From the Service de Biochimie et de Génétique Moléculaire, CEA/Saclay, F-91191 Gif-sur-Yvette Cedex, France
Received for publication, December 9, 2002, and in revised form, January 16, 2003
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ABSTRACT |
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The yeast transcription factor IIIC (TFIIIC) is
organized in two distinct multisubunit domains, TFIIIC1 is a
multisubunit DNA binding factor that serves as a dynamic platform to
assemble pre-initiation complexes on class III genes. Once bound to
tDNA intragenic promoter elements, TFIIIC directs the assembly of
TFIIIB on the DNA, which in turns recruits the RNA polymerase III (pol
III) and activates multiple rounds of transcription (1).
Biochemical and genetic studies have yielded a detailed
characterization of Saccharomyces cerevisiae TFIIIC factor.
Yeast TFIIIC consists of six subunits (named The The In this work, we pursued the characterization of the DNA Constructions and TFC1 Random Mutagenesis--
The 2.8-kb
XhoI/NotI fragment from plasmid YcpCS7 (CEN, URA,
wt HA-TFC1) (27), containing the promoter region and a
modified open reading frame of TFC1 allowing addition of an
HA tag sequence after the initiation codon, was cloned into the
polylinker region of plasmid pRS314 (28) creating pYSJ1 (CEN, TRP, wt
HA-TFC1).
Two oligonucleotides SJt95-5 (5'-AGTTGTTTGTTCATGATCTT) and SJt95-7
(5'-ATATTTATGTCCCCATCAGC) were used to amplify an 811-bp DNA fragment
from bp 806 to 1616 of the TFC1 open reading frame under
mild mutagenic PCR conditions (3 mM MgCl2).
Gapped pYSJ1 containing extremities homologous to both ends of the PCR
products was produced by an NruI-NcoI digestion
followed by agarose purification and was cotransformed with the PCR
products into YCS7 haploid strain (ade2-101 ura3-52
lys2-801 his3- TFIIIC Purification--
TFIIIC was purified starting from ~15
g of YSJ1 or mutant YSJ1-E447K cells following a two-step
chromatographic procedure (heparin HyperD followed by MonoQ
purification) as described previously (10). Both Western blot analysis
with anti- DNA Binding and in Vitro Transcription Assays--
TFIIIC·DNA
interactions were monitored by gel shift assays as described previously
(13). A 32P-labeled DNA fragment (3-10 fmol; 4,000-10,000
cpm) carrying the SUP4 tRNATyr (345 bp) gene was
incubated for 10 min at 25 °C in a reaction mixture containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10% glycerol, 2 mg/ml bovine serum albumin (Sigma), DNA competitor, TFIIIC (MonoQ fraction) at a final KCl concentration of 120 mM. To
analyze
The apparent dissociation constants (Kapp) of
wild type and mutant TFIIIC-DNA complexes were determined, using the
above binding conditions as described (4, 29).
Kapp was derived from Equation 1,
To assemble TFIIIB·TFIIIC·DNA (B·C·DNA) complexes,
Ultrogel-heparin TFIIIB fraction (27) was added to the TFIIIC·DNA
binding reaction described above together with additional DNA
competitor and further incubated for 30 min at 25 °C. The final KCl
concentration was adjusted to 120 mM. To form TFIIIB·DNA
complexes, TFIIIC was stripped by addition of 500 ng of heparin (5 min
at 25 °C) or by raising the incubation temperature for 5 min at
50 °C. To form pol III·TFIIIB·DNA complexes, highly purified RNA
polymerase III (100 ng) was added to a heat-treated TFIIIB·DNA
complex and further incubated for 45 min at 25 °C. Protein·DNA
complexes were analyzed by non-denaturing electrophoresis on 5%
polyacrylamide gel.
Standard in vitro transcription assays were performed as
described previously (10, 27) using Ultrogel-heparin-purified TFIIIB,
MonoQ-purified TFIIIC, and highly purified RNA polymerase III (100 ng).
The final KCl concentration was 100 mM. Plasmid templates
harbored either the SUP4 tRNATyr gene or a
TATA-containing yeast tRNAAsp(GTC) gene (30). Transcription
reactions were allowed to proceed for 45 min at 25 °C, and
transcripts were analyzed by electrophoresis on 6% polyacrylamide gel
under denaturing conditions (8 M urea).
Primer Extension Analysis--
Transcripts were produced by
in vitro specific transcription as described above using 0.6 mM each ATP, CTP, GTP, and UTP and 150 ng of plasmid
template carrying SUP4 tRNATyr gene,
precipitated with ammonium acetate and 10 µg of carrier Escherichia coli tRNA (Sigma), and resuspended in 16 µl of
diethyl pyrocarbonate, water. 8 µl were then mixed with 8 units of RNasin (Amersham Biosciences), 0.5 pmol of an internal 5'
end-labeled oligonucleotide primer (5'-GTGATAAATTAAAGTCTTGCGCCTTAAACC,
complementary to the coding region of SUP4
tRNATyr gene between positions +29 and +59), and NaCl to a
final concentration of 0.3 M. After DNA denaturation for 5 min at 85 °C, primer annealing was allowed to proceed for 90 min at
40 °C. Reaction mixtures were then adjusted to a final volume of 80 µl and contained 50 mM Tris-HCl (pH 8), 50 mM
KCl, 10 mM MgCl2, 5 mM spermidine,
10 mM dithiothreitol, 1 mM of each dNTP
(Invitrogen), 40 units of RNasin (Amersham Biosciences). Addition of 10 units of avian myeloblastosis virus-reverse transcriptase (Promega)
allowed primer extension to proceed for 90 min at 42 °C. Resulting
cDNAs were precipitated with sodium acetate/ethanol. Pellets were
resuspended in 10 µl of denaturing loading buffer (90% (v/v)
formamide, 10 mM Tris-HCl (pH 8), 1 mM EDTA,
bromphenol blue, and xylene cyanol), heated for 5 min at 90 °C, and
loaded onto a 7% (w/v) polyacrylamide-urea sequencing gel in parallel
with 32P-labeled DNA kb ladder (Invitrogen).
Sequence Homology Searches--
S. cerevisiae
Immunoprecipitation Experiments--
Cell extracts were prepared
as described (35). Ascitic fluid (0.5 µl) containing mouse monoclonal
12CA5 anti-HA antibody was incubated for 30 min at 10 °C with 20 µl of magnetic beads coated with human monoclonal antibodies directed
against mouse immunoglobulin G and Dynabeads Pan Mouse IgG from Dynal
(8 × 108 beads/ml, in phosphate-buffered saline
containing 0.1% bovine serum albumin). After extensive washing first
in phosphate-buffered saline containing 0.1% bovine serum albumin and
then in phosphate-buffered saline, 20% glycerol, 0.05% Nonidet P-40,
the beads were incubated for 180 min with gentle shaking at 10 °C
with cell crude extracts (50 µl, 120 µg of proteins) from cells
expressing or not expressing HA-tagged
Next, we investigated whether both forms of stripped complexes were
able to recruit RNA polymerase III using gel shift assays (Fig.
2B). Control B·DNA complexes formed by heat treatment at 50 °C (lane 3) were able to recruit pol III (at 25 °C)
to form a supershifted complex (lane 4) migrating
approximately like B·C·DNA complexes (lane 2).
Similarly, the two forms of complexes obtained by stripping mutant
TFIIIC by heat treatment were both supershifted upon incubation with
pol III suggesting that both complexes were able to recruit the RNA
polymerase, although the slow-migrating complex appeared somewhat less
efficient (lanes 7 and 8).
The above results prompted us to compare wild type and mutant TFIIIC
for their ability to direct specific transcription in vitro.
TFIIIC, TFIIIB, and purified pol III fractions were used to transcribe
the SUP4 tDNATyr or the tRNAAsp(GTC)
gene that harbors a TATA element at
A primer extension analysis of in vitro synthesized
SUP4 tRNA is shown in Fig. 4.
Whereas wild type TFIIIC directed essentially RNA chain initiation at
position +1, chain initiation directed by mutant TFIIIC was more
polydisperse, with a major start site at position +1 and additional
start sites at positions +4 and +8 and to a lesser extent at +13 and
+15. As shown a decade ago by Geiduschek and colleagues (42), TFIIIB is
the main factor responsible for initiation by pol III. Accurate
initiation depends on the proper placement of TFIIIB on the DNA,
placement that depends on TFIIIC and TBP even on genes deprived of a
canonical TATA box like SUP4 tDNA (7). Therefore, the
present results suggested that mutant TFIIIC had misplaced a proportion
of TFIIIB·DNA complexes resulting in shorter transcripts.
Upstream Border of TFIIIC·DNA Complexes--
Site-specific
DNA-protein photocross-linking and protein-protein interaction studies
implicated
The apparent dissociation constant (Kapp) of
TFIIIC·DNA complexes was determined by titrating a constant amount
(as determined by Western blot) of wild type or mutant TFIIIC with
increasing concentrations of a SUP4 DNA probe at permissive
temperature (25 °C), under optimal binding conditions (4). The
concentration of TFIIIC·DNA complex [TFIIIC·DNA] and free DNA
probe [DNA] was determined using the gel shift assay (Fig.
6). When the SUP4 tRNATyr
binding data were plotted for mutant TFIIIC and wild type TFIIIC (Fig.
6, A and B), good fits to the respective
theoretical curves were obtained (see "Experimental Procedures").
The linearity in the Scatchard representation (Fig. 6B)
suggested the presence of a single binding component in the wild type
and mutant TFIIIC fractions. The apparent dissociation constant and
concentration of active TFIIIC in the binding assay
[TFIIIC0] were determined by fitting the data to Equation 2 (see "Experimental Procedures") (4, 29) by linear regression. The
slope of the curve (Fig. 6B) yielded a value for
Kapp of
Next, we investigated the stability of mutant TFIIIC·DNA complexes.
TFIIIC fractions were preincubated at varying temperatures, for 10 min,
before complex formation with the SUP4 DNA probe for 10 min
at 25 °C. TFIIIC·DNA complexes were then analyzed by gel shift
assays (Fig. 7A). Wild type
TFIIIC was resistant to heat treatments at 35-40 °C and retained
most of its DNA binding activity (Fig. 7A, upper
panel, lanes 3 and 4, see also
lane 13). In contrast, mutant TFIIIC lost much of its DNA
binding activity upon incubation at 35 °C and was totally
inactivated at 40 °C (Fig. 7A, lower panel, lanes 3 and 4). In other experiments
(Fig. 7B), factor·DNA complexes were preformed at 25 °C
then subjected to heat treatments at different temperatures before gel
electrophoresis. Preformed TFIIIC·DNA complexes were more resistant
to heat denaturation, but again the stability of mutant TFIIIC·DNA
complexes decreased drastically at 40 °C (Fig. 7B,
lower panel, lane 10), a temperature that did not
affect control TFIIIC·DNA complexes that were resistant to heat
treatment up to 45 °C (Fig. 7B, upper panel,
lane 11).
It was unexpected to observe such a thermolability for TFIIIC·DNA
complexes mutated in a In this work, we show that the A and
B, that are
respectively responsible for TFIIIB assembly and stable anchoring of
TFIIIC on the B block of tRNA genes. Surprisingly, we found that the removal of
A by mild proteolysis stabilizes the residual
B·DNA complexes at high temperatures. Focusing on the well conserved
95 subunit that belongs to the
A domain, we found that
the
95-E447K mutation has long distance effects on the
stability of TFIIIC·DNA complexes and start site selection. Mutant
TFIIIC·DNA complexes presented a shift in their 5' border, generated
slow-migrating TFIIIB·DNA complexes upon stripping TFIIIC by heparin
or heat treatment, and allowed initiation at downstream sites. In
addition, mutant TFIIIC·DNA complexes were highly unstable at high
temperatures. Coimmunoprecipitation experiments indicated that
95 participates in the interconnection of
A with
B
via its contacts with
138 and
91 polypeptides. The
results suggest that
95 serves as a scaffold critical for
A·DNA spatial configuration and
B·DNA stability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
138,
131,
95,
91,
60, and
55)
distributed between two globular domains named
A and
B based on
their ability to bind the internal tDNA promoter elements, the A and B
blocks, respectively (2, 3).
B·block B binding is predominant over
the low affinity
A·block A interaction (4, 5). The link between
the
A and
B domains displays a remarkable flexibility as it can
stretch across a range of distances separating the A and B blocks in
natural tRNA genes, regardless of their helical phasing (6). The
A
domain is essential for transcription activation and start site
selection as it mediates TFIIIB assembly, in cooperation with the
TATA-binding protein (TBP), strongly directing its DNA binding position
upstream of the transcribed region (6, 7).
B subassembly is composed of
138,
91, and
60 (8-10). Protein-DNA cross-linking analyses of
TFIIIC·DNA complex have localized the
138 subunit over the B block
and
91 over the transcriptional terminator further
downstream (11, 12). Both subunits cooperate in DNA binding. A point
mutation in the TFC3 gene encoding
138 was found to
severely alter TFIIIC·DNA complex stability and to be suppressed by
an amino acid substitution in
91 (9, 13). The
B domain
also contains
60, which does not seem to be involved in DNA
binding. Instead, in vivo and in vitro analysis
of a mutant form of TFIIIC provided evidence in favor of a functional
interaction of
60 with TBP. This result suggested that
60 could participate in TFIIIB recruitment, despite its
downstream localization, and possibly link the
A and
B domains
(10).
A domain contains the three other TFIIIC subunits,
131,
95, and
55 (11, 14-17). Genetic and
biochemical evidence point to the central role of
131 in TFIIIB
assembly on the DNA.
131 interacts with Brf1 (18, 19) and with Bdp1,
previously known as B" or TFIIIB90 (20), and extends far upstream
within the TFIIIB binding region (11). Several lines of evidence
indicate that recruitment of TFIIIB is a complex process that involves important conformational changes of
131 (as well as TFIIIB
components). First, the efficiency of
131 photocross-linking to DNA
was found to vary during the TFIIIB assembly process (21).
Second, two-hybrid experiments with internal deletion mutants
also suggested that
131 flips between different states that expose
or mask TFIIIB-binding sites (18). Third, several dominant
mutations in the N-terminal part of
131 increased TFIIIB recruitment
(22, 23). Finally, circular dichroism spectra analysis directly
revealed a conformation change following
131·Brf1 interaction that
may concern both proteins (24). In contrast, little is known about the
function of
95 and
55. Both polypeptides can be
found in a subcomplex potentially containing other proteins (17). In
the TFIIIC·DNA complex,
95 and
55 are both
located over the A block (11). The 95-kDa subunit is very conserved
through evolution and is thought to be responsible for the
A·block
A interaction (8, 11, 25, 26).
A domain to get
further insight into TFIIIC function. The results indicate that
95 holds a key position in TFIIIC exerting both upstream and downstream influence on the TFIIIC·DNA complex via its
interactions with
A and
B components.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
200 trp1-
1 tfc1-
::HIS3 YcpCS7) (27). Transformants containing recombinant gap-repaired plasmids were plated on 5-fluoro-orotic acid media at 24 °C to chase
the wild type copy of TFC1 gene harbored on YcpCS7.
Thermosensitive mutants were isolated by comparing growth at 30, 34, and 37 °C. One mutant showing slow growth at 34 °C and no growth
at 37 °C was selected and analyzed by sequencing. The mutated
tfc1 gene harbored two point mutations, one neutral (G888T)
and another (G1339A) causing the substitution of Glu to Lys at
position 447. The strain containing this mutated tfc1 gene
was named YSJ1-E447K. The control strain, YSJ1, contains wild type
HA-tagged TFC1 harbored on plasmid pYSJ1.
55 and anti-HA antibodies and gel retardation
experiments showed that mutant TFIIIC fractions contained three times
less factor than wild type fractions.
B·tDNA interaction, TFIIIC·tDNA complexes were subjected
to limited proteolysis for 10 min at 25 °C with 40 ng of
-chymotrypsin (Sigma), and digestion was stopped by addition of 40 ng of aprotinin (Sigma).
where [TFIIIC0], [DNA], and [TFIIIC·DNA]
indicate, respectively, the concentration of active TFIIIC protein and
the free and complexed SUP4 tRNATyr gene.
Equation 1 describes a hyperbola with [TFIIIC0] as the asymptotic value. Kapp was determined using the
Scatchard representation shown in Equation 2,
(Eq. 1)
The slope of the plot gives the apparent dissociation constant
Kapp, and the y intercept yields
TFIIIC concentration [TFIIIC0] in the assay.
(Eq. 2)
Exonuclease Digestion--
TFIIIC·DNA complex was
assembled on a NotI-XhoI DNA fragment (307 bp)
from pRS316-SUP4 tRNATyr, labeled by filling in
the XhoI site (3' end of the non-coding strand) with 0.2 mM of each dATP, dGTP, dTTP, [
-32P]dCTP
(20 µCi), and the Klenow fragment of E. coli DNA
polymerase (Amersham Biosciences, 5 units) with appropriate buffer. The
TFIIIC·DNA binding reaction (15 µl) was adjusted to 50 µl to
reach the following conditions: 40 mM Tris-HCl (pH 8), 0.3 mM EDTA, 0.6 mg/ml of bovine serum albumin, 10% glycerol,
and 20 mM MgCl2. Digestion was started with
addition of 1 unit of
exonuclease (Amersham Biosciences), followed
by a 1-min incubation at 25 °C, and stopped by heating samples at
80 °C for 10 min. DNA fragments were precipitated with sodium
acetate/ethanol and 20 µg of glycogen as a carrier. Pellets were
resuspended in 10 µl of denaturing loading buffer and heated for 5 min at 90 °C, and samples were loaded on a 7% (w/v)
polyacrylamide-urea sequencing gel. To map the 5' border site, a G + A
sequence reaction was performed on the same labeled DNA fragment
following Maxam and Gilbert procedure (31).
95 protein sequence (GenBankTM accession
number A39711) was searched against several data bases using the BLAST
(32) server at NCBI (similar proteins were exhaustively searched
against the same data bases until all similar proteins were
identified). Protein sequences were aligned using Clustal X (33) based
on the BLOSUM 62 scoring matrix (34). Complete
95-like
proteins were identified in Homo sapiens
(GenBankTM accession number NP_036219);
Drosophila melanogaster (GenBankTM
accession AAF53767); Caenorhabditis elegans
(GenBankTM accession CAA84675); Arabidopsis
thaliana (GenBankTM accession AAG52180);
Schizosaccharomyces pombe (GenBankTM
accession CAB11095); Neurospora crassa
(GenBankTM accession CAC28811); and Candida
albicans (unfinished sequence obtained at the Stanford Genome
Technology Center).
95, FLAG-tagged
138, or
91. The beads were washed twice with 200 µl of
phosphate-buffered saline, 20% glycerol, 0.05% Nonidet P-40.
Immunopurified proteins were eluted by heating for 3 min at 95 °C in
SDS loading buffer and then analyzed by SDS-PAGE on a 8%
polyacrylamide gel and revealed by Western blotting with 12CA5 anti-HA,
M2 anti-FLAG (Sigma), or anti-
-91 antibodies, using an Amersham
enhanced chemiluminescence kit.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
95 Subunit of TFIIIC Is Highly Conserved--
The
strong conservation of
95 sequence from yeast to human is
illustrated in Fig. 1. In addition to
human TFIIIC63 (25), six additional protein sequences found in data
bases presented significant homology with S. cerevisiae
95. As shown in Fig. 1, six regions of sequence similarity
including an acidic tail are colinearly conserved in each of the eight
protein sequences. The role of the acidic tail is unknown, but it has
been shown recently (36) that this domain is not required for yeast
cells viability. The central region D (bordered by amino-acids 317-392 in
95 sequence) was the most remarkably conserved. When
comparing the yeast and human pol III transcription system, it appears
that the most conserved components are those engaged in protein-protein interactions critical for transcription complex assembly, as is the
case for
131, for example (37, 38). Thus, the conservation of
95 suggested some critical functions in addition to its
postulated role in the A-block binding of TFIIIC. We therefore set out
to mutagenize a DNA fragment encompassing the conserved regions D and E
of
95. As described under "Experimental Procedures,"
PCR mutagenesis generated one thermosensitive point mutant showing a
decreased growth rate at 34 °C and lethality at 37 °C. The
mutation E447K (indicated by the star in Fig. 1) is mapped
just upstream of the E region of sequence similarity. As seen by
in vivo RNA labeling with tritiated uracil, the mutation
caused a marked decrease in tRNA synthesis after 3 h of incubation
of mutant cells at 37 °C (data not shown). Among all the genes of
the pol III transcription system, high dosage of TFC1,
BRF1, and, to a lesser extent, of TFC5 (encoding
Bdp1) was able to improve the growth rate of mutant cells at 34 °C,
but only TFC1 suppressed the lethality at 37 °C (data not
shown).
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Fig. 1.
95 subunit of TFIIIC
and potential orthologs. Complete
95-like proteins
were identified in H. sapiens (Hs), D. melanogaster (Dm), C. elegans
(Ce), A. thaliana (At), S. pombe (Sp), N. crassa (Nc),
C. albicans (Ca), and S. cerevisiae
(Sc). Conserved domains are boxed and labeled by
letters A-D (the most conserved) and E (the
least conserved) on the upper panel. A star
indicates the E447K mutation. Sequence alignments of each conserved
domain (A-E) are shown on the lower panel. Amino
acids conserved in at least two sequences are boxed.
95-E447K Influences Start Site Selection--
In view
of the genetic interaction noted above between
95 and
TFIIIB components, we examined the ability of mutant TFIIIC to recruit
TFIIIB. Mutant and wild type TFIIIC were partially purified by two
chromatographic steps (a heparin Hyper D column and a MonoQ column as
described in Ref. 10). Based on Western blotting assays with anti-HA or
anti-
55 antibodies and gel shift analysis, TFIIIC
preparation from mutant cells was estimated to contain one-third as
much factor as wild type fractions. In the following experiments, care
was taken to use similar amounts of mutant and wild type factor. TFIIIB
recruitment was monitored by gel shift assay (39). TFIIIC·DNA
complexes were first formed by preincubating TFIIIC with a
SUP4 DNA probe for 10 min at 25 °C and then further
incubated with TFIIIB to form B·C·DNA complexes that can be
separated by gel electrophoresis. As shown in Fig. 2A, TFIIIB supershifted all
the C·DNA complexes formed with wild type TFIIIC (lane 2).
The supershifted band was probably composed of a mixture of B·C·DNA
and B'·C·DNA complexes, the latter containing TBP and Brf1 but
lacking Bdp1 (40, 41). Mutant TFIIIC assembled TFIIIB less efficiently
(compare lanes 2 and 6) as evidenced by the large
proportion of unshifted C·DNA complexes. TFIIIC can be selectively
stripped from B·C·DNA complexes by heparin treatment to yield the
heparin-resistant B·DNA complexes (42). Interestingly, although
B·C·DNA complexes formed with wild type TFIIIC generated, as
expected, B·DNA complexes of characteristic fast migration, the
complexes formed with mutant TFIIIC generated, after stripping TFIIIC
with heparin, the characteristic B·DNA complex plus a more slowly
migrating form of complex (Fig. 2A, compare lanes
4 and 8). A similar result was obtained upon heating
the B·C·DNA complexes for 5 min at 50 °C (lanes 3 and
7). The slow-migrating complex (see arrow in Fig.
2A) was presumed to be a distinct form of B·DNA complex
because B'·DNA complexes are heparin-sensitive (43) and should be
expected to migrate faster than B·DNA complexes. A less likely
possibility could be that the slow-migrating complex had retained a
fraction of TFIIIC to yield a bigger complex (that should be resistant
to heparin and heat treatment). This possibility cannot be excluded
even though we found that anti-
55 antibodies did not
supershift the two forms of complexes (data not shown).
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Fig. 2.
95-E447K affects
TFIIIB·DNA complex formation. Protein·DNA complex formation
with wild type (WT) or mutant (E447K) TFIIIC
(C), TFIIIB (B), and RNA polymerase III
(Pol) was analyzed by gel retardation assays. The protein
components incubated with the SUP4 tDNATyr probe
are indicated above the lanes. A,
lanes 4 and 8, B·C·DNA complexes were first
assembled, and then heparin (500 ng) was added in order to strip TFIIIC
(CBHEP). A and B, lanes 3 and 7, B·C·DNA complexes were heated for 5 min at
50 °C before loading on the gel (CB50). B,
lanes 4 and 8, RNA polymerase III was added after
heating the B·C·DNA complex at 50 °C, and the mixture was
further incubated for 45 min at 25 °C before gel analysis. The
positions of TFIIIB·DNA and RNA polymerase III·TFIIIB·DNA
complexes are shown. The slow migrating complexes obtained with mutant
TFIIIC are indicated by arrows.
30 but requires TFIIIC to be
transcribed efficiently (30). As shown in Fig.
3, equal amounts of wild type or mutant
TFIIIC factor yielded similar amounts of transcripts. However, mutant
TFIIIC-directed transcription of both genes generated new RNA species
(indicated by arrows in the autoradiograms and in the scans
shown in Fig. 3) shorter than the full-size pre-tRNA. The same
additional RNA species were observed when TFIIIC was stripped from the
DNA before transcription (data not shown).
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Fig. 3.
Mutant TFIIIC affects the transcript
pattern. The SUP4 tRNATyr or the
tRNAAsp(GTC) gene were transcribed as described under
"Experimental Procedures" in the presence of wild type
(WT) or mutant (E447K) TFIIIC. Left
panel, electrophoretic analysis of the transcripts;
right panel, PhosphorImager (Amersham Biosciences)
quantification of radiolabeled transcripts. The continuous
and dotted line profiles correspond to wild type and mutant
TFIIIC, respectively. The new RNA species generated in the presence of
mutant TFIIIC are shown by arrows.
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Fig. 4.
Mutant TFIIIC affects start site
selection. The SUP4 tRNATyr gene was
transcribed in the presence of wild type (WT) or mutant
(E447K) TFIIIC. Start site selection was determined by
primer extension of the RNA transcripts and analyzed on a sequencing
gel as described under "Experimental Procedures." Sites of
transcription initiation are indicated on the right.
131 as the TFIIIB-assembling subunit of TFIIIC;
131
projects far upstream within the TFIIIB binding region to contact Brf1
and Bdp1 (18, 19, 35, 40, 44). To investigate whether
95-E447K affected the placement of
131, we analyzed the
upstream border of TFIIIC·DNA complexes by
exonuclease digestion.
Wild type or mutant TFIIIC were preincubated with SUP4 DNA
probe labeled at the 3' end of the non-coding strand, and TFIIIC·DNA
complexes were digested by
exonuclease for 1 min at 25 °C.
Resulting DNA fragments were analyzed on a sequencing gel alongside G + A sequencing reaction products from the same probe (Fig.
5). When compared with the pattern
obtained in the absence of factor, wild type TFIIIC arrested
exonuclease digestion at two positions,
53 and
47, as evidenced by
the accumulation of two DNA fragments of similar labeling intensity
(lanes 3 and 4). In contrast, mutant TFIIIC·DNA
complexes showed only the
47 upstream border (lanes 5 and
6). As the TFIIIC factors used in these experiments were
partially purified, we tested several highly purified preparations of
TFIIIC to ascertain that these observed exonuclease arrests were due to
TFIIIC and not to spurious contaminants. Immunopurified or DNA
affinity-purified TFIIIC caused predominantly the
53 digestion arrest
and the
47 arrest to a variable extent (lanes 1 and
2, and results not shown). The loss of the
53 blockage site in mutant TFIIIC complexes therefore indicated that the
95 mutation influenced
131 projection on upstream
DNA.
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Fig. 5.
95-E447K influences
the 5' border of TFIIIC·DNA complexes. The SUP4
tDNATyr probe labeled at the 3' end of the non-coding
strand was complexed with TFIIIC and digested with
exonuclease, and
the digestion products were analyzed on a sequencing gel as described
under "Experimental Procedures." Nuclease-resistant DNA fragments
in reactions without factor (lanes 3 and 6), with
wild type TFIIIC (lane 4), or mutant TFIIIC (lane
5) were separated on a sequencing gel, alongside G + A cleavage
products of the same probe (G+A lane). DNA cleavage products
obtained with immunopurified TFIIIC (T1) or with DNA
affinity highly purified TFIIIC (T2) were analyzed,
respectively, on lanes 1 and 2. Positions of the
transcription start site (+1) and of the A block are indicated on the
left. The arrows show the direction and extent of
digestion for the main nuclease-resistant DNA fragments in the presence
of bound TFIIIC.
A Affects TFIIIC·DNA Binding--
As the above observations
could not account for the strong thermosensitivity and the drop of
in vivo tRNA synthesis occurring in the YSJ1-E447K strain,
we looked for other major defects in mutant TFIIIC.
95 is
thought to be involved in block A binding (8, 11), but the overall DNA
affinity of TFIIIC is predominantly governed by the interaction of
B
domain with the B block (5). Nevertheless, we investigated whether the
95 mutation could affect the apparent dissociation constant
of TFIIIC·DNA complexes.
0.33 × 10
10
M for the wild type and
1.33 × 10
10
M for mutant TFIIIC·SUP4
tDNATyr complexes. The concentration of active TFIIIC
in the binding assay given by the y intercept of the curve
was
0.75 × 10
10 M for wild type
TFIIIC and
0.73 × 10
10 M for mutant
TFIIIC. Therefore, the weaker binding of mutant TFIIIC (at permissive
temperature) was due to its 4-fold lower DNA binding affinity.
Remarkably, the tsv115 mutation in
138, a
B subunit,
caused a similar (5-fold) reduction in DNA binding affinity at the
permissive temperature of 25 °C (13). Note, however, that due to the
weak binding signal, the DNA affinity of the mutant factor was
estimated with lower accuracy than for the wt factor.
View larger version (22K):
[in a new window]
Fig. 6.
tDNA affinity of mutant TFIIIC.
A, TFIIIC·DNA complexes formed with varying amounts
of labeled SUP4 tDNATyr probe and equal amount
of wild type (WT) or mutant (E447K) TFIIIC were
analyzed by the gel retardation assay. The concentrations of TFIIIC
complexes [TFIIIC·DNA] and free probe [DNA] formed in each
reaction were determined, and the data were plotted according to
Equation 1 (see "Experimental Procedures"). B,
determination of TFIIIC·tDNA apparent dissociation constant by
Scatchard representation. The curve slope gives the apparent
dissociation constant (Kapp) of TFIIIC·tDNA
complexes formed with wild type (WT) or mutant
(E447K) factor. Mutant (E447K) and wild type factors are
plotted as black and white circles,
respectively.
View larger version (109K):
[in a new window]
Fig. 7.
95-E447K affects
TFIIIC·DNA binding stability. A, wild type
(WT) or mutant (E447K) TFIIIC was preincubated
for 10 min at different temperatures as indicated. After addition of
the labeled SUP4 tDNATyr probe, the mixture was
further incubated for 10 min at 25 °C and then subjected to gel
electrophoresis. B, temperature sensitivity of
TFIIIC·DNA complexes. TFIIIC fractions were preincubated for 10 min
at 25 °C in standard binding buffer with the SUP4
tDNATyr probe and competitor DNA and then incubated for 10 min at different temperatures, as indicated, before the mixtures were
analyzed by gel retardation assay. C,
B, the
protease-resistant domain of TFIIIC, was obtained by digestion of the
wild type or mutant (E447K) TFIIIC·SUP4
tDNATyr complexes with 40 ng of
-chymotrypsin for 10 min
at 25 °C. Digestion was stopped by addition of 40 ng of aprotinin,
and
B·DNA complexes were further incubated for 10 min at different
temperatures, as indicated, before gel analysis. Lane
14, undigested TFIIIC·DNA complex. Lane C,
control.
A component because
B·DNA interaction is
known to be dominant in TFIIIC. The same thermolability was observed in
gel shift assays using a short (55 bp) DNA probe harboring only the B
block sequence (data not shown). Therefore the binding of the mutant
A domain on the DNA appears not to be a prerequisite for the
destabilization of the
B·DNA complexes. We then explored the
temperature sensitivity of
B·DNA complexes produced by limited proteolysis of wild type or mutant TFIIIC·DNA complexes (3). TFIIIC
fractions were preincubated with the SUP4 DNA probe for 10 min at 25 °C before the
-chymotrypsin treatment. The resultant protein·DNA complexes were then subjected to 10 min of heat treatment as indicated in Fig. 7C. It was remarkable that
B·DNA
complexes derived from wild type or mutant factor exhibited the same
thermostability and were more resistant to heat denaturation at
50 °C than the native TFIIIC·DNA complexes (compare Fig.
7C, lane 19, upper and lower
panels with Fig. 7B, lane 11,
upper and lower panels). These results suggested
that in the context of native TFIIIC, the
A domain indirectly
affected
B·DNA interaction and that the
95 mutation
exacerbated this negative influence.
95 Interacts Directly with
91 and
138--
To understand how
95, a
A subunit, could
influence the distal
B domain, we used a coimmunoprecipitation assay
to investigate possible interactions between
95 and the
individual components of
B,
138,
91, and
60. Epitope-tagged
95 and
138
(HA-
95 and FLAG-
138) were used to facilitate the
identification and the purification of the protein complexes on
anti-HA-coated magnetic beads. High five insect cells were coinfected
with different combinations of recombinant baculovirus to express
HA-tagged
95 alone or in combination with
B components.
SDS-PAGE analysis of crude extracts revealed that all recombinant
proteins display the expected size (Fig.
8A, lanes 2,
3, and 5). The level of expression was
variable and generally better when only one recombinant protein was
expressed (compare Fig. 8A, lanes 2,
3, and 5 with Fig. 8A,
lanes 4, 6, and 7).
B
components selectively retained on the beads were analyzed by SDS-PAGE
and immunoblotting, using 12CA5 anti-HA, M2 anti-FLAG, or
anti-
91 antibodies (Fig. 8B). The anti-HA
antibody revealed a single band of about 100 kDa in crude extracts from
cells expressing HA-
95 (Fig. 8B, lanes
2, 4, 6, and 7). This
protein was absent from control extracts (Fig. 8B,
IP, lanes 1, 3, and
5), was properly immunopurified on the beads (Fig.
8B, IP, lanes 2, 4,
6, and 7), and therefore corresponded to
HA-
95. Interestingly, FLAG-tagged
138 or
91
were found to be detectably retained by the anti-HA-coated beads only
when coexpressed with HA-
95 (Fig. 8B, see Co-IP
of
138 and
91 for lanes 4 and 6).
The coexpression of the three proteins (
138,
95, and
91) markedly decreased the expression of
91 but
yielded qualitatively the same coimmunopurification results (Fig.
8B, see Co-IP of
138 and
91 for
lane 7). These results suggested that
95 engages
multiple contacts with
B domain. Due to a high background binding of
recombinant
60 on magnetic beads coated with the 12CA5
antibody, it was not possible to study the interaction between
95 and
60 (data not shown).
View larger version (34K):
[in a new window]
Fig. 8.
95 interacts with
138 and
91.
High five insect cells were coinfected with various combinations of
recombinant baculovirus, as indicated. A, recombinant
proteins. Coomassie Blue staining of an 8% polyacrylamide gel.
Positions and molecular masses (in kilodaltons) of marker bands are
indicated on the left. 15 µg of crude extracts from cells
expressing HA-tagged
95 (lanes 2, 4,
6, and 7), FLAG-tagged
138 (lanes
3, 4, and 7), or
91
(lanes 5-7) were loaded in each lane. The positions of
FLAG-tagged
138 and HA-tagged
95 and
91 are
indicated by arrows on the right. B,
crude extracts (120 µg of proteins) from cells expressing HA-tagged
95 (lanes 2, 4, 6,
and 7), FLAG-tagged
138 (lanes 3,
4, and 7), or
91 (lanes
5-7) were mixed with magnetic beads coated with 12CA5 anti-HA
antibodies. The beads were washed, and bound proteins were eluted by
boiling in loading buffer and analyzed by SDS-PAGE. The input (only 15 µg of the 120 µg used for the immunoprecipitation) and the entire
bound protein fraction were analyzed by Western blotting using 12CA5
anti-HA (
95), M2 anti-FLAG (
138), or anti-
91
antibodies. For clarity, the polypeptides revealed by specific
antibodies are identified on the left. IP,
immunoprecipitation; Co-IP, coimmunoprecipitation. The beads
coated with anti-HA antibodies retained specifically HA-
95
(IP, lanes 2, 4,
6, and 7) and FLAG-
138 or
91
copurified with HA-
95 (Co-IP, lanes
4, 6, and 7), whereas background
retention of FLAG-
138 and
91 by the beads was
undetectable (Co-IP, lanes 3 and
5).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
95 subunit of TFIIIC
that lies over the A block of tRNA genes influences start site
selection and has a strong indirect effect on TFIIIC·DNA binding
stability. The observations suggest that
95 influences the
positioning of the TFIIIB assembling subunit (
131) and participates
in the interconnection of the A block and B block binding domains of
TFIIIC (see Fig. 9).
View larger version (15K):
[in a new window]
Fig. 9.
Schematic representation of the upstream and
downstream effects of 95-E447K
mutation. The preinitiation complex B·C·DNA is represented,
with the A and B blocks, the
B domain, the TFIIIB-assembling subunit
131,
95-E447K, and TFIIIB. The
95 mutation
is proposed to affect
131 placement resulting in a different
placement of TFIIIB and consequently in downstream initiation sites
(dotted lines). The TFIIIC·DNA destabilization effect of
A deletion or the
95 mutation also suggests a downstream
effect on
B·block B interaction which is the major determinant of
TFIIIC·DNA affinity.
95 is thought to participate in A block binding based on
its DNA cross-linking and positioning over the A block (8, 11). The A
block contributes to TFIIIC DNA binding affinity that is, however,
predominantly dependent on B block binding (4), and most importantly,
its location dictates the transcription start sites (6, 45) in
cooperation with a preferential TBP-binding site (7). It was
interesting, therefore, to find that the mutant factor assembled two
distinct forms of B·DNA complexes, as seen by gel electrophoresis
after removing TFIIIC by heparin or heat treatment. Both complexes were
able to recruit pol III and were likely to be transcriptionally
competent. The contention that both complexes had properly assembled
TFIIIB was supported by their heparin resistance property which is
typical of fully assembled TFIIIB·DNA complexes (43). As TFIIIB has
been shown to bend DNA sharply (46-48), we presumed that the different
migration rate of both complexes in gels was due to a different
placement of TFIIIB on the DNA probe. Distinctly placed TFIIIB implied
distinct initiation sites that were indeed evidenced by transcript
analysis: mutant TFIIIC-directed transcription produced additional RNA
species shorter than the full size pre-tRNA, with two different
templates, and primer extension analysis mapped multiple start sites on
SUP4, essentially at positions +1, +4, and +8 but also
further downstream, at +13 and +15. The +4 and +8 sites were only
weakly observed in the control transcription reaction with wild type
TFIIIC. These alternative start sites have already been mentioned, and
their relative utilization was shown to depend on distinct TFIIIB
placements upon modification of the upstream promoter sequence (7).
How could a 95 point mutation influence TFIIIB placement?
There is no evidence that
95 interacts with TFIIIB
components but its human homolog hTFIIIC63 has been reported to
interact with TBP, hBRF/TFIIIB90, and with pol III (25). As we supposed that
95 mutation could affect the upstream placement of the
TFIIIB assembling subunit
131, we mapped the upstream border of
TFIIIC·DNA complex. Highly purified TFIIIC blocked
exonuclease
digestion of SUP4 DNA predominantly at positions
53 and
also
47. Remarkably, the mutant factor lost the
53 arrest site and
displayed only the
47 block. It is likely that
131 is responsible
for
exonuclease blockage as it is the TFIIIC subunit that protrudes
the most upstream, over the TFIIIB binding region (40).
131 was
found to be cross-linked weakly to SUP4 DNA at positions
47/
48 of the transcribed strand, and this cross-linking was
markedly enhanced upon TFIIIB binding (44). This far upstream
interaction is probably facilitated by DNA bending (46). Note that
previous
exonuclease digestion experiments on a tRNAGlu
gene could not reveal such a far upstream border because the probe was
truncated too close from the transcription start site (49). In
contrast, photocross-linking of
95 was restricted over and
closely around the A block (11). Therefore, as schematically illustrated in Fig. 9, we concluded that mutant
95
influenced the placement of
131 far upstream of the start site,
thereby affecting TFIIIB placement and start site selection.
In the above experiments it should be noted that the mutant factor
often imposed, or favored, particular states of C·DNA and B·C·DNA
complexes or of the initiation complexes that can be weakly observed
with wild type TFIIIC (like the 47 border of TFIIIC·DNA or the +4
or +8 start sites), as if the mutation favored certain conformations of
the TFIIIC·DNA complex (for TFIIIC flexibility, see Ref. 7).
The second unexpected effect of the 95-E447K mutation was
on TFIIIC·SUP4 DNA binding stability; a point mutation in
the A block binding subunit was not expected to cause the dissociation of TFIIIC·DNA complexes. As shown by Baker et al. (4), the interaction between A block and TFIIIC contributes relatively little to
the overall stability of the TFIIIC·SUP4 DNA complex. Mutations within the A block decreased the equilibrium constant (Ks) for TFIIIC·SUP4 DNA binding
less than 2-fold, whereas point mutations within the B block decreased
the Ks values several hundredfold. Even the total
deletion of the A block reduced the Ks for TFIIIC
binding at most 5-fold (4). The dissociation of mutant TFIIIC·DNA
complexes at 38 °C therefore suggested a long distance effect of the
mutated subunit on the DNA binding properties of
B, the B-block
binding module of TFIIIC (Fig. 9). Composed of three polypeptides
138,
91, and
60, two of which,
138 and
91, cooperate in DNA binding (9, 10, 13),
B can be
isolated by mild proteolysis of TFIIIC·DNA complexes. As shown in
Fig. 7C, the mutant factor was normally susceptible to
proteolysis, and
B·DNA complex generated from the mutant factor displayed the same remarkable heat resistance as the control
B·DNA complex. To interpret this paradoxical observation, one could imagine
that the mutation causes a conformation change in
95 that
is transmitted to
B domain through the hinge that connects
A and
B. Our coimmunopurification experiments suggested that
95 participates in the interconnection of
A with
B
via its contacts with
138 and
91. So
95
might directly influence
B·DNA binding.
131 and
60
also appear as good candidates for this hinge function,
131 because
this protein can be cross-linked between the A and B blocks (11, 44)
and
60 because it belongs to
B and participates in
TFIIIB assembly (10). However, there is no evidence that
131 contact
B components. Concerning
60, we want to retract the
reported suppression of the ts phenotype of N-terminally tagged
60 by overexpression of
95 (10). The suppression was due to an unfortunate mishandling of plasmids and was
not reproducible with bona fide
TFC1.2
Our findings attribute to 95, and more globally to the
A
module, the ability to influence
B·DNA stability. A conformation change of TFIIIC facilitating the dissociation of
B may naturally occur during transcription where the RNA polymerase has to dissociate TFIIIC to transcribe the B block. Among he mechanisms that can be
invoked to explain the release of TFIIIC by the advancing RNA polymerase (50), one could imagine a connection between the A block and
B block binding domains of TFIIIC that would favor B block dissociation
after the A block region is transcribed.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Christine Conesa for suppressor verifications, to Emmanuel Favry for technical advice and protein preparations, and to Giorgio Dieci (University of Parma) for kindly providing tRNAAsp(GTC) gene. We thank Gérald Peyroche, Christine Conesa, and Isabelle Callebaut (CNRS, UMR 7590) for helpful comments and stimulating discussions. We also thank Victor Fazakerley for improving the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the Fondation pour la Recherche Médicale Fellowship FDT20000915040 (to S. J.).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.
Present address: Neurodegeneration Research, Neurology-CEDD,
GlaxoSmithKline, Third Ave., Harlow, Essex CM19 5AW, UK.
§ To whom correspondence should be addressed: Service de Biochimie et Génétique Moléculaire, Bâtiment 144, CEA/Saclay, F-91191 Gif-sur-Yvette Cedex, France. Tel.: 33-1-69-08-59-57; Fax: 33-1-69-08-47-12; E-mail: Olivier.Lefebvre@Cea.Fr.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M213310200
2 C. Conesa, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TF, transcription factor; HA, hemagglutinin; wt, wild type; pol III, polymerase III; TBP, TATA-binding protein.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Geiduschek, E. P., and Kassavetis, G. A. (2001) J. Mol. Biol. 310, 1-26[CrossRef][Medline] [Order article via Infotrieve] |
2. | Schultz, P., Marzouki, N., Marck, C., Ruet, A., Oudet, P., and Sentenac, A. (1989) EMBO J. 8, 3815-3824[Abstract] |
3. | Marzouki, N., Camier, S., Ruet, A., Moenne, A., and Sentenac, A. (1986) Nature 323, 176-178[Medline] [Order article via Infotrieve] |
4. |
Baker, R. E.,
Gabrielsen, O. S.,
and Hall, B. D.
(1986)
J. Biol. Chem.
261,
5275-5282 |
5. | Stillman, D. J., and Geiduschek, E. P. (1984) EMBO J. 3, 847-853[Abstract] |
6. | Baker, R. E., Camier, S., Sentenac, A., and Hall, B. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8768-8772[Abstract] |
7. | Joazeiro, C. A. P., Kassavetis, G. A., and Geiduschek, E. P. (1996) Genes Dev. 10, 725-739[Abstract] |
8. |
Gabrielsen, O. S.,
Marzouki, N.,
Ruet, A.,
Sentenac, A.,
and Fromageot, P.
(1989)
J. Biol. Chem.
264,
7505-7511 |
9. |
Arrebola, R.,
Manaud, N.,
Rozenfeld, S.,
Marsolier, M. C.,
Lefebvre, O.,
Carles, C.,
Thuriaux, P.,
Conesa, C.,
and Sentenac, A.
(1998)
Mol. Cell. Biol.
18,
1-9 |
10. |
Deprez, E.,
Arrebola, R.,
Conesa, C.,
and Sentenac, A.
(1999)
Mol. Cell. Biol.
19,
8042-8051 |
11. | Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-2205[Abstract] |
12. | Braun, B. R., Bartholomew, B., Kassavetis, G. A., and Geiduschek, E. P. (1992) J. Mol. Biol. 228, 1063-1077[Medline] [Order article via Infotrieve] |
13. |
Lefebvre, O.,
Rüth, J.,
and Sentenac, A.
(1994)
J. Biol. Chem.
269,
23374-23381 |
14. | Marck, C., Lefebvre, O., Carles, C., Riva, M., Chaussivert, N., Ruet, A., and Sentenac, A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4027-4031[Abstract] |
15. | Rameau, R., Puglia, K., Crowe, A., Sethy, I., and Willis, I. M. (1994) Mol. Cell. Biol. 14, 822-830[Abstract] |
16. |
Conesa, C.,
Swanson, R. N.,
Schultz, P.,
Oudet, P.,
and Sentenac, A.
(1993)
J. Biol. Chem.
268,
18047-18052 |
17. |
Manaud, N.,
Arrebola, R.,
Buffin-Meyer, B.,
Lefebvre, O.,
Voss, H.,
Riva, M.,
Conesa, C.,
and Sentenac, A.
(1998)
Mol. Cell. Biol.
18,
3191-3200 |
18. |
Chaussivert, N.,
Conesa, C.,
Shaaban, S.,
and Sentenac, A.
(1995)
J. Biol. Chem.
270,
15353-15358 |
19. | Khoo, B., Brophy, B., and Jackson, S. P. (1994) Genes Dev. 8, 2879-2890[Abstract] |
20. |
Willis, I. M.
(2002)
Genes Dev.
16,
1337-1338 |
21. | Kassavetis, G. A., Blanco, J. A., Johnson, T. E., and Geiduschek, E. P. (1992) J. Mol. Biol. 226, 47-58[Medline] [Order article via Infotrieve] |
22. |
Moir, R. D.,
Puglia, K. V.,
and Willis, I. M.
(2002)
Mol. Cell. Biol.
22,
6131-6141 |
23. | Moir, R. D., Sethy-Coraci, I., Puglia, K., Librizzi, M. D., and Willis, I. M. (1997) Mol. Cell. Biol. 17, 7119-7125[Abstract] |
24. |
Moir, R. D.,
Puglia, K. V.,
and Willis, I. M.
(2000)
J. Biol. Chem.
275,
26591-26598 |
25. |
Hsieh, Y.-J.,
Wang, Z.,
Kovelman, R.,
and Roeder, R. G.
(1999)
Mol. Cell. Biol.
19,
4944-4952 |
26. |
Huang, Y.,
Hamada, M.,
and Maraia, R. J.
(2000)
J. Biol. Chem.
275,
31480-31487 |
27. | Huet, J., Manaud, N., Dieci, G., Peyroche, G., Conesa, C., Lefebvre, O., Ruet, A., Riva, M., and Sentenac, A. (1996) Methods Enzymol. 273, 249-267[Medline] [Order article via Infotrieve] |
28. |
Sikorski, R. S.,
and Hieter, P.
(1989)
Genetics
122,
19-27 |
29. |
Vignais, M. L.,
Huet, J.,
Buhler, J.-M.,
and Sentenac, A.
(1990)
J. Biol. Chem.
265,
14669-14674 |
30. | Dieci, G., Percudani, R., Giuliodori, S., Bottarelli, L., and Ottonello, S. (2000) J. Mol. Biol. 299, 601-613[CrossRef][Medline] [Order article via Infotrieve] |
31. | Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 65, 499-560[Medline] [Order article via Infotrieve] |
32. |
Altschul, S. F.,
Madden, T. L.,
Schaffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402 |
33. |
Thompson, J. D.,
Gibson, T. J.,
Plewniak, F.,
Jeanmougin, F.,
and Higgins, D. G.
(1997)
Nucleic Acids Res.
25,
4876-4882 |
34. | Henikoff, S., and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10915-10919[Abstract] |
35. | Dumay-Odelot, H., Acker, J., Arrebola, R., Sentenac, A., and Marck, C. (2001) Mol. Cell. Biol. 22, 298-308 |
36. |
Aye, M.,
Dildine, S. L.,
Claypool, J. A.,
Jourdain, S.,
and Sandmeyer, S. B.
(2001)
Mol. Cell. Biol.
21,
7839-7851 |
37. | Chédin, S., Ferri, M. L., Peyroche, G., Andrau, J. C., Jourdain, S., Lefebvre, O., Werner, M., Carles, C., and Sentenac, A. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 381-389[Medline] [Order article via Infotrieve] |
38. |
Huang, Y.,
and Maraia, R. J.
(2001)
Nucleic Acids Res.
29,
2675-2690 |
39. | Kassavetis, G. A., Riggs, D. L., Negri, R., Nguyen, L. H., and Geiduschek, E. P. (1989) Mol. Cell. Biol. 9, 2551-2566[Medline] [Order article via Infotrieve] |
40. | Kassavetis, G. A., Joazeiro, C. A. P., Pisano, M., Geiduschek, E. P., Colbert, T., Hahn, S., and Blanco, J. A. (1992) Cell 71, 1055-1064[Medline] [Order article via Infotrieve] |
41. | Kassavetis, G. A., Nguyen, S. T., Kobayashi, R., Kumar, A., Geiduschek, E. P., and Pisano, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9786-9790[Abstract] |
42. | Kassavetis, G. A., Braun, B. R., Nguyen, L. H., and Geiduschek, E. P. (1990) Cell 60, 235-245[Medline] [Order article via Infotrieve] |
43. | Kassavetis, G. A., Bartholomew, B., Blanco, J. A., Johnson, T. E., and Geiduschek, E. P. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7308-7312[Abstract] |
44. | Bartholomew, B., Kassavetis, G. A., and Geiduschek, E. P. (1991) Mol. Cell. Biol. 11, 5181-5189[Medline] [Order article via Infotrieve] |
45. | Fabrizio, P., Coppo, A., Fruscoloni, P., Benedetti, P., Di Segni, G., and Tocchini-Valentini, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8763-8767[Abstract] |
46. |
Léveillard, T.,
Kassavetis, G. A.,
and Geiduschek, E. P.
(1991)
J. Biol. Chem.
266,
5162-5168 |
47. |
Braun, B. R.,
Kassavetis, G. A.,
and Geiduschek, E. P.
(1992)
J. Biol. Chem.
267,
22562-22569 |
48. | Grove, A., Kassavetis, G. A., Johnson, T. E., and Geiduschek, E. P. (1999) J. Mol. Biol. 285, 1429-1440[CrossRef][Medline] [Order article via Infotrieve] |
49. | Camier, S., Gabrielsen, O. S., Baker, R. E., and Sentenac, A. (1985) EMBO J. 4, 491-500[Abstract] |
50. | Bardeleben, C., Kassavetis, G. A., and Geiduschek, E. P. (1994) J. Mol. Biol. 235, 1193-1205[CrossRef][Medline] [Order article via Infotrieve] |