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(Received for publication, October 30,
1995) From the
Both 45- and 47-kDa subunits of TFIIK, a subcomplex of RNA
polymerase II general transcription factor TFIIH, are encoded by the
yeast cyclin gene CCL1. In all likelihood, these two subunits
individually form cyclin-dependent kinase/cyclin dimers with Kin28
protein, a key enzyme in phosphorylation of the C-terminal domain of
RNA polymerase II concomitant with transcription.
The initiation of RNA polymerase II transcription requires the
five general transcription factors TFIIB, -IID, -IIE, -IIF, and -IIH.
Yeast TFIIH has been described as a holoenzyme (holoTFIIH), comprising
Ssl2 protein, a five-subunit core complex (core TFIIH), and
polypeptides of 33, 45, and 47 kDa, collectively referred to as TFIIK
(Svejstrup et al., 1994, 1995; Feaver et al., 1993).
Ssl2 protein and core TFIIH are required for both transcription and DNA
repair (Svejstrup et al., 1995; Wang et al., 1994).
TFIIK is a protein kinase which may play dual roles as well (Feaver et al., 1994b; Svejstrup et al., 1995): in addition
to phosphorylation of the C-terminal domain of RNA polymerase II, TFIIK
has been shown in vertebrate systems (as the cyclin-dependent
kinase-activating kinase) to phosphorylate cell cycle control protein
kinases (Fesquet et al., 1993; Poon et al., 1993;
Solomon et al., 1993; Fisher and Morgan, 1994; Makela et
al., 1994; Roy et al., 1994; Serizawa et al.,
1995; Shiekhattar et al., 1995). The 33-kDa subunit of yeast
TFIIK is encoded by the KIN28 gene (Feaver et al.,
1994b), originally studied on the basis of its homology to the
cyclin-dependent kinase CDC28 (Simon et al., 1986). KIN28 was further shown to interact with a cyclin homolog, CCL1, in vivo (Valay et al., 1993). Here we
demonstrate the association of Ccl1 with TFIIK in vitro.
Three approaches were taken to reveal CCL1 gene
products in TFIIK. Surprisingly, both the 45- and the 47-kDa subunits
of TFIIK were identified in this way. First, affinity-purified
anti-Ccl1 antibodies, raised against recombinant Ccl1 protein produced
in bacteria, reacted with the 45- and 47-kDa polypeptides in
immunoblots (Fig. 1). Second, these polypeptides, revealed both
by silver staining and by immunoreactivity (Fig. 2), precisely
coeluted with Kin28, with the other subunits of TFIIH, and with
C-terminal domain kinase activity (Svejstrup et al., 1994) in
the final step of TFIIH purification. Finally, tryptic peptides derived
from both 45- and 47-kDa subunits corresponded identically with the
deduced amino acid sequence of the CCL1 gene (Table 1).
Figure 1:
Affinity-purified anti-Ccl1 antibodies
specifically cross-react with the 45- and 47-kDa subunits of holoTFIIH.
Purified holoTFIIH (60 µl of Mono S fraction 46 (Svejstrup et
al., 1994)) was analyzed in a 10% SDS-polyacrylamide gel and
visualized by staining with silver (Silver) or transferred to
nitrocellulose and detected with affinity-purified anti-Ccl1 antibody (CCL1ab) or preimmune serum (Preimmune). The subunits
of holoTFIIH are indicated at the left. Protein bands
migrating slower than Ssl2 are the only apparent contaminants of the
fraction.
Figure 2:
Ccl1 protein copurifies with subunits p45
and p47 and with Kin28 in highly purified holoTFIIH. Consecutive
fractions (60 µl) from Mono S (Svejstrup et al., 1994)
were analyzed in a 10% SDS-polyacrylamide gel and visualized by
staining with silver (top panel) or transferred to
nitrocellulose and probed with affinity-purified anti-Ccl1 antibodies
(panel labeled CCL1) or with affinity-purified anti-Kin28
antibodies (panel labeled KIN28).
How might both 45- and 47-kDa subunits of TFIIK derive from the
single CCL1 gene? Two possibilities, phosphorylation and
proteolytic degradation, were rendered unlikely by further analysis.
Treatment with calf intestinal alkaline phosphatase had no effect on
the mobility of either subunit in SDS-PAGE, although the same treatment
did cause a mobility shift of Kin28, indicative of its phosphorylation,
as described previously (Feaver et al., 1994b). Likewise, both
forms of Ccl1 protein were detected at similar levels in crude extracts
from cells disrupted with hot SDS, arguing against proteolysis during
the course of purification. An alternative explanation for the
multiple forms of Ccl1 protein may lie in the occurrence of a second
AUG, 19 codons downstream from the first AUG in the open reading frame
of the CCL1 gene. Translation initiation from this second AUG
would give rise to a protein product 2199 daltons smaller than that
from the first AUG, consistent with the apparent difference in
molecular mass of the two forms of Ccl1 revealed by SDS-PAGE. While the
``first AUG-rule'' of eukaryotic translation posits that the
AUG codon closest to the 5`-end of the mRNA is a unique site of
initiation (Kozak, 1987), one of two escape mechanisms that account for
most exceptions to this rule (Kozak, 1991) may apply in the case of CCL1: the sequence context for 40 S ribosomal subunit binding
appears far more favorable at the second AUG (GCUACCAUGU,
matches to the vertebrate consensus sequence, boldfaced) than at the
first (GAUAGAAUGA). Another feature of the region
upstream from the CCL1 gene which might result in dual sites
of translational initiation is the short distance (less than 80 bases)
from the putative TATA-box of the promoter to the first ATG. As the
site of transcriptional initiation in yeast is between 40 and 120 bases
downstram from the TATA-box, the 5`-leader might be too short for
translational initiation exclusively at the first AUG. Deletion
analysis of the yeast HIS4 translational initiator region has
shown that a short leader (20 bases from 5`-end to position +1)
can result in bypass of the first AUG and usage (up to 20% depending on
sequence context surrounding the translational start sites) of a
downstream AUG (Cigan et al., 1988). Attempts to confirm
translation initiation at both AUGs in CCL1 by N-terminal
sequencing of the 45- and 47-kDa polypeptides have so far proved
unsuccessful, probably due to blocked N termini of the proteins. These findings alter our view of the oligomeric state of TFIIK. It
is most likely a two-subunit protein, comprising Kin28 and either 45-or
47-kDa forms of Ccl1, rather than a complex of all three polypeptides
as originally surmised. It would seem, then, to differ from its
mammalian counterpart, the MO15/cyclin H pair, which are found in a
complex, termed cyclin-dependent kinase-activating kinase, with a
distinct, third polypeptide. Recently, however, we and others have
found that the third subunit of cyclin-dependent kinase-activating
kinase is homologous to a subunit of the yeast TFIIH core complex (
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 643-645
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
HoloTFIIH Purification and
Microsequencing
HoloTFIIH was purified from yeast whole cell
extracts essentially as described (Svejstrup et al., 1994)
except that the Phenyl HR5/5 step was omitted. HoloTFIIH was at least
50% pure in the final (Mono Q) fractions. Purified holoTFIIH (100
µg) was separated in a 9% SDS-polyacrylamide gel, the polypeptides
transferred to a polyvinylidene difluoride membrane (Bio-Rad), and
tryptic peptides from the 45- and 47-kDa bands were microsequenced by
the Harvard Microchemistry Facility.Expression and Purification of Recombinant
CCL1
The open reading frame encoding Ccl1 was amplified by the
polymerase chain reaction from yeast genomic DNA by the
``touchdown'' method as described (Feaver et al.,
1994a). Primers were used which introduced HindIII and XhoI sites at the 5` and 3` ends of the reading frame,
respectively. Primer sequences were:
5`-ATATCCCGGGAAGCTTACGGATATTCAACTAAATGG-3` and
5`-ATATGAATTCCTCGAGTGTTTTTTGCTTTTTCTCAA-3`. The HindIII-XhoI fragment was cloned into the
corresponding sites of the bacterial expression plasmid pET-20b
(Novagen) introducing a leader peptide and a six-histidine tag at the
5` and 3` ends of the open reading frame, respectively. Use of this
vector enabled expression of the otherwise toxic Ccl1 protein in
bacteria, by virtue of leader peptide-mediated export to the
periplasmic space. Recombinant Ccl1 protein was expressed in BL21/DE3
cells (Novagen) by growth at room temperature to an A of 0.6, and then addition of
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 0.1 mM. Cells were grown for 4 h after
induction. Recombinant Ccl1 was only partially soluble and was purified
from the insoluble fraction essentially as described previously for
recombinant Kin28 protein (Feaver et al., 1994b). The yield
was about 3 mg/liter starting culture.Preparation of Ccl1 Antisera and Affinity
Purification
Purified, recombinant Ccl1 protein was fractionated
by SDS-PAGE (
)and visualized by staining with 0.1% Coomassie
Blue R-250. The protein band was excised and used to inoculate rabbits
(Berkeley Antibody Co.). The antibody was affinity-purified on a
recombinant Ccl1-Sepharose column, essentially as described previously
for anti-Kin28 antibody (Feaver et al., 1994b).Other Methods
Immunoblots were performed as
described (Chasman and Kornberg, 1990). The affinity-purified anti-Ccl1
antibody and the affinity-purified anti-Kin28 antibody were both used
at a final dilution of 1/250, whereas the secondary antibody/detection
reagent was a goat anti-rabbit alkaline phosphatase conjugate
(Bio-Rad). Silver staining was done as described (Blum et al.,
1987).
)(
)(Fisher et al., 1995), so the
difference between the yeast and mammalian systems may only reflect
relative affinities of subunits within holoTFIIH and be of no
functional significance.
)))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
