(Received for publication, August 17, 1995; and in revised form, September 25, 1995)
From the
Mammalian ribonucleotide reductase shows S-phase specific
expression and consists of two non-identical subunits, proteins R1
(large subunit) and R2 (small subunit). A comparison between the human
and mouse TATA-less R1 gene promoters revealed four highly conserved
DNA regions, while the remaining sequence showed a low degree of
conservation. Two regions, and
, were earlier identified as
protein binding regions in the mouse R1 promoter by using DNase
footprinting technique. The two new regions are located to the
transcription start and to a DNA sequence about 40 base pairs
downstream from the start. Gel shift assays using TFII-I antibodies and
competition with an oligonucleotide representing the terminal
deoxynucleotidyl transferase initiator element identified the start
region as a TFII-I binding initiator element. The conserved downstream
region, called
, also formed specific DNA-protein complexes in gel
shift assays. Functional studies, using synchronized cells stably
transformed by R1 promoter-luciferase reporter gene constructs,
indicated that the initiator and the
elements together were
necessary for cell cycle-regulated R1 promoter activity. Earlier
published data, indicating Sp1 binding to the R1
/
regions,
could not be confirmed, suggesting that the R1 initiator element may
function independent of Sp1.
Ribonucleotide reductase is a key enzyme in the synthesis of DNA precursors, catalyzing the reduction of the four ribonucleotides to their corresponding deoxyribonucleotides. The mammalian enzyme belongs to the class I ribonucleotide reductases(1, 2) . In this class, the active enzyme consists of two nonidentical homodimeric subunits, proteins R1 and R2, each inactive alone. The large subunit (R1) binds the ribonucleoside diphosphate substrates and the nucleoside triphosphate allosteric effectors as well as harboring the active site, containing redox-active cysteine residues. The small subunit (R2) contains binuclear non-heme iron centers, which generate a tyrosyl free radical required for catalysis. The crystal structures of the corresponding Escherichia coli proteins were recently published(3, 4) .
Ribonucleotide reductase activity is cell cycle regulated showing maximal levels during the S-phase. This regulation is imposed by de novo synthesis and breakdown of the R2 protein while the R1 protein is present in constant and excess amounts during the cell cycle(5, 6) .
The levels of
R1 and R2 mRNA in serum-synchronized cells and in elutriated cells vary
in parallel with undetectable levels in G/G
cells and maximal values in S-phase
cells(7, 8) .
In mouse cells, the R1 gene is
localized to chromosome 7 (9) and the active R2 gene to
chromosome 12(10) . The mouse R1 gene covers 26 kilobases and
contains 19 exons. In contrast to the R2 promoter, the R1 promoter
lacks a TATA box. DNase I footprinting assays identified two
protein-binding regions in the mouse R1 promoter, (nt (
)-98 to -76) and
(nt -189 to
-167), that are identical except for one base pair. Three protein
complexes bound to each 23-mer, and one showed S-phase specific
binding(11) . The sequence of the human R1 gene promoter was
recently published(12) .
In common promoters the TATA box is the binding site for the general transcription factor TBP, which in turn promotes the assembly of the transcription initiation complex(13, 14) . Other promoters lack TATA box and instead contain an initiator element at the transcription start. This element directs the transcription start to a certain nucleotide, as in the case of terminal deoxynucleotidyl transferase (TdT)(15) . The mouse thymidylate synthase gene, lacking both a TATA box and an initiator element, has a region that displays a random start of transcription. However, the insertion of a TdT initiator element in the thymidylate synthase promoter will direct the start of transcription to a major start site(16) . Finally, there are promoters that contain both a TATA box and an initiator element, e.g. the adenovirus-major late promoter (Ad-ML). A model has been presented, based on the Ad-ML promoter, suggesting different pathways for the formation of the preinitiation complex(17) . The pathway of choice is decided by the presence or absence of a TATA box or an Inr element.
The transcription factor TFII-I, a 120-kDa polypeptide, binds to the initiator elements of the Ad-ML promoter, the TdT promoter, and the human immunodeficiency virus-1 promoter (HIV-1)(18) . TFII-I, in combination with other proteins, has been shown to promote the assembly of a transcription initiation complex containing TBP on the Ad-ML initiator element(17) . In this paper, we present data showing that the TATA-less mouse ribonucleotide reductase R1 gene promoter contains an initiator element of the TdT type. The initiator binding protein TFII-I binds to the R1 initiator element. We also show that the R1 initiator element is important for the regulation of promoter activity.
Figure 1: R1 promoter-luciferase reporter gene constructs used to stably transform BALB/3T3 cells for functional promoter analyses. The boxed regions show the different protein binding elements, and the luciferase gene is represented by a filled-in box. The solid arrow indicates the start of transcription.
The plasmids p19lucR1 0.49 and p19lucR1 0.30 were created by digesting p19lucR1 1.0 with, respectively, EcoRV and AccI (AccI filled in by the Klenow fragment of DNA polymerase I) together with SphI (in the luciferase gene). The resulting fragments were ligated into p19luc opened with HindIII (filled in by the Klenow fragment of DNA polymerase I) and SphI (in the luciferase gene). The promoter fragments in p19lucR1 0.49 and p19lucR1 0.30 started at nt -241 and nt -53, respectively, and both ended at nt +242.
Overlap extension polymerase chain reaction (20) was used to create the mutations of the Inr and the
regions in the two constructs p19lucR1 0.30-Inr and p19lucR1
0.30-
. Briefly, to construct p19lucR1 0.30-Inr, we first amplified
the region upstream from the Inr, from -53 to -8, with the
oligonucleotides 5`-TCCCAAGCTTGGGTCTACTGCTCAGTTTCCGCC-3` (P3) and
5`-AACCAGATATCCAGAACAGACGTTTCAACGCCGGGAGT-3` (P4).
5`-TGTTCTGGATATCTGGTTTCGCGTTGCTCTGCACGTCA-3` (P5) and P2 were used to
amplify the region downstream from the Inr, from nt +19 to nt
+242. These two fragments with overlapping 3` termini were mixed
in a polymerase chain reaction together with P3 and P2. A non-protein
binding region of the R1 promoter (nt -249 to nt -232) had
replaced the Inr element -7 to +18 in the new fragment. The
new fragment was digested with HindIII (underlined in P3) and StuI (underlined in P2) and ligated into the HindIII
and SmaI sites of p19luc. The construct p19lucR1 0.30-
was made by the same technique with the following modifications. The
primers 5`-AACCAGATATCCAGAACATGCAGAGCAACGCGACGGAC-3` (P6) and
5`-TGTTCTGGATATCTGGTTTTCGTAATTCGGTTAGTCTG-3` (P7) were used instead of
primers P4 and P5. The same region that replaced the Inr in the
previous construct had now replaced the region (from nt +34 to nt
+59).
All constructs were verified by restriction enzyme analysis or dideoxyribonucleotide sequencing.
A typical reaction mixture included 1.4 fmol end-labeled oligonucleotide (7100 cpm/fmol), 0.78 µg of poly(dI-dC), 10% glycerol, 10 mM Hepes (pH 7.9), 60 mM KCl, 1 mM dithiothreitol, 4.5 µg of bovine serum albumin, and 10 µg of nuclear extract in a volume of 15 µl(24) . In competition experiments, unlabeled oligonucleotides were added to the reaction mixes at different concentrations prior to the addition of nuclear extract. A 15-min incubation of the mixture at 30 °C was followed by separation on a 4% native low-ionic strength polyacrylamide gel, which was dried and autoradiographed on Kodak X-Omat AR film(11) .
Figure 2: A, comparison between the mouse and human R1 promoters. The arrows indicate the transcription start. The conserved regions are boxed, and the small letters indicate non-conserved nucleotides. The mouse and human sequences are published(11, 12) . B, comparison between the nucleotide sequence around the R1 transcription start and the published sequence of some initiator elements(18) . The transcription start is underlined.
The strong homology between the human and mouse R1 genes at the transcription start suggested the presence of an initiator element, directing the start of transcription. A comparison of the R1 Inr with known initiator elements (Fig. 2B) showed some homology to the initiator element of the mouse TdT gene(15) , where six nucleotides at the transcription start were identical to the R1 Inr element.
Figure 3: Competition between the R1 transcription start oligonucleotide and TdT Inr oligonucleotide analyzed by gel shift assays. Lane 1, end-labeled R1 oligonucleotide without nuclear extract and lanes 2-8 with nuclear extract. In lanes 3-5 a 100-, 200-, and 500-fold molar excess, respectively, of unlabeled R1 oligonucleotide was added. In lanes 6-8, a 100-, 200-, and 500-fold molar excess, respectively, of unlabeled TdT Inr oligonucleotide was added. In lane 9, a 500-fold molar excess of unlabeled Sp1 oligonucleotide was added.
Figure 4: Competition between the TdT Inr oligonucleotide and the R1 transcription start oligonucleotide analyzed by gel shift assays. Lane 1, end-labeled TdT Inr oligonucleotide without nuclear extract and lanes 2-8 with nuclear extract. In lanes 3-5, a 100-, 200-, and 500-fold molar excess, respectively, of unlabeled TdT Inr oligonucleotide was added. In lanes 6-8, a 100-, 200-, and 500-fold molar excess, respectively, of unlabeled R1 oligonucleotide was added. In lane 9, a 500-fold molar excess of unlabeled Sp1 oligonucleotide was added, and in lane 10, a 1000-fold molar excess of unlabeled AP-1 oligonucleotide was added.
Figure 5: Effects of anti-TFII-I antibodies in gel shift assays using end-labeled R1 Inr oligonucleotide. Lane 1, R1 Inr oligonucleotide without nuclear extract; lanes 2-8, R1 Inr oligonucleotide plus nuclear extract; lane 3, the same as lane 2 but with 1 µl preimmune serum. Lane 4, 1 µl of TFII-I antibodies was added; lane 5, 2 µl of TFII-I antibodies was added; lane 6, 3 µl of TFII-I antibodies was added. Lane 7, supershift antibodies against USF were added; lane 8, supershift antibodies against YY1 were added.
Figure 6: Effects of anti-TFII-I antibodies in gel shift assays using end-labeled TdT Inr oligonucleotide. Lane 1, TdT Inr oligonucleotide without nuclear extract; lanes 2-8, TdT Inr oligonucleotide plus nuclear extract; lane 3, the same as lane 2 but with 1 µl preimmune serum. Lane 4, 1 µl of TFII-I antibodies was added; lane 5, 2 µl of TFII-I antibodies was added.
Figure 7:
Gel
shift assays using end-labeled R1 oligonucleotide. Lane
1, oligonucleotide without nuclear extract; lane 2,
oligonucleotide with nuclear extract; lanes 3-5, the
same as lane 2 but with 100-, 200-, and 500-fold molar excess,
respectively, of unlabeled
oligonucleotide; lanes
6-8, the same as lane 2 but with 100-, 200-, and
500-fold molar excess, respectively, of unlabeled R1
footprint
oligonucleotide added.
Figure 8:
Comparison of protein binding to the R1
footprint oligonucleotide and an Sp1 consensus oligonucleotide
studied by gel shift assays. Lane 1, free R1
oligonucleotide; lane 2, free Sp1 consensus oligonucleotide; lane 3, R1
oligonucleotide plus nuclear extract; lane 4, the same as in lane 3 but with the addition
of Sp1 antibodies; lane 5, end-labeled Sp1 oligonucleotide
plus nuclear extract; lanes 6 and 7, the same as in lane 5 but with the addition of Sp1 antibodies; lane
8, Sp1 oligonucleotide plus 3 footprinting units recombinant human
Sp1 (Promega); and lane 9, the same as in lane 8 but
with the addition of Sp1 antibodies.
Figure 9:
Upper panel, luciferase activity in
serum-synchronized BALB/3T3 cells stably transformed with R1
promoter-luciferase gene constructs. The luciferase activity in each
time point has been divided by the luciferase activity in extracts from
the quiescent cells (O h). The values for quiescent cells varied from
13 to 187 light units/µg protein in the different experiments. Lower panel, cell cycle phase composition, determined by DNA
flow cytometry. , G
-phase cells;
, S-phase
cells;
, G
+ M-phase cells. A represents a clone containing the construct p19lucR1 0.49, B represents a clone containing the construct p19lucR1 0.30, C represents a clone containing the construct p19lucR1 0.30-Inr, and D represents a clone containing the construct p19lucR1
0.30-
.
The conserved R1 promoter initiator element binds TFII-I
specifically but does not fully conform to the pyrimidine-rich Inr
consensus YYAN(T/A)YY(31) . Furthermore, the TdT, the Ad-ML,
and the HIV-1 promoters all contain two initiator elements, one located
around the transcription start and the other about 40 base pairs
downstream from the first one(28) . The position of the
conserved R1 region fits the position of a second Inr element,
but the
region is purine rich and has no sequence similarity to
the R1 Inr element.
Many Inr-containing promoters bind Sp1, which
greatly stimulates transcription from the Inr(14) . For
example, it was shown that Sp1 stimulated a synthetic promoter that
only contained an Inr more effectively than a promoter that only
contained a TATA box(32) . There is one putative Sp1 binding
element in reverse orientation in the mouse R1 promoter (nt -38
to -33), but no Sp1 binding could be demonstrated by using DNase
I footprinting experiments(11) . In contrast, earlier UV
cross-linking experiments suggested that Sp1 binds to the
footprint (11) . However, our gel shift assays in this paper
with Sp1 supershift antibodies did not reveal any interaction between
Sp1 and the
/
footprints. Therefore, the transcription from
the R1 Inr element seems to be regulated independently of Sp1, in
contrast to the earlier described pyrimidine-rich Inr elements.
TFII-I stimulates Inr element-dependent transcription from the Ad-ML
promoter in a cell-free system(17) . We now demonstrate that
the TFII-I binding R1 Inr element is important for R1 promoter
activity. Earlier results had demonstrated that a protein binds
specifically to the conserved /
footprints when cells enter
S-phase(11) . Two additional DNA-protein complexes bind to the
/
footprints, but this binding was constant during the cell
cycle. The two cell cycle-invariant DNA-protein complexes contained
different proteins since they precipitate at different ammonium sulfate
concentrations (data not shown).
Unexpectedly, our R1
promoter-reporter gene constructs showed a very similar cell
cycle-dependent pattern of luciferase expression when the /
footprints were present and when they were absent. Therefore, the
region nt -53 to +242, containing the Inr element plus the
element, appears to be sufficient for a cell cycle-regulated
activation of the R1 promoter. In our experiments using synchronized
clones of stably transformed cells, it is difficult to compare absolute
promoter strength due to variations in levels of synchrony and
positional effects. Therefore, protein binding to the
/
footprints may increase the expression seen with the Inr and
elements alone. We hope to be able to measure this using in vitro transcription assays.
In promoters containing a TATA box, it has been shown that TBP binds to the TATA box. TBP stabilizes the formation of a functional preinitiation complex in vitro (cf. 14), but to mediate an activation of transcription from upstream elements, the TBP-associated factors are required(33) . TATA-less promoters have also been shown to be dependent on TBP for the formation of the preinitiation complex (cf. 34). However, it is still unclear how the components of this complex are recruited to a TATA-less promoter. One idea is that an initiator binding protein (i.e. TFII-I(18) , YY1(29) , HIP-1(35) , USF(18, 28, 30) ), with specificity for a certain type of initiator element first recruits TBP and then other proteins to the preinitiation complex(17) . However, sequence analyses of synthetic Inr-containing promoters downstream from Sp1 sites suggested that a universal protein should recognize all initiator sequences(31) .
Recent data from experiments with the TATA-less TdT promoter suggested an alternative pathway for preinitiation complex formation involving TATA-less, Inr-containing promoters where TBP-associated factors participate in TBP recruitment. In this reaction the TATA binding activity of TBP is not required(34) . It has also been reported that RNA polymerase II has some affinity for sequences around the transcription start(36) . Our data give no information about additional proteins binding to the R1 Inr, but our gel shift experiments indicate that if more proteins are involved, they are dependent on TFII-I.
We
cannot explain why the major rise in luciferase activity in our p19luc
R1 1.0/0.49/0.30 constructs appears first in late S-phase and not in
early S-phase as the R1 mRNA. This may indicate that we still have not
identified all elements required for correct cell cycle-regulated R1
gene expression. Furthermore, we do not understand the transient early
G increase in luciferase activity we observe with all our
constructs since no early increase of R1 mRNA was observed in BALB/3T3
cells synchronized by serum starvation(7) .