From the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014
Received for publication, August 3, 2000, and in revised form, October 12, 2000
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ABSTRACT |
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The human adenovirus type 2 E2 early (E2E)
transcriptional control region contains an efficient RNA polymerase III
promoter, in addition to the well characterized promoter for RNA
polymerase II. To determine whether this promoter includes intragenic
sequences, we examined the effects of precise substitutions introduced
between positions +2 and +62 on E2E transcription in an RNA polymerase III-specific, in vitro system. Two noncontiguous sequences
within this region were necessary for efficient or accurate
transcription by this enzyme. The sequence and properties of the
functional element proximal to the sites of initiation identified it as
an A box. Although a B box sequence could not be unambiguously located, substitutions between positions +42 and +62 that severely impaired transcription also inhibited binding of the human general initiation protein TFIIIC. Thus, this region of the RNA polymerase III E2E promoter contains a B box sequence. We also identified previously unrecognized intragenic sequences of the E2E RNA polymerase II promoter. In conjunction with our previous observations, these data
establish that RNA polymerase II and RNA polymerase III promoter sequences are superimposed from approximately positions -30 to +20 of
the complex E2E transcriptional control region. The alterations in
transcription induced by certain mutations suggest that components of
the RNA polymerase II and RNA polymerase III transcriptional machines
compete for access to overlapping binding sites in the E2E template.
Human subgroup C adenoviral proteins are synthesized in a strict
temporal sequence during productive infection as a result of sequential
activation of viral RNA polymerase II transcription units (see Refs. 1
and 2). The viral promoters recognized by components of the cellular
RNA polymerase II machinery are analogous to those of cellular genes,
indeed served as important models in early studies of RNA polymerase II
transcription, but their activity is regulated by virus-specific
mechanisms. The viral E1A proteins induce efficient transcription from
early promoters by altering the activity or availability of a variety
of cellular, sequence-specific transcriptional regulators or general
initiation proteins (see Refs. 3, 4). Expression of viral late genes requires viral DNA synthesis in the infected cell and
sequence-specific, transcriptional activators, including the
IVa2 protein (5). Adenoviruses also depend on cellular RNA
polymerase III (responsible for synthesis of tRNA, 5 S rRNA, and other
small, host cell RNA species (see Refs. 6-8)), to transcribe
the viral VA RNA I and VA RNA II genes. The
promoters of these genes comprise two intragenic sequences closely
related to similarly located promoter sequences of tRNA
genes and termed A and B boxes (see Refs. 6, 9). These internal
sequences are recognized by the RNA polymerase III-specific initiation
protein TFIIIC (10-15). This type of RNA polymerase III promoter
architecture is designated type 2 (see Refs. 6, 8, 16). In infected
cells, VA RNA I accumulates to a very high concentration,
~108 molecules per cell (17). This RNA is required for
efficient synthesis of viral late proteins in productively infected
cells (18), for it counters a cellular, anti-viral defense mechanism (see Refs. 19, 20).
Although the adenoviral promoters recognized by the cellular RNA
polymerase II and RNA polymerase III machines are generally typical of
those of cellular genes, the E2 early
(E2E)1 transcriptional
control region is unusual. The E2 RNA polymerase II transcription unit,
which encodes the viral replication proteins, is expressed from a well
characterized promoter (see Fig. 1) that contains a TATA-like sequence,
two inverted binding sites for E2F, and an ATF recognition site
(3, 4). Each of these sequences is required for both basal
transcription, and E1A protein-mediated stimulation of transcription in
infected cells (21, 22). However, RNA polymerase III also transcribes
the 5'-end of this transcription unit both in vitro and in
infected cells (23, 24) to produce small RNA species of some 45 and 90 nucleotides (see Fig. 1). Efficient E2E transcription by RNA polymerase
III requires sequences 5' to the site of initiation, notably the
TATA-like sequence (23).2
Thus, promoters directing transcription by these two cellular enzymes
are at least partially superimposed. An analogous arrangement of RNA
polymerase III and II transcription units had been observed previously
at the P1 and P2 promoters of the human c-myc gene (25-27).
These RNA polymerase III promoters support efficient transcription in vitro, but c-myc transcription by this enzyme
is much less efficient than that by RNA polymerase II in
Xenopus oocytes microinjected with the gene, and cannot be
detected from the endogenous human genes of HeLa or HL60 cells (25, 27,
28). Thus, the adenoviral E2E transcriptional control region was the
first documented to support transcription by both these cellular RNA
polymerases in a normal biological context.
Also atypical is the low concentration attained by each small E2E RNA
made by RNA polymerase III: 10-20 copies per infected cell (24). This
unusual property explains why E2E RNA polymerase III transcripts were
not detected in the many previous studies of adenoviral gene
expression. It also suggests that these RNAs are not likely to function
in the same manner as the typical, abundant RNAs synthesized by RNA
polymerase III. As a necessary prelude to genetic analyses of the
function of the unorthodox adenoviral E2E RNA polymerase III
transcription unit and its small RNA products, we are defining the
sequences that comprise the RNA polymerase III promoter and their
relationship to those of the overlapping (and essential) RNA polymerase
II promoter. Here we report the results of mutational and biochemical
analyses of intragenic sequences common to the E2E transcription units
of RNA polymerases II and III.
Templates for in Vitro Transcription and Their
Mutagenesis--
The wild-type E2E template for in vitro
transcription was the plasmid pEII', which contains the human
adenovirus type 2 (Ad2) E2E sequence from position -249 to position
+200. This plasmid was constructed by first inserting a NarI
fragment comprising bp 26,893 to 27,187 of the viral genome into the
ClaI site of the polylinker of pSP73 (Promega). A DNA
fragment corresponding to positions 27,105 to 27,341 in the Ad2 genome,
generated by PCR, was then introduced via BsshII and
XhoI sites within the E2E DNA fragment and the pSP73
polylinker, respectively. The pEII'
Plasmid DNA templates for in vitro transcription reactions
were purified using the Qiagen Maxi-prep protocol. The
concentrations of the different templates to be compared, as well as
their quality (i.e. presence of largely supercoiled DNA),
were confirmed by ethidium bromide staining of DNA, following
electrophoresis in 1.4% agarose gels, cast, and run in 0.04 M Tris acetate, pH 8.0, containing 1 mM EDTA.
In Vitro Transcription by RNA Polymerase III--
Transcription
of E2E templates by RNA polymerase III was analyzed using nuclear
extracts of HeLa cells prepared according to the procedure of Dignam
et al. (31), in which at least 90% of the E2E transcripts
are synthesized by RNA polymerase III (23). All transcription reactions
contained 2 µg/ml In Vitro Transcription by RNA Polymerase II--
Transcription
of wild-type and mutant E2E templates by RNA polymerase II was examined
using whole cell extracts prepared as described previously (34) from
HeLa cells infected with 15 plaque-forming units/cell Ad2 for 12 h, or from uninfected cells. Transcription reactions contained the
quantities of template DNA listed in the figure legends, 0.1 pmol of
the major late G-less cassette template, pML(C2AT) (35) as
an RNA polymerase II-specific internal control, 5.5 mg/ml Ad2-infected
or uninfected cell extract protein, 67 mM KCl, 6.7 mM MgCl2, and other components as described
above for RNA polymerase III transcription. The reactions also
contained 40 µM tagetitoxin, a concentration empirically
determined to inhibit both E2E and VA RNA I transcription by RNA
polymerase III (see "Results"). Transcripts synthesized in
vitro were analyzed by reverse transcription from the primer
described above or a primer complementary to the luciferase sequence
corresponding to positions +94 to +113 of the pEII' Binding of TFIIIC to RNA Polymerase III Promoter
Sequences--
Human TFIIIC was partially purified from nuclear
extracts prepared from 3-4 × 1010 HeLa cells by
sequential chromatography on phosphocellulose, DEAE-cellulose, and
double-stranded DNA cellulose according to the procedure of Carey
et al. (36). Binding of TFIIIC to VA RNA I or E2E RNA
polymerase III promoters was examined using DNA fragments corresponding
to positions +6 to +74 or positions -15 to +100, respectively, of these
transcription units. The VA RNA I DNA fragment was obtained by
annealing complementary, purified oligonucleotides, while the E2E DNA
fragment was generated by PCR from the pEII' template with appropriate
primers. Mutated E2E DNA fragments used as competitors in some
experiments were made from mutated DNA templates with the same primers.
All PCR products were purified by electrophoresis in 12%
polyacrylamide gels cast, and run in 1× TBE buffer (0.09 M
Tris borate, pH 8.0, containing 2 mM EDTA). The
concentration of DNA fragments were determined by ethidium bromide
staining of the DNA following electrophoresis in 8% polyacrylamide
gels with a DNA mass ladder (Life Technologies, Inc.) as standard. DNA
fragments were end-labeled as described previously (33). Binding
reactions contained the quantities of 32P-labeled VA RNA I
or E2E DNA fragments, human TFIIIC, and competitor DNA listed in the
figure legends, 10 mM Hepes-KOH, pH 7.9, 50 mM
KCl, 2.5 mM MgCl2, and 25 ng of
poly(dI-dC)·(dI-dC) in a total volume of 20 µl. Free and
protein-bound DNA were separated by electrophoresis in 4%
polyacrylamide (60:1, acrylamide:bis-acrylamide gels) cast in 0.5× TBE
buffer and run in 0.25× TBE buffer at 150 V for 2 h at 4 °C,
following incubation of binding reactions at 30 °C for 45-60 min.
The quantities of specific TFIIIC·DNA formed in the presence
of increasing concentrations of competitor complexes were measured
using a Molecular Dynamics PhosphorImager. The molar excess
concentrations of competitor required for 50% competition were then
determined from plots of these values, averaged from several assays,
versus competitor concentration, and used to calculate the
affinity with which TFIIIC bound to the mutant DNAs relative to that
for binding to wild-type E2E DNA set at 1.00.
Elimination of the RNA Polymerase III t1 Termination Site Does Not
Alter the Efficiency of Transcription by This Enzyme--
The
termination of E2E transcription by RNA polymerase III at two sites
(Fig. 1) complicates analysis of E2E
transcription by the method of primer extension, which allows both
transcription efficiency and the accuracy of initiation to be evaluated
in a single assay. We therefore examined the effects of mutation of the
t1 termination site (Fig. 1) on the products made by RNA polymerase III
and the efficiency of transcription by this enzyme. Termination of
transcription by vertebrate RNA polymerase III can be affected by a
variety of signals (see Ref. 37 for a discussion). Nevertheless, a run
of four (or more) Ts in the nontranscribed strand is commonly present.
The last two of the four such T-A base pairs of the E2E t1 site (23)
were therefore replaced by A-T base pairs, to create the pEII'
This template and its wild-type parent were transcribed in the RNA
polymerase III-specific in vitro system described under "Experimental Procedures," and the E2E RNAs made from each template were compared using an RNase T1 protection assay with a complementary RNA probe specifically end-labeled at position +2, where +1 is defined
as described in the legend to Fig. 1. As illustrated in Fig.
2A, RNA polymerase III
transcription of the wild-type E2E template produced approximately
equal quantities of the two predicted RNase T1 protection products
corresponding to RNA I and RNA II. However, no 55-nucleotide protection
product corresponding to RNA I was observed when RNA polymerase III
transcripts of the E2E Comparison of the Sites of Initiation of E2E Transcription by RNA
Polymerases II and III--
To map accurately the site(s) at which RNA
polymerase III initiates E2E transcription, located previously in the
vicinity of the major initiation site recognized by RNA polymerase II
(23), primer extension was used to compare the 5'-ends of transcripts synthesized from the E2E Identification of Intragenic Elements of the E2E RNA Polymerase III
Promoter--
Previous studies established that E2E sequences 3' to
position +62 are not necessary for efficient RNA polymerase III
promoter activity in vitro (23), whereas deletion of
sequences downstream of position +2 or of positions +22 to +62 reduced
transcription to undetectable levels (data not shown). We therefore
examined the effects of precise substitutions within the region +2 to
+62 on the efficiency and accuracy of transcription. Both the 10- and
5-bp substitutions shown in Fig. 3, which
were named according to the positions of the mutated base pairs, were
introduced into the
These results indicated that the E2E RNA polymerase III promoter
includes two blocks of internal sequence, occupying positions +22 to
+26 and positions +42 to +62 of the transcription unit. This
organization resembles that of type 2 RNA polymerase III promoters (see
the introduction) as well as some that comprise both intragenic and
essential upstream sequences, such as the promoter of the EBV
EBER RNA 2 gene (40, 41). We therefore searched the intragenic E2E
RNA polymerase III promoter sequences for consensus A and B box
sequences (6). As illustrated in Fig. 5,
the functionally defined promoter sequence +22 to +26 is included
within a sequence that resembles the A box consensus sequence of tRNA
genes: both the inhibitory substitutions mut22-26 and mut22-31 (Fig.
4C) reduced the match to the A box consensus sequence from
7/12 (Fig. 5) to 4/12 (Fig. 3) positions. The alterations in initiation
specificity to include the +1 and +3 sites induced by all substitutions
within this intragenic E2E sequence (Fig. 4A, lanes
4 and 5; Fig. 4B, lanes 5-8)
provide strong support for its classification as an A box, for such
change in initiation specificity is characteristic of mutations within
the A box sequence of type 2 RNA polymerase III promoters
(e.g. Refs. 42-44). Furthermore, as described in the next
section, substitutions within the A box consensus sequence shown in
Fig. 5 impaired binding of human TFIIIC to the E2E promoter. Although
templates carrying the mut14-19 and mut17-21 substitutions that alter
the 5' portion of the A box consensus sequence were transcribed more
efficiently than the wild-type E2E template (Fig. 4C), these
mutations did not improve the match to the consensus. Rather, both
these substitutions reduced the match, to 6/12 positions, and each
eliminated one of the two invariant base pairs of the consensus
sequence, both of which are represented in the wild-type E2E sequence
(Figs. 3 and 5). An alternative explanation for these observations is discussed below. In contrast to the ready identification of an A box
consensus sequence, it was not possible to locate a B box by inspection
of the more downstream E2E sequence required for efficient RNA
polymerase III transcription: this 21-bp segment contains two adjacent
sequences that are weak matches to the B box consensus sequence (Fig.
5). Such ambiguity prompted us to evaluate more directly the question
of whether the E2E RNA polymerase III promoter contains a B box
sequence.
Human TFIIIC Components Recognize the Internal RNA Polymerase
III Promoter Sequences--
The A and B box sequences of type 2 RNA
polymerase III promoters are recognized by components of TFIIIC (see
the introduction). Thus, if the internal elements of the RNA polymerase
III promoter described above included functional A and B sequences,
TFIIIC should bind specifically to the E2E promoter. To test this
prediction, the interactions of human TFIIIC with the internal
sequences of the Ad2 VA RNA I and E2E RNA polymerase III promoters were
compared using electrophoretic mobility shift assays (45, 46). The human TFIIIC used in these experiments was partially purified by a
previously described procedure (36), but was not subjected to B-box
DNA-affinity chromatography (10). The latter procedure separates the B
box binding component, TFIIIC2, from a second component, TFIIIC1, which
is required for RNA polymerase III transcription and interacts strongly
with termination signals and weakly with A box sequences (10, 13-15).
Because a B box sequence had not been unambiguously identified in the
E2E promoter, it seemed prudent to avoid separation of the
promoter-binding proteins of TFIIIC.
As expected, human TFIIIC bound specifically to a DNA fragment spanning
the A and B box sequences of the VA RNA I gene: formation of
a pair of protein·DNA complexes was inhibited by homologous unlabeled
DNA but not by an oligonucleotide unrelated in sequence (Fig.
6A, lanes 1-3).
The mobility of these two complexes relative to free DNA and to one
another suggests that they represent previously described TFIIIC2·DNA
complexes containing transcriptionally active and inactive forms of
this initiation protein (12, 13). An E2E DNA fragment containing all
sequences transcribed by RNA polymerase III also inhibited binding of
TFIIIC to the internal promoter sequences of the VA RNA I
gene (Fig. 6A, lanes 1 and 4). When this E2E DNA fragment was used as a probe, a set of specific
protein·DNA complexes, two of which exhibited the same mobilities as
the TFIIIC·VA I DNA complexes, was observed (Fig. 6A,
lanes 5, 6, and 8). Their formation
was inhibited by both homologous, unlabeled DNA and the DNA containing
the VA RNA I promoter (Fig. 6A, lanes 5,
7, and 8). These results established that human
TFIIIC specifically recognizes internal sequences of the E2E RNA
polymerase III promoter.
We next examined the relationship between binding of TFIIIC to the
internal E2E promoter sequence and the initiation of transcription by
RNA polymerase III, by determining whether mutations that inhibited E2E
transcription altered the binding of the protein. The abilities of
wild-type DNA and mutant DNA fragments carrying specific substitutions to act as competitors of binding of TFIIIC to 32P-labeled
E2E DNA were compared as a function of competitor concentration. The
mut27-31 substitution, which did not decrease the efficiency of E2E
transcription by RNA polymerase III (Fig. 4, B and
C), did not impair binding of TFIIIC to wild-type E2E DNA
(Fig. 6B, compare lanes 1-5 and
11-15). Mutations within the portion of the E2E sequence
resembling an A box inhibited binding of TFIIIC, but by only a modest
degree (Table I). In contrast,
substitutions within the +42 to +62 sequence required for efficient
transcription by this enzyme significantly impaired binding of TFIIIC
(Fig. 6B, lanes 6-10; Table I). The correlation
between the effects of the substitution mutations on E2E transcription
by RNA polymerase III (Fig. 4) and on binding of TFIIIC components to
E2E DNA (Table I) indicated that binding of TFIIIC to internal promoter
sequences is required for RNA polymerase III transcription. Thus, the
intragenic sequences required for E2E transcription by RNA polymerase
III appear to be functionally equivalent to those of the well
characterized type 2 promoters recognized by this enzyme.
Identification of Internal Sequences Required for RNA
Polymerase II Transcription--
As the contribution of
transcribed sequences to the E2E RNA polymerase II promoter had never
been examined, we also determined the effects of the intragenic
mutations on transcription by this enzyme. Initial surveys of E2E
transcription by RNA polymerase II and RNA polymerase III,
distinguished by their sensitivities to 2 µg/ml
Substitution of sequences between positions +32 and +62 did not
significantly alter the efficiency of RNA polymerase II transcription (Fig. 7B, compares lanes 1 and 5-8;
Fig. 7C). However, mutation of the sequence adjacent to the
initiation site (+2 to +11), as well as a more downstream segment (+14
to +21), resulted in inhibition of transcription by up to a factor of 8 (Fig. 7A, lanes 1-3 and 5-7; Fig.
7C). Thus, the E2E RNA polymerase II promoter extends for
some 20 bp beyond the initiation site. Although substitution of
positions +12 to +16 did not inhibit transcription (Figs.
7A, lanes 1 and 4, and 7C),
direct inhibitory effects on RNA polymerase II transcription might be
offset by relief of competition from RNA polymerase III transcription
(see "Discussion"), for this substitution impinges on the A box of
the RNA polymerase III promoter (Fig. 5). Substitution of positions +27
to +31 increased the efficiency of RNA transcription, by some 2.5-fold
(Figs. 7B, lanes 1 and 4, and
7C). None of these intragenic sequence mutations altered the
specificity of initiation of E2E transcription by RNA polymerase II
(Fig. 7, A and B), indicating that this parameter
is determined by RNA polymerase II promoter sequences lying upstream of
the initiation site.
The promoters of many cellular and viral genes transcribed by RNA
polymerase III include intragenic A and B boxes (see Refs. 6, 8, 16 for
reviews). The adenoviral E2E RNA polymerase III promoter also contains
two intragenic sequences required for efficient or accurate
transcription by this enzyme (Fig. 4). The sequence of the initiation
site-proximal promoter element (Fig. 5) and the alterations in
initiation specificity observed when it was mutated (Fig. 4,
A and B) allow it unambiguous identification as
an A box. The specific binding of human TFIIIC to internal sequences of
the E2E RNA polymerase III transcription unit (Fig. 6) confirms that
this viral promoter contains a B box (10, 13-15). The observation that
mutations within the sequence +42 to +62 strongly impaired both
transcription (Fig. 4) and TFIIIC binding (Fig. 6B; Table I)
locate the E2E B box to within this 21-bp segment of the transcription
unit. However, two nonoverlapping candidate sequences lie within this
region (Fig. 5). Several arguments suggest that the E2E sequence
located between positions +51 and +61 is the binding site for TFIIIC2.
This sequence includes three times as many of the invariant base pairs
of the B box consensus sequences as the functionally defined sequence
immediately 5' to it (Fig. 5). Furthermore, a B box at positions +51 to
+61 would yield a separation of E2E A and B box sequences of 26 bp,
closer to the range typically observed (see Refs. 6, 8, 16, 47) and
required for optimal type 2 promoter function (e.g. Refs. 42, 48). Specific binding of the TFIIIC1 component of TFIIIC to the
termination regions of the Ad2 VA RNA I gene and a
Xenopus leavis
tRNA1Met gene has been reported
to stabilize binding of TFIIIC2 to the promoters to support maximally
efficient transcription (14). The E2E t1 termination site for RNA
polymerase III transcription (Fig. 1) is included within the 10-bp
sequence lying 5' to the putative B box. Thus, TFIIIC1 may bind to a
region that includes this sequence to stabilize otherwise low affinity
binding of TFIIIC2 to the adjacent B box. Further experiments will be
required to determine whether such a mechanism of TFIIIC binding
accounts for the requirement for an unusually long, initiation
site-distal, intragenic sequence.
The E2E RNA polymerase III promoter does not belong to one of the three
types now generally recognized (see Refs. 6, 8, 16), but rather closely
resembles that of the EBV EBER RNA 2 gene: both include
intragenic A and B boxes and upstream TATA-like sequences (Fig. 5 (23,
40))2 and binding sites for sequence-specific activators of
RNA polymerase II transcription that are necessary for maximal activity
of the promoters (23, 40). Despite such similarities, the EBV
EBER RNA 2 gene is transcribed only by RNA polymerase III. One
important determinant of such exclusivity is its TATA-like sequence,
designated ETAB, located at positions Mutations introduced between positions +14 and +21, a sequence that
includes most of the A box of the RNA polymerase III promoter (Fig. 5),
significantly inhibited E2E transcription by RNA polymerase II (Fig.
7). Thus, a single intragenic E2E sequence functions as part of both
the RNA polymerase II and the RNA polymerase III promoters (Fig. 5).
This arrangement seems likely to account for the initially surprising
observation that mutations introduced into the 5'-end of the A box
altered initiation specificity but either did not reduce, or modestly
increased, the efficiency of transcription by RNA polymerase III (Fig.
4). In the reactions specific for this enzyme, RNA polymerase II was
inhibited by addition of an appropriate concentration of Substitutions between positions +2 and +11 that inhibited RNA
polymerase II transcription did not stimulate RNA polymerase III
transcription (Figs. 4 and 7). Conversely, when the B box-containing region of the RNA polymerase III promoter was crippled, no increase in
transcription was observed in the RNA polymerase II-specific system, in
which tagetitoxin inhibits elongation by RNA polymerase III with no
apparent effect on preinitiation complex assembly (50). It therefore
appears that binding of TFIIIC to the RNA polymerase III promoter
neither interferes directly with recognition of the RNA polymerase II
promoter, nor is the reaction rate-limiting for assembly of stable
preinitiation complexes committed to transcription by RNA polymerase
III under the conditions used in these experiments. A number of
parameters, including absolute and relative concentrations of
initiation proteins specific for each RNA polymerase, their affinities
for binding sites in the template or in other proteins, and, in the
cell, the relative concentrations of the RNA polymerase holoenzymes
that are believed to mediate initiation of transcription (see Refs.
51-53), seem likely to govern which reactions will be in competition
under different conditions. It will therefore be important to establish
which sequences of the RNA polymerase II and RNA polymerase III
promoters set the balance of E2E transcription by the two RNA
polymerases in adenovirus-infected cells.
Because E2E RNA I contains only the 5'-half of the sequence of the
longer E2E RNA II (Fig. 1), it is unlikely that these RNA species
conform to a common structure necessary for a common function. In fact,
the run of four T-A base pairs of the E2E t1 termination site of Ad2 or
Ad5 is not conserved even in the genomes of all human members of the
Mastadenoviridae, and elimination of the t1 site does not
impair replication of subgroup C
adenoviruses.4 These
properties of the small E2E RNAs, and the low concentrations at which
they are present in infected cells (24), suggest that production of
functional RNA species may not be the primary purpose of transcription
of E2E sequences by RNA polymerase III. Rather, such transcription
might negatively regulate expression of the viral replication proteins
encoded in the RNA polymerase II E2E transcription unit. The
competition for access to superimposed sequences of the two E2E
promoters discussed above is also consistent with such a regulatory
role. Analogous function as regulators of RNA polymerase II
transcription has been proposed (e.g. Refs. 26, 54, 55) or
demonstrated (56) for specific RNA polymerase III transcription units
present in cellular genomes. The identification of sequences that
contribute to one or both of the adenoviral E2E promoters reported here
will allow the role of the E2E RNA polymerase III promoter in the
adenoviral genome to be tested, using genetic approaches, to determine
whether the small E2E RNA species synthesized by RNA polymerase III
perform a specific function during the adenoviral infectious cycle or
are by-products of a less well understood regulatory mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
t1 template, which was used as
the parent in mutational analyses of intragenic promoter sequences (see
"Results"), contained two T to A substitutions at positions +48 and
+49 within the RNA polymerase III t1 termination site (see Fig. 1).
Precise substitutions were introduced into the pEII'
t1 template by
the unique site elimination method (29). A control "maxigene"
template, to serve as an internal control for in
vitro transcription reactions, was constructed by ligating a 30-bp
random DNA sequence flanked by PvuII sites into the
PvuII site (+62) of pEII'. A second series of templates, comprising the E2E DNA sequence from position -249 to position +62, or
specifically mutated derivatives thereof, linked to the Photinus
pyralis luciferase coding sequence was constructed for analysis of E2E transcription in whole cell extracts prepared from
Ad2-infected cells. A DNA fragment encompassing the first 535 bp of the
P. pyralis luciferase gene was amplified from
pGL2 (Promega) using primers designed to introduce a PvuII
and a BglII site at the 5'- and 3'-ends, respectively, of
the PCR product. The PvuII/BglII-digested DNA was then
ligated into similarly digested pEII'
t1 DNA to place the E2E
promoter region from -249 to +62 immediately upstream of the luciferase
sequence. The substitution mutations created in the pEII'
t1
background were then introduced by swapping XhoI to
PvuII fragments, or by the unique site elimination method.
The nucleotide sequences of all pEII'
t1 and
t1/Luc templates were
confirmed by the chain termination DNA sequencing method (30).
-amanitin to inhibit RNA polymerase II, 0.1 pmol
of template DNA, 0.05 pmol of the internal control template described
in the previous section, 1.33 mg/ml nuclear extract protein, 25 mM Hepes-KOH (pH 7.9), 30 mM KCl, 6 mM MgCl2, 600 µM each GTP, ATP,
UTP, and CTP, 12% glycerol, and 2 mM dithiothreitol.
Transcripts synthesized in 60 min at 30 °C were purified and
analyzed by primer extension, as described previously (32, 33), using a
saturating quantity (20 fmol) of a DNA primer complementary to
positions +68 to +89 of the E2E transcription unit. The concentrations
of the primer extension products of E2E and internal control
transcripts were determined using a Molecular Dynamics PhosphorImager.
Initial analyses of transcription from the pEII' and pEII'
t1
templates employed an RNase T1 protection assay with an E2E riboprobe
specifically end-labeled at positions +2, as described previously
(24).
t1/Luc
transcription unit.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
t1
mutant template.
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Fig. 1.
Features of the Ad2 E2E transcription
units. The region of the human Ad2 genome spanning the 5'-end of
the RNA polymerase II transcription unit is depicted by the
horizontal line at the top, on which features of
the RNA polymerase II and III transcription units and promoters are
indicated. The major site of initiation by RNA polymerase II, which is
denoted by the jointed arrow, is defined as +1. The RNA
polymerase III termination sites t1 and t2, each of which comprise 4 or
more TA base pairs flanked by GC-rich sequences (23), are denoted by
the inverted triangles. The vertical arrowhead at
+68 represents the 5'-splice site between exon 1 and intron 1 of E2E
pre-mRNA. The pre-mRNA and mRNA products of RNA polymerase
II transcription and the small RNA species (E2E RNA I and
RNA II) transcribed by RNA polymerase III are represented
below.
t1 template were examined in parallel (Fig.
2A, compare lanes 1 and 2).
Concomitantly, this mutation increased the concentration of E2E RNA II
synthesized by RNA polymerase III in vitro (Fig. 2A, lanes 1 and 2). Indeed, the
concentration of the E2E RNA II transcribed from the E2E
t1 template
was 106.5% (±1.5%) of the total concentration of the small E2E RNA
species synthesized from the wild-type template. Because the
t1
mutation prevented termination at the t1 site but did not inhibit RNA
polymerase III transcription, the
t1 template was used in all
subsequent transcription assays as a surrogate wild-type.
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Fig. 2.
Transcription of the
E2E t1 template by the RNA polymerase III.
A, transcription reactions contained 0.2 pmol of the
wild-type (wt) or pEII'
t1 (
t1)
templates, 1.33 mg/ml HeLa cell nuclear extract, and 2 µg/ml
-amanitin. Transcription reactions and purification of the
transcription products were as described under "Experimental
Procedures." The RNA was examined by RNase T1 protection following
hybridization to an antisense E2E RNA specifically end-labeled at
position +2. The protection products observed following electrophoresis
in an 8% sequencing gel are identified at the left. DNA and
RNA markers were run in lanes 3 and 4,
respectively. The lengths of the RNA markers are listed at the
right. B, transcription reactions contained no
template (lane 1) or 2.0 pmol of E2E
t1Luc (lanes
2-5), no drugs (lanes 1 and 2), 40 µM tagetitoxin (lane 3), 2 µg/ml
-amanitin (lane 4), or both drugs (lanes 5)
and Ad2-infected whole HeLa cell extract protein. End-labeled
HaeIII fragments of pBR322 DNA of the lengths indicated were
run in lane 6. The positions of cDNA copied from RNA
polymerase II transcripts initiating at position +1 or from RNA
polymerase III transcripts with 5'-ends at positions +2 and
1 are
indicated by the asterisk or arrows,
respectively, at the left. These products were quantified
using a PhosphorImager. The values obtained were corrected for
variations in recovery of products by quantification of signals from a
single-stranded, 32P-labeled oligonucleotide was added to
each reaction once transcription was complete and used to calculate the
contributions of each RNA polymerase to the total transcription from
the E2E transcriptional control region (shown at top).
t1 template in reactions in which only one
of the two enzymes was active. The fungal toxin
-amanitin (at a low
concentration, 2 µg/ml) and the bacterial phytotoxin tagetitoxin (40 µM) were exploited to inhibit RNA polymerases II and III,
respectively. The latter compound has been reported to be a specific
inhibitor of RNA polymerase III transcription from the type 1 and type
3 promoters of the 5 S RNA and U6 genes, respectively (38).
It inhibited completely RNA polymerase III transcription from both the
Ad2 VA RNA I and E2E promoters by a concentration of 30 µM, with no effect on RNA polymerase II transcription
from the adenoviral major late promoter (data not shown). When RNA
polymerase III was inhibited by the addition of tagetitoxin to the
reaction, initiation was observed at a single site (Fig. 2B,
lane 3) corresponding to the major cap site of E2E mRNAs
made by RNA polymerase II in infected cells (39), by definition
position +1. In contrast, RNA polymerase III transcription of the E2E
template, initiated predominantly from position +2, although
transcripts beginning at position
1 were also detected (Fig. 2B,
lane 4). Thus, the two enzymes initiated
transcription from distinct sites.
t1 background, as described under
"Experimental Procedures." The mutant and wild-type (
t1)
templates were transcribed in vitro by RNA polymerase III,
and the products were analyzed by primer extension. To allow accurate
quantification of the effects of the mutations on the efficiency of
transcription, an E2E' maxigene, which contains an insertion of a 30-bp
random DNA sequence at position +62, was included in all transcription
reactions. Typical results of these analyses are shown in Fig.
4, A and B, and the effects of the mutations upon the efficiency of RNA polymerase III
transcription are summarized in Fig. 4C. None of the
substitutions introduced immediately downstream of the initiation
sites, such as mut2-6, mut7-11, or mut7-16 (Fig. 4A,
lanes 3 and 4; Fig. 4B, lanes
3-5), or between positions +27 and +42 (Fig. 4A,
lanes 3 and 7; Fig. 4B, lanes
3 and 10) resulted in significant inhibition of RNA
polymerase III transcription (Fig. 4C). In contrast, the quantities of E2E transcripts detected in reactions containing the
mut22-31, mut42-51, and mut52-62 templates were severely reduced, to
values of 0.22, 0.04, and 0.06, respectively, relative to the wild-type
concentration set at 1.00 (Fig. 4A, lanes 6,
8, and 9; Fig. 4C). None of these
changes in the transcribed sequence altered the stability of the RNA
made in the in vitro transcription reactions (data not
shown), indicating that these substitutions inhibited transcription.
Various 5-bp substitutions made between positions +42 and +62 also
severely inhibited transcription, as did the mut48-57 substitution,
which overlapped the 10-bp alterations initially introduced and
analyzed (Fig. 4B, compare lanes 3,
11, 12, and 13; Fig. 4C).
These properties suggest that the 21-bp segment occupying positions +42
and +62 comprises a single, functional promoter element. The data
reported in Fig. 4, in conjunction with previous analysis of E2E
templates carrying 3'-deletions (23), indicate that its 3'-boundary is
close to position +62, whereas its 5'-boundary can be placed between
positions +42 and +47. Mutation of the sequence +22 to +26 also
resulted in inhibition of transcription (Fig. 4B, lane
10; Fig. 4C), confirming the presence of a second
intragenic promoter sequence. In contrast, templates carrying mutations
between positions +12 and +21 directed somewhat more efficient
transcription by RNA polymerase III, with the greatest increase (a
factor of 2.5) observed when the sequence between positions +14 and +19
was altered (Fig. 4B, lane 6; Fig.
4C).
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Fig. 3.
Sequences of mutated E2E templates. The
sequence of the surrogate wild-type E2E t1 template from positions +1
to +68 is shown at the top, with the mutation in the t1
termination site shown in boldface and
italics. The A box sequence is highlighted
in gray. Positions that match the consensus A box sequence
in Fig. 5 are shown in boldface. The sequences altered in
the mutant templates listed at the right are shown below,
with boldface indicating changes that maintain a match to
the A box consensus.
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Fig. 4.
Identification of intragenic sequences
required for E2E transcription by RNA polymerase III.
Transcription of the wild-type E2E template t1 or its mutated
derivatives carrying 10-bp (A) or 5-bp (B)
substitutions between positions +2 and +62 was carried out under the
RNA polymerase III-specific conditions described under "Experimental
Procedures." Transcription reactions contained 0.05 pmol of the
internal control E2E maxigene template and no test template (lane
2 in A and B) or 0.1 pmol of wild-type
E2E
t1 DNA (A and B, lane 3) or the
mutant templates mut7-16, mut17-26, mut22-31, mut32-41, mut42-51,
and mut 52-62 (A, lanes 4-9, respectively) or
mut2-6, mut7-11, mut12-16, mut14-19, mut17-21, mut22-26,
mut27-31, mut42-47, mut48-57, and mut58-62 (B,
lanes 4-13, respectively), which are designated m7-16 and
so on. Transcripts were detected by primer extension, followed by
electrophoresis of cDNA products under denaturing conditions and
autoradiography (see "Experimental Procedures"). The positions of
the cDNAs synthesized from the E2E (E2E) and internal
control (IC) templates are indicated at the
right, and the lengths of DNA markers (5'-end-labeled
HaeIII fragments of pBR322 DNA run in lane 1 in
each of the gels shown) are listed at the left. The specific
E2E cDNAs were quantified directly using a Molecular Dynamics
PhosphorImager, and values obtained for each mutant template were
corrected using those of products of transcription of the internal
control template. In C, these corrected values are expressed
relative to that obtained from the wild-type E2E
t1 template, set at
1.0. The relative activity shown for each mutant template represents
the mean of three independent experiments.
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Fig. 5.
Comparison of intragenic E2E RNA polymerase
III promoter sequences to tRNA consensus A and B boxes. The
sequence of the Ad2 E2E transcription units extending to position +68
is shown at the top. Sequences required for efficient or accurate
transcription by RNA polymerase III are underlined, whereas
those necessary for maximally efficient transcription by RNA polymerase
II are indicated by the line above the sequence. Consensus A
and B box sequences (6, 44) and typical distances between them are
shown below, with invariant nucleotides in larger
sized font, where R = a purine, Y = a pyrimidine, and N = any nucleotide. The best
matches of E2E sequences within the functionally defined promoter
regions are indicated below, with conserved nucleotides in
boldface.
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Fig. 6.
Specific binding of human TFIIIC to internal
sequences of the VA RNA I and E2E promoters. A, binding
reactions were as described under "Experimental Procedures," and
contained 0.35 fmol of a 32P-labeled DNA fragment
comprising positions +6 to +74 of the Ad2 VA RNA I gene
(VAI, lanes 1-4) or positions -15 to +100 of the
Ad2 E2E transcription unit (E2E, lanes 5-10),
0.58 µg of partially purified human TFIIIC and no competitor
(lanes 1 and 5), or 100-fold molar excess of the
competitors indicated at the top, where NS = an unrelated DNA competitor. Protein·DNA complexes and free DNA were
separated by electrophoresis, as described under "Experimental
Procedures." Specific TFIIIC·DNA complexes are indicated by the
arrows. B, binding of human TFIIIC to
32P-labeled E2E DNA was as described in A,
except that the reactions contained increasing concentrations (from 10- to 100-fold molar excess) of homologous, unlabeled E2E DNA
(E2E), or E2E DNA fragments containing the substitution
mutations designated as described in the legend to Fig. 4 as
indicated.
Relative affinities of TFIIC for mutated E2E promoter sequences
-amanitin and 40 µM tagetitoxin, respectively, indicated that RNA
polymerase III transcription invariably predominated in both nuclear
and whole HeLa cell extracts (data not shown). However, two conditions
that increased the efficiency of E2E transcription by RNA polymerase II
relative to that by RNA polymerase III were identified. Under in
vitro conditions typically used for RNA polymerase II
transcription, the proportion of the total E2E RNA synthesized by this
polymerase was 2- to 3-fold greater in whole HeLa cell extracts
prepared from cells harvested 8-12 h after Ad2 infection than in
uninfected cell extracts prepared in parallel (data not shown),
reaching some 30% of the total (Fig. 2B). This difference could be the result of stimulation of RNA polymerase II transcription from the E2E promoter by the viral E1A proteins (see Ref. 4). We also
observed that the fraction of E2E RNA made by RNA polymerase II in
uninfected cell extracts increased to values like those supported by
Ad2-infected cell extracts when the concentration of template DNA was
reduced (data not shown). To avoid possible complications that might be
introduced by effects of viral proteins, the latter condition was used
to examine the effects of the E2E mutations described previously on RNA
polymerase II transcription. A G-less cassette under the control of the
viral ML promoter served as an internal control (see "Experimental
Procedures"). Typical results of these experiments are shown in Fig.
7, A and B, and the
quantification of several is summarized in Fig. 7C.
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Fig. 7.
Effects of intragenic E2E substitutions on
RNA polymerase II transcription. In vitro transcription
reactions were as described under "Experimental Procedures" and
contained 0.2 pmol of E2E t1 luciferase (
t1)
template DNA or the derivatives carrying the substitutions mut2-6,
mut7-11, mut12-16, mut14-19, mut17-21, mut17-26, mut22-26,
mut22-31, mut27-31, mut32-41, mut42-51, mut52-62, and mut48-62
(A, lanes 2-14, respectively), designated as in
Fig. 4. The positions of the primer extension products of the E2E
(E2E) and the pML(C2AT) internal control
(ML) transcripts are indicated at the right. The
results of quantification of the effects of these mutations on the
efficiency of RNA polymerase II transcription, corrected using the ML
internal control and normalized to the E2E
t1 template as described
in the legend to Fig. 4, are shown in B. Each value is the
mean of at least three independent determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
25 to
30 (41). The E2E
TATA-like sequence, TTAAGAGT, is noncanonical, for it includes GC base
pairs at positions analogous to those of the ETAB sequence. However, in
contrast to this latter sequence, the E2E TATA sequence is also part of
a functional RNA polymerase II promoter (see the introduction and Fig.
1) and behaves as a typical RNA polymerase II TATA sequence in assays
for TBP binding and transcriptional function.3 It will be of
considerable interest to identify the features of the E2E TATA sequence
that allow recognition by TBP-containing initiation proteins specific
for both RNA polymerase II and RNA polymerase III.
-amanitin.
This toxin binds to the largest subunit of RNA polymerase II and
inhibits transcription after assembly of preinitiation complexes and
formation of the first phosphodiester bond in nascent RNA (see Ref.
49). Consequently, when a mutation alters both the RNA polymerase III
and the RNA polymerase II promoters, such as several that impinge on
the A box, direct inhibition of RNA polymerase III transcription may be
offset by inhibition of formation of RNA polymerase II initiation complexes. Thus, RNA polymerase III transcription would appear unchanged or stimulated, as indeed observed (Fig. 4). The properties of
the E2E template with a substitution at the 3'-end of the A box
(positions +22 to +26) provide additional support for the hypothesis
that proteins mediating recognition of the RNA polymerase II and the
RNA polymerase III promoters are in competition for superimposed
binding sites: This mutation had no significant effect on the
efficiency of transcription by RNA polymerase II (Fig. 7, B
and C), and inhibited E2E transcription by RNA polymerase III by some 90% (Fig. 4, B and C).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Alison McLane for initial analyses of binding of TFIIIC to VA-RNA I and E2E promoters, and Jana Kiefer and Dara Whalen for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was funded by a grant from the National Institutes of Health.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.
Supported by a fellowship from the Medical Research Council of
Canada. Present address: University of Colorado Health Sciences Center,
Pediatric Hematology-Oncology Laboratories, Dept. of Pediatrics, 4200 East Ninth Ave., Denver, CO 80262.
§ To whom correspondence should be addressed: Tel.: 609-258-6113; Fax: 609-258-2759; E-mail: sjflint@molbio.princeton.edu.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M007036200
2 W. Huang and S. J. Flint, unpublished observations.
3 H. Chen and S. J. Flint, unpublished observation.
4 R. L. Finnen, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: E2E, E2 early; Ad2, adenovirus 2; PCR, polymerase chain reaction; bp, base pair(s).
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