotides was carried out, and the RNA products were purified and
analyzed by primer extension with radiolabeled oligonucleotide
GLESS_PE. With the same oligonucleotide chain-terminating cycle,
sequencing of the plasmid pTBU2_GLb was conducted using the
Taq
cycle sequencing kit (United States Biochemical
Corp.) according to the manufacturer's protocol.
The U2 G-less Cassette Gene
In the past, we
repeatedly attempted to prepare cell-free T. brucei extracts
active in transcription and capable of supporting correct transcription
initiation of tRNA and 5 S rRNA genes. One of the major problems we
encountered was the abundance of nonspecific labeling activities in our
extracts which made it impossible to detect specific transcription
signals. As an alternative strategy, we decided to apply the G-less
cassette approach (11) to the RNA polymerase III-transcribed U2
snRNA gene, since this approach is very efficient in removing
nonspecifically labeled RNAs and has been previously employed to
establish an in vitro transcription system for the human U1
snRNA gene(11) . A G-less cassette gene lacks cytosine residues
in the transcribed strand and thus gives rise to guanosine-less
(G-less) RNAs. Transcription reactions are supplemented with the RNA
chain-terminating nucleotide 3`-OMeGTP to suppress nonspecific
transcription either from cryptic promoters in the template DNA or from
genomic DNA contamination present in the extract and with RNase T1 to
further degrade nonspecific transcripts and to trim RNAs generated by
readthrough transcription (see Fig. 2for a more detailed
description). Since the first 24 nucleotides of the U2 snRNA coding
region are essential for in vivo expression(7) , we
first determined whether the three guanosine residues in this element
could be replaced without affecting transcription efficiency. We
therefore randomly substituted the guanosine residues at position
+11, +12, and +20 relative to the transcription start
site with adenosine, thymidine, and cytidine residues and analyzed the
effect of these mutations in vivo by transient transfection of
trypanosome cells. As shown in Fig. 1, this analysis revealed
that the six different combinations of G replacements tested did not
significantly reduce the transcription efficiency of the U2 snRNA gene.
This result made it possible to construct the G-less U2 snRNA gene
schematically outlined in Fig. 2A. In the U2 snRNA
gene, all Gs were replaced by As from position +1 to +74 of
the RNA sequence. In addition, to prevent possible premature
termination by RNA polymerase III, we disrupted stretches of three Ts
by one T to C transition. To the 3` end of the coding region we
attached a gene-specific tag which included five 3`-terminal thymidine
residues for efficient transcription termination by RNA polymerase III.
Finally, the guanosine residues immediately upstream of the
transcription start site up to position -9 were replaced by
adenosine residues, since we have previously shown that these sequences
are not essential for U2 snRNA gene expression in
vivo(7) . These substitutions will allow us to distinguish
between correct initiation at position +1, which should give rise
to a transcript of 97 nt, and nonspecific readthrough transcription,
which instead will produce a 106-nt-long transcript (Fig. 2A). The final U2 G-less cassette gene was then
tested in vivo by transient DNA transfection. Northern blot
analysis revealed that the transcript is indeed 97 nt long (Fig. 2C, lane U2_GLa). In the process of
cloning the U2 G-less cassette, we isolated a second construct,
TbU2_GLb, which differed only at the very 3` end (Fig. 2B) and produced an approximately 8-nt longer
transcript (Fig. 2C, lane 3), most likely
terminating at a stretch of Ts at positions +157 to +160 of
the U2 snRNA gene(16) . Primer extension with an
oligonucleotide complementary to the tag resulted in a product whose
size is consistent with transcription initiation at position +1 (Fig. 2D). Thus, both U2 G-less cassette genes are
expressed in vivo and initiate at the correct site.
A Cell-free Extract Active in RNA Polymerase III
Transcription
Based on our experience with permeabilized
trypanosome cells and on our previous attempts to prepare
transcriptionally active extracts(17) ,(
)we introduced several modifications to the protocol
of Dignam et al.(12) . Most notably, we replaced
glycerol in all our buffers by sucrose. Extracts containing glycerol
were not able to maintain a stable ATP pool even in the presence of an
ATP regenerating machinery. Next, we broke trypanosome cells by
douncing in an isotonic solution. Douncing of T. brucei cells
in hypotonic conditions results in substantial leakage of nuclear
contents into the cytoplasmic fraction (16, data not shown). Finally,
potassium chloride was substituted with potassium glutamate in all
buffers, because transcription in permeabilized cells is not inhibited
by a wide range of salt concentrations when the transcription buffer
contained potassium glutamate (data not shown, see also (18) ).
(Fig. 3).
Changing the potassium glutamate concentration from 10 to 75 mM did not significantly affect transcription of the U2 G-less
cassette gene, whereas a further increase to 100 mM reduced
the signal by 70%. However, in agreement with data obtained with the
permeabilized cell system(17) , KCl concentrations above 30
mM reduced the transcription signal by more than 80% (data not
shown). Specific transcription was dependent upon addition of exogenous
DNA, and the strongest signal was obtained at a concentration of 40
µg/ml (data not shown). Finally, we determined that adding 5
µM unlabeled UTP enhanced the transcription signal by
about one-third (data not shown). Taken together, these optimizations
increased the transcription reaction efficiency more than a 100-fold.
, and the RNA was analyzed on a 6%
polyacrylamide-50% urea gel. The appearance of a second band just above
the specific product is most likely due to variable 3` ends of the U2
G-less cassette RNA since analysis of the 5` end by primer extension
revealed only one signal of the correct size. M, MspI-digested pBR322 marker. B, histogram showing the
relative strengths of the specific transcription signals. The strongest
signal at 2.5 mM MgCl
was set to
100%.
Fig. 4shows an array of reaction conditions that illustrate
the effects of the addition of template, 3`-OMeGTP, and RNase T1. The
control reaction lacking all three components gives rise to bands
superimposed on a smear on the autoradiograph (lane 1). The
generation of these RNA molecules may be due to end-labeling of RNAs or
to endogenous transcription from chromatin present in the extract.
Although the effect of template addition is hard to detect because of
the high background, a faint band of 105 nt, which is not present in
the control reaction, can be seen when TbU2_GLb was added (lane
2). RNase T1 removes the labeling background by degrading all but
the G-less cassette RNAs (lane 3). The two remaining bands
correspond to correctly initiated (97 nt) and readthrough transcripts
(106 nt). The further addition of the chain terminator 3`-OMeGTP
blocked nonspecific readthrough transcription and enhanced the specific
product (lane 4). Finally, high magnesium concentrations
prevent correct transcription initiation and enhance nonspecific
readthrough transcription, so that the 106-nt product becomes
detectable even in the presence of 3`-OMeGTP (lane 5).
on the in vitro transcription reaction. In vitro transcription reactions
were carried out in the presence of
-
P-labeled UTP,
and RNA products were analyzed on a 6% polyacrylamide-50% urea gel. DNA
template, RNase T1, 3`-O-methyl-GTP, and MgCl
were
added as indicated above the lanes. M, MspI-digested
pBR322.
Accurate Transcription Initiation
To determine the
transcription start site, the in vitro transcription reaction
was conducted in the absence of labeled UTP, and total RNA was
subjected to primer extension analysis with an oligonucleotide
complementary to the U2 G-less cassette tag. As shown in Fig. 5,
this analysis gave rise to one major band corresponding to correct
initiation at position +1(16) .
Kinetic of RNA Synthesis
Fig. 6shows a
time course of the in vitro transcription reaction. There is a
distinct lag phase of about 10 min after which a faint signal can be
detected on a long exposure. The lag phase most likely reflects the
time needed to assemble transcription initiation complexes. A linear
increase then occurs between 20 and 90 min, when a plateau is reached.
RNA Polymerase III Transcription
Studies with
permeabilized T. brucei cells showed that a concentration of 2
µg/ml -amanitin had only a moderate effect on transcription of
the U2 snRNA gene by RNA polymerase III, whereas 100 µg/ml
-amanitin inhibited U2 snRNA synthesis by more than
90%(7) . In the in vitro system,
-amanitin
concentration of 2 µg/ml did not significantly affect transcription
of the U2 G-less cassette gene (an average decrease of 13%), but a
concentration of 100 µg/ml reduced transcription to undetectable
levels (Fig. 7, lanes 2-4). To verify this
result, we used the RNA polymerase III-specific inhibitor
tagetitoxin(19) . Similar to what was observed in permeabilized
cells(7) , tagetitoxin concentrations of 15 µM and
60 µM inhibited in vitro transcription of the U2
snRNA gene by 52% and 75%, respectively (Fig. 7, lanes
5-7). Hence, we conclude that in vitro transcription of the U2 G-less cassette gene is mediated by RNA
polymerase III.
-amanitin (lanes 2-4). In a
second set of reactions, the RNA polymerase III-specific inhibitor
tagetitoxin was added to final concentrations of 0, 15, and 60
µM (lanes 5-7). Lane 1, the vector
without U2 gene sequences served as template. Lane 8, the U2
G-less cassette template was replaced by a mutant which carried six
point mutations in the upstream B box promoter element. The RNAs were
analyzed on a 6% polyacrylamide-50% urea gel. B, quantitation
results of the transcription signals from three independent experiments
with
-amanitin. In each case, the signal of the control reaction
was set to 100%. C, quantitation results of two experiments
with tagetitoxin.
Effect of B Box Mutations
To further test the
validity of the in vitro system, we examined the effect of
mutations in a known regulatory element of the U2 snRNA gene. Similar
to what was observed in vivo(7) , six point mutations
within the upstream B box promoter element completely abolished U2
transcription in vitro (Fig. 7, lane 8),
further indicating the close resemblance of the in vitro and in vivo systems.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.