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INTRODUCTION |
The synthesis of accurately initiated messenger RNA in eukaryotic
organisms requires the assembly of RNA polymerase II and the general
transcription factors
(TFIIA,1 B, D, E, F, and H)
at core promoters (1, 2). Human TFIIA is composed of 35- (
), 19- (
), and 12- (
) kDa subunits encoded by the hTFIIA
/
(3, 4)
and hTFIIA
(5-7) cDNAs, and corresponding cDNAs have been
characterized in yeast (yTOA1 and yTOA2) (8) and
Drosophila (dTFIIA-L and dTFIIA-S) (9-11). In humans and
Drosophila, a large subunit precursor encoded by
hTFIIA
/
or dTFIIA-L is post-translationally processed to form the
(N-terminal) and
(C-terminal) subunits of the mature factor (3,
4, 9). Together, the subunits of TFIIA form a six strand
-barrel
that interacts with the TATA-binding protein (TBP) and a separate
four-helix bundle positioned just upstream of the TBP-TATA complex (12, 13).
TFIIA has multiple roles in transcription initiation by RNA polymerase
II. First, the association of TFIIA and TBP stabilizes the TBP-TATA
element interaction (14-17). In so doing, TFIIA also stimulates
transcription by displacing TBP-associated repressors such as Dr1/NC2,
Dr2/Topo1, HMG1, and DSP1 (18-22) and counteracts the ability of
ADI/MOT1, hTAFII172, yTAFII145, and
hTAFII250 to inhibit TBP binding to DNA (23-27). Second,
TFIIA serves as a cofactor for the AP-1, Gal4-AH, Zta, VP16, CTF, NTF,
and Sp1 activators (4-7, 10, 28-31) and for the PC4 and HMG-2
coactivators (32, 33). Third, TFIIA is required for the isomerization
and extension of TFIID-promoter contacts (34, 35) and for stabilizing
interactions between TFIID and initiator sequences (36). Indeed,
efficient transcription from the TATA-less human TdT promoter (37) and preferential utilization of the distal Drosophila Adh core
promoter (38) require TFIIA.
Previous studies have revealed the presence of two genes for TBP-like
proteins in Arabidopsis (At-1 and At-2) (39),
Drosophila (TBP and TRF) (40), and humans (TBP and TLF) (41)
and raise the idea that TBP homologs may contribute to tissue- and
gene-specific regulation. For example, the Drosophila TRF
protein is expressed in the central nervous system and gonads and is
localized to a limited number of sites on polytene chromosomes (40,
42). In this work we extend the idea of multiplicity within the general transcription factors to include human TFIIA. In particular, we identify a cDNA clone that encodes a novel human factor, ALF
(TFIIA
/
-like factor), that is
expressed almost exclusively in testis. In addition, ALF sequences are
present as part of another cDNA, termed SALF (Stoned
B/TFIIA
/
-like factor), that
contains an N-terminal domain homologous to Drosophila
Stoned B (43) and clathrin adapter proteins µ1 (AP47) and
µ2 (AP50) (44, 45). Results are also presented that show
ALF is a functional counterpart of TFIIA
/
that, together with
TFIIA
(p12), can stabilize TBP-TATA element interactions and support
RNA polymerase II-dependent transcription in
vitro. Finally, we discuss the significance of ALF as a
testis-specific general transcription factor.
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EXPERIMENTAL PROCEDURES |
cDNA Cloning--
NCBI data base searches (tBLASTn) with
human TFIIA
/
identified an incomplete human placental cDNA
sequence (I.M.A.G.E. Consortium Clone ID 259637) (46) homologous to
amino acids 315-376. This clone was obtained from Genome Systems and
sequenced. Human cDNAs that correspond to this EST clone were
initially identified by PCR amplification (35 cycles) of 1.1- and
0.9-kb products from human placenta, liver, and testis "Marathon"
cDNA libraries (CLONTECH) using 25 pmol of the
upstream primer 2a2-1 (5'-AGAAATTCCCTCTGATTG-3') and the downstream
primers 2a2-6 (5'-AGTAACCCGAATGCTTAA-3') and 2a2-8
(5'-ATGCTAGCTGAACCACTG-3'). The 1.1- and 0.9-kb products derived from
the liver cDNA library were subcloned into pGEM-T Easy (Promega)
and sequenced.
The 5'-end of the SALF cDNA was isolated by PCR (40 cycles) using 4 µl of the human placental cDNA library with primer 2a2-6 and a
library-specific adapter primer AP1 (5'-CCATCCTAATACGACTCACTATAGGGC-3') (CLONTECH), according to the manufacturer's
instructions. The resulting products were reamplified (35 cycles) with
primer 2a2-8 and the nested library-specific adaptor primer AP2
(5'-ACTCACTATAGGGCTCGAGCGGC-3'), and the resulting 2930-bp product was
subcloned into the pCRII cloning vector (Invitrogen) to form
pRACE4. The overlapping sequences of pRACE4 and EST ID259637 were
combined to form the composite SALF sequence.
The 5'-end of the ALF cDNA was isolated by PCR (35 cycles) using 4 µl of the human testis cDNA library
(CLONTECH) with the gene-specific primer 2a2-20
(5'-CCAGAAGGTAGAATTGCGGGTTGCTGTAGC-3') and primer AP1 and reamplified
with 2a2-22 (5'-GGAGTTTGAAGTGCCCAGGTCTGCTGTGG-3') and primer AP2. The
369-bp amplification product was subcloned into pGEM-T Easy to form
pRACE22. A full-length PCR product was amplified (35 cycles) from 4 µl of the testis library using primer 2a2-17
(5'-GGTGCTGTCATGGCCTGCCTCAACCCGG-3'), located within the unique 5'-end
of ALF, and primer AP1. The resulting ~1.7-bp fragment was subcloned
into pGEM-T Easy to form pRACE17. The overlapping sequences of pRACE22
and pRACE17 were combined to form the composite ALF sequence.
Northern and Dot Blot Analyses--
Multiple tissue Northern
blots containing 2 µg of poly(A) mRNA from 16 human tissues and a
dot blot containing 89-514 ng of poly(A) mRNA from 50 adult and
fetal tissues were obtained from CLONTECH.
Gene-specific probes for hybridization experiments were as follows:
ALF, a 621-bp NcoI-KpnI fragment or an 899-bp
HincII-BglII fragment from region II (see Fig.
1B); 5'-SALF, a 1002-bp EcoRI-EcoRI fragment from pRACE4 containing the 5'-UTR and nucleotides encoding the
first 282 residues (see Fig. 1B); hTFIIA
/
, a
full-length 1.1-kb EcoRI-EcoRI fragment from
11 (3) or a 282-bp HaeIII-HaeIII fragment from
region II; hTFIIA
, a full-length 355-bp
NdeI-BamHI fragment or a 262-bp
NdeI-EcoRI fragment from pRSEThp12 (7); and actin
and ubiquitin controls provided by CLONTECH. DNA
fragments were typically labeled with [
32P]dCTP using
Ready-To-Go DNA Labeling Beads (Amersham Pharmacia Biotech).
Northern blots were hybridized for 1 h in ExpressHyb solution
(CLONTECH) and washed at 68 °C for 1 h.
Membranes were typically exposed for 1-2 days to either XAR-5 film
(Kodak) or a PhosphorImager screen (Molecular Dynamics). The results in
the left hand column in Fig. 5, C and D, were
obtained with different blots; other results within each column were
obtained by reusing a single blot. The actin control in the left hand
column is a representative result.
For experiments using the dot blot, labeled DNA was combined with 30 µg of Cot-1 DNA (Roche Molecular Biochemicals) and 100 µg of salmon
sperm DNA, denatured, and allowed to renature in 200 µl of 5× SSC at
68 °C for 30 min prior to the addition. After hybridization in 5 ml
of ExpressHyb solution at 65 °C overnight, the blot was washed in
0.1× SSC at 55 °C. Membranes were exposed as follows: Fig.
6A, 19 h; 6B, 2 h 45 min;
6C, 14 h; 6D, 25 h, and 6E,
30 min. For reprobing, Northern and dot blots were stripped twice with
0.5% SDS at 100 °C, cooled to room temperature, and exposed
overnight to confirm the loss of the previous signals. Quantitation of
hybridization signals was performed using ImageQuaNT (Molecular
Dynamics), and relative transcript levels in testis were determined by
comparison to an average level from non-testis tissues.
Genomic Blot Analysis--
Genomic DNA (10 µg) from HeLa cells
was digested with the indicated restriction enzymes (BglII
and EcoRI), electrophoresed on 0.7% agarose gels, and
transferred overnight to nitrocellulose membranes (Schleicher & Schuell). Hybridization was performed under stringent conditions at
42 °C in 50% formamide, or at 68 °C, in hybridization buffer
(6× SSC, 0.5% SDS, 5× Denhardt's solution, and 100 µg/ml salmon
sperm DNA). The probes included a full-length 1.1-kb
EcoRI-EcoRI TFIIA
/
fragment from
11 (3),
a full-length NdeI-BamHI ALF fragment described
in the following section (pRSET-ALF), or an
NdeI-BamHI TFIIA
fragment from pRSEThp12 (7).
Blots were typically washed at 65 °C in 0.1× SSC and 0.5% SDS and
exposed at
80 °C to XAR-5 film.
Protein Expression--
To express recombinant ALF polypeptides,
primers A1
(5'-ACTACTCATATGGCACACCATCACCATCACCATGTACCTAAACTCTACAGATCT-3') and A2 (5'-AGTAGTGGATCCTTACCACTCTGCATCACC-3') were used to create a 1445-bp NdeI-BamHI PCR fragment whose reading frame
begins with the N-terminal extension MHHHHHHV and terminates with the
natural TAA stop codon. This construct does not encode the first six
amino acids (MACLNP) found in the intact testis-derived ALF cDNA.
After subcloning into pRSETC (Invitrogen), the resulting construct
(pRSET-ALF) was transformed into Escherichia coli
BL21(DE3)pLysS (Novagen) and was expressed and purified essentially as
described in protocols supplied by Qiagen. Specifically, cells were
grown in LB medium at 37 °C to an A600 of
~0.5, and production of the 69-kDa recombinant protein was induced
with 2 mM isopropyl-b-D-thiogalactopyranoside. Cells were harvested 3 h post-induction, solubilized in Buffer A
(0.1 M NaH2PO4, 0.01 M
Tris, pH 8.0, and either 6 M guanidine or 8 M
urea), and sonicated five times for 30 s. The denatured cell
lysate (~20 ml) was then incubated with 2 ml of nickel-NTA-agarose resin (Qiagen) at room temperature for 1 h. The resin was washed successively with Buffer A containing 8 M urea at pH 8.0, 6.3, and 5.9, and bound polypeptides were eluted at pH 3.5. Preparation of expression constructs for rat TFIIA
/
and rat TFIIA
subunits (GenBank Accession numbers AF000943 and AF000944, respectively) and
purification of the corresponding 55- and 12-kDa recombinant proteins
were performed similarly. For transcription experiments the recombinant
p69 and p12 proteins were codialyzed to prevent precipitation of the
p12 subunit.
Human TBP was expressed from pET11d (Novagen), induced with 2 mM isopropyl-b-D-thiogalactopyranoside at
A600 0.3, and purified at 4 °C from the
soluble fraction of the bacterial lysate over nickel-NTA-agarose.
Purification was performed by washing the resin with D700 buffer (20 mM HEPES, 20% glycerol, 0.2 mM EDTA, 10 mM
-mercaptoethanol, 0.5 mM
phenylmethylsulfonyl fluoride, and 700 mM KCl) that
contained 5, 10, and 15 mM imidazole and eluting bound
polypeptides with D700 buffer that contained 100 mM
imidazole. Recombinant proteins were dialyzed against Buffer C (10 mM Tris, pH 7.9, 2 mM dithiothreitol, 20%
glycerol, and 0.5 mM phenylmethylsulfonyl fluoride)
containing 100 mM KCl prior to use.
To express SALF, primers NN1 (5'-TACTGCTCGAGCAACTTTAGAGT-3') and 2a2-8
were used to generate a 2988-bp product from pRACE4. An internal
2207-bp XhoI-BglII fragment (amino acids 1-716)
derived from this PCR product was then inserted into the
XhoI-BglII-digested pT7T3D vector that contained
EST ID259637. Because an internal BglII-BglII
fragment that spans amino acids 717-1084 was excised during the
preparation of this vector, this fragment was later reinserted in the
appropriate orientation to create a full-length SALF open reading frame
(ORF) (pT7T3-SALF). This construct (0.8 µg) was used to program
rabbit reticulocyte lysates in the presence of
[35S]methionine as described by the manufacturer
(Promega). Labeled polypeptides were separated on 8%
SDS-polyacrylamide gels.
Mobility Shift Assays--
Mobility shift assays were performed
using 10 fmol of a [
-32P]ATP kinase-labeled
TATA-containing oligonucleotide (5'-AAGGGGGGCTATAAAAGGGGTGGG-3') that
spanned
40 to
16 of the adenovirus major late (AdML) promoter. Binding reactions (25-µl final volume) were performed in 10 mM HEPES (pH 7.9), 2% polyethylene glycol-8000 (w/v), 60 mM KCl, 5 mM dithiothreitol, 0.2 mM
EDTA, 5 mM ammonium sulfate, 4 mM MgCl2, and 8% glycerol essentially as described (3, 47). Recombinant rat p55 (30 ng, 29 nM), rat p12 (1.1 µg, 3.5 µM), human ALF (180 ng, 137 nM), and human
TBP (125 ng, 133 nM) were added to reactions as indicated.
Reactions were incubated for 30 min at room temperature, and complexes
were separated on native 5% polyacrylamide gels containing 0.5× TBE
and 5% glycerol. Competition experiments contained either cold AdML
TATA or SP1 (5'-TTCGATCGGGGCGGGGCGAG-3') oligonucleotides, and antibody
supershift reactions contained 2-4 µl of rabbit polyclonal antiserum
raised against the 55-kDa hTFIIA
/
polypeptide (3).
In Vitro Transcription--
HeLa cell nuclear extracts were
depleted of TFIIA essentially as described (3-5, 7). In brief, 200 µl of extract was incubated with 100 µl of nickel-NTA-agarose resin
for 30 min at 4 °C in the presence of 400 mM KCl.
Control extracts were processed similarly, except that no
nickel-NTA-agarose was present. After microcentrifugation for 5 min,
supernatants were removed and dialyzed for 3 h against Buffer C
that contained 100 mM KCl. Transcription reactions were performed using a template (pMLC2AT) that contains the AdML
promoter upstream of a G-free cassette (48). The template was
linearized at a SmaI site just beyond the G-free cassette
prior to use. Reactions (20 µl) contained 8 µl of nuclear extract
(~60 µg protein), 2 µl (550 ng) of recombinant p69 (0.22 µM) and p12 (0.9 µM) proteins, 1 µg of
pMLC2AT, 10 mM HEPES (pH 7.5), 25 mM KCl, 6 mM MgCl2, 625 µM UTP, 625 µM ATP, 35 µM
CTP, 200 µM O-methyl-GTP, 3% glycerol, 0.7 µl of [
-32P]CTP, and 37.3 units of RNAguard
(Amersham Pharmacia Biotech). After incubation at 30 °C for 45 min,
reactions were terminated by adding 270 µl of stop solution (0.25 M NaCl, 1% SDS, 20 mM Tris, pH 7.5, 5 mM EDTA, and 66.7 µg/ml tRNA) and extracted with an equal
volume of phenol/chloroform (1:1). Ethanol-precipitated transcripts
were resuspended in formamide-containing loading dye and
electrophoresed on 5% acrylamide gels containing 1× TBE and 8 M urea.
Other Procedures--
Sequencing reactions were performed at The
University of Texas Southwestern Medical Center. Sequence comparisons
and alignments were done with Lasergene (DNASTAR). Custom
oligonucleotides were obtained from Operon.
 |
RESULTS |
Isolation of SALF--
Computer searches of expressed sequence
tags (dbEST) with a human TFIIA
/
query identified a homologous
placental cDNA sequence (I.M.A.G.E. Consortium Clone ID 259637).
Sequence analysis showed that this 1885-bp clone (Fig.
1A) encodes 471 amino acids
similar in sequence and organization to TFIIA
/
. Surprisingly, the
homology with TFIIA
/
ends several residues short of where the
initiating methionine was expected to be. Instead, the ORF continues
for an additional 93 amino acids at the N terminus and bears similarity to a Drosophila protein called Stoned B (43) and to the
mammalian clathrin adaptor proteins (AP) µ1 (AP47) and
µ2 (AP50) (44, 45) involved in membrane trafficking
(reviewed in Refs. 49 and 50).

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Fig. 1.
Strategy for isolation of the ALF and SALF
cDNA clones. A, I.M.A.G.E. Consortium
Clone ID 259637 contains 1885 bp that encode connected
TFIIA / -like (black area) and Stoned B-like (gray
area) domains. The locations of primers 2a2-1, 2a2-6, and 2a2-8
(black boxes) and the corresponding PCR products
(single lines) are indicated. The bold line
indicates UTR sequences. B, the composite SALF cDNA
includes TFIIA / -like sequences and an upstream Stoned B-like
region and is shown with selected restriction enzyme sites. The 5'-end
of SALF was isolated in PCR reactions using gene-specific primers
2a2-6 and 2a2-8 and library-specific primers AP1 and AP2. The
resulting clone (pRACE4) is shown as a single line that is
interrupted by a 141-bp internal deletion. C, the composite
ALF sequence consists of TFIIA / -like sequences only. The 5'-end
of ALF was identified using gene-specific primers 2a2-20 and 2a2-22
and library-specific primers AP1 and AP2. The resulting clone, pRACE22,
is shown as a single line. A PCR product that spans the
entire ALF sequence (pRACE17) was obtained using the gene-specific
primer 2a2-17 and the library-specific primer AP1. D, PCR
products that contain the junction between the Stoned B- and
TFIIA / -like domains of SALF were amplified from human placenta
and liver cDNA libraries. The primers used are indicated above each
lane (2a2-1, 2a2-6, and 2a2-8). Lanes 5 and 6 are control reactions to which no cDNA template was added.
M, marker.
|
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To determine whether this EST reflects the actual structure of a
corresponding human mRNA, we performed PCR analysis. Primers within
the TFIIA
/
-like sequence (2a2-6 and 2a2-8) and the upstream Stoned B-like sequence (2a2-1) amplified products of expected size
(1.1- and 0.9-kb) from both human placenta and liver cDNA libraries
(Fig. 1, A and D, lanes 1-4). Similar
results were obtained using a human testis cDNA library (data not
shown). Analysis of the liver-derived products showed that they contain
sequences identical to those present in EST ID259637, confirming that
the EST is a real, albeit incomplete, human cDNA clone.
To isolate the full-length 5'-end of this species, we performed PCR
reactions on a human placental cDNA library using gene-specific primers within the TFIIA
/
-like sequence (2a2-6 and 2a2-8) and adapter primers AP1 and AP2. The resulting PCR product (pRACE4) (Fig.
1B) contains 3'-sequences that are identical to EST
ID259637, except for a 141-bp deletion that removes 47 amino acids
(787-833) within region II of the TFIIA
/
-like domain. pRACE4
also contains a 1968-nucleotide 5'-end that extends the region of
Stoned B homology by 344 amino acids and continues for 274 amino acids
further upstream.
The results of these experiments reveal a novel human mRNA, SALF,
that is composed of both Stoned B/clathrin AP-like and
TFIIA
/
-like sequences. The 3853-bp composite sequence contains a
114-nucleotide 5'-UTR and a 161-nucleotide 3'-UTR with a poly(A)
addition signal and a 29-nucleotide poly(A) tract (Fig. 1B).
The deduced ORF commences with a putative start codon
(AAGATGT) that is preceded by an in-frame stop codon 27 nucleotides upstream and predicts a 1182-residue polypeptide (Fig.
2) with a molecular mass of 132 kDa and a
pI of 5.1.

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Fig. 2.
Deduced amino acid sequence of the ALF and
SALF proteins. The 478 amino acid ALF sequence begins with the
residues MACLNPV (shown in the center) and continues with
the bottom sequence. The entire sequence is
boxed, and conserved regions I and IV are denoted by
dark boxes. The 1182-amino acid SALF sequence contains the
top sequence followed by the bottom sequence but
does not include MACLNPV. Shading of amino acids in the
top sequence denotes homology with Stoned B. EST ID259637
begins with the underlined Val619 residue. The
numbers to the left correspond to the ALF ORF and
those to the right correspond to the SALF ORF.
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|
Isolation of ALF--
Northern blot analysis of human mRNA
using a probe from the TFIIA
/
-like region of SALF revealed a
1.8-kb mRNA in testis (see Fig. 5), indicating that this sequence
is also present in an mRNA that is substantially shorter than that
predicted by SALF. To isolate the 5'-end of the corresponding cDNA,
we performed PCR on a human testis cDNA library using gene-specific
primers 2a2-20 and 2a2-22 and adapter primers AP1 and AP2. The
resulting clone (pRACE22) (Fig. 1C) contains 298 bp that are
identical to SALF and a 35-bp 5'-end that is unique. This new 5'-end
consists of a 15-bp UTR, a putative initiation codon
(GTCATGG) that conforms to the Kozak concensus
((A/G)NNATGG) (51), and 17 bp downstream of the ATG that predict six
amino acids (ACLNPV) not present in SALF. These residues
include the conserved valine found at the N terminus of all other TFIIA
large subunits (Fig. 3B). To
isolate a complete cDNA, we then performed PCR with the
gene-specific primer 2a2-17 and primer AP1. The sequence of the
resulting clone (pRACE17) (Fig. 1C) is identical to the
composite SALF sequence except for its unique 5'-end and a longer
poly(A) tail (~90 nucleotides), which begins four nucleotides
downstream of the poly(A) tail in SALF. Together, pRACE22 and pRACE17
form a 1617-bp composite cDNA, ALF, that predicts a 478-amino acid
polypeptide (Fig. 2) with a molecular mass of 52 kDa and a pI of
4.4.

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Fig. 3.
Schematic diagram and alignment of the
ALF and SALF sequences. A, ALF contains conserved
regions I, III, and IV and an internal nonconserved region II. Beneath
ALF are diagrams of TFIIA large subunits from human (hTFIIA / ),
Arabidopsis (aTFIIA-L), Drosophila (dTFIIA-L),
and yeast (yTOA1). B, alignment of regions I and IV from ALF
with TFIIA large subunit sequences from hTFIIA / , dTFIIA-L,
aTFIIA-L (GenBank Accession number X98861), and yTOA1. Amino acids
conserved in at least four of five sequences, including ALF, are
shaded. Gaps in the alignment are indicated by dashes.
C, a diagram of the N terminus of SALF is shown, indicating
an upstream serine, threonine, and proline-rich domain and a downstream
domain that is homologous to Drosophila Stoned B and the
clathrin APs µ1 (AP47) and µ2 (AP50).
aa, amino acid. D, alignment of SALF with
Drosophila Stoned B, a C. elegans Stoned
B-related protein C27H6.1 (53), mouse clathrin AP µ1
(AP47), and rat clathrin AP µ2 (AP50). Amino acids
similar in at least three sequences, including SALF, are
shaded.
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Two ESTs that contain partial ALF sequences connected at nucleotide
1344 to an alternative 261-bp 3'-end were also identified (I.M.A.G.E.
Consortium Clone ID 785133 and 1657721). These clones both predict a C
terminus in which the last 35 amino acids of ALF are replaced with the
residues "AFPRRTSFNT" followed by a stop codon and a 3'-UTR that
contains a poly(A) addition signal and a poly(A) tail. PCR analysis has
verified that both ALF and SALF cDNAs that contain this alternative
3'-end can be amplified from human cDNA libraries (data not shown).
However, because the conserved C terminus is required for TFIIA
activity, it is not clear whether these clones will encode functional polypeptides.
Description of the Predicted ORFs--
A schematic comparison of
ALF and other TFIIA large subunit sequences from humans (3, 4),
Drosophila (9), Arabidopsis (GenBank Accession
number X98861), and yeast (8) is shown in Fig. 3A. These
sequences share a common organization consisting of conserved regions I
and IV, acidic region III, and an internal nonconserved region II. ALF
is similar to its human TFIIA
/
counterpart in region I (amino
acids 1-54, 67%) and region IV (amino acids 417-478, 73%), as shown
in Fig. 3B, and in the negatively charged region III (amino
acids ~340-414, 42% D/E residues). In contrast, region II shares no
homology with the corresponding region in hTFIIA
/
(or other TFIIA
large subunits) and is approximately 100 residues longer. The
importance of the conserved regions I, III, and IV for TFIIA structure
and function has been demonstrated by mutagenesis (52) and by x-ray
crystallographic studies (11, 12). The presence of similar domains in
ALF (and in SALF) suggests that these sequences encode functional TFIIA
large subunits.
The unique N terminus of SALF is 711 amino acids in length (Fig.
3C) and contains a region between amino acids 44 and 150 that is rich in proline (20%), serine (21%), and threonine (9%) residues. Residues between 275 and 692 display 47% similarity to the
Drosophila Stoned B protein (43) and 46% similarity to an
uncharacterized Stoned B-like ORF in Caenorhabditis
elegans, C27H6.1 (53) (Fig. 3D). The
Drosophila stoned locus was first identified as a class of
mutations that caused neurological defects such as
temperature-sensitive paralysis (54), and it has been suggested that
Stoned B functions in membrane trafficking in neurons (43). In
addition, residues from 410 to 692 within the Stoned B homology region
are 33 and 37% similar to the mouse µ1 (AP47) and rat
µ2 (AP50) clathrin APs, respectively (Fig. 3D)
(44, 45). The µ1 (AP47) and µ2 (AP50)
clathrin APs are subunits of the AP-1 and AP-2 complexes associated
with the trans-Golgi and plasma membranes, respectively, and
function in the internalization, sorting, and recycling of receptors
and other membrane proteins (49, 50). Thus, the N terminus of SALF is
related to a family of proteins involved in membrane trafficking.
Genomic DNA Blot Analysis--
To confirm that the ALF sequences
were derived from a distinct human gene, we performed genomic DNA
hybridization (Fig. 4). Hybridization
with an ALF probe revealed bands of 8.6, 6.9, 5.0, and 1.0 kb
(BglII, lane 1) or 11.5, 8.4, 6.0, and 4.5 kb
(EcoRI, lane 2). In contrast, hybridization with
a TFIIA
/
probe revealed bands of 15, 8.0, and 4.2 kb
(BglII, lane 3) or 12.0, 8.0, 5.4, and 4.2 kb
(EcoRI, lane 4). Hybridization with a TFIIA
probe showed bands of 4.2 and 3.8 kb (BglII, lane
5) or 10.0, 8.0, 6.6, 2.5, and 1.7 kb (EcoRI,
lane 6). The presence of multiple bands in some lanes
(e.g. lane 6) is due in part to the presence of restriction
enzyme sites in the corresponding cDNAs. These results show that
sequences complimentary to ALF and TFIIA
/
are present on
different sized human genomic DNA fragments and indicate that the
corresponding mRNAs are encoded by separate genes.

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Fig. 4.
Genomic blot analysis of ALF and
TFIIA / . Human
genomic DNA was digested with either BglII or
EcoRI and hybridized with a probe for either ALF,
TFIIA / , or TFIIA . The enzymes are indicated above each lane
(BglII, lanes 1, 3, and 5;
EcoRI, lanes 2, 4, and 6).
The positions of the kilobase size markers are indicated to the
left of each panel.
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Northern Blot Analysis--
To evaluate the abundance and
distribution of the ALF and SALF mRNAs, we performed Northern blot
analysis of mRNA from 16 different human tissues. Hybridization
with a probe from the TFIIA
/
-like region of SALF revealed a
1.8-kb mRNA that was present in testis but not in other tissues
(Fig. 5A, lane 12).
The isolation of the ALF cDNA that corresponds to this species is
illustrated in Fig. 1C. Surprisingly, the predicted 3.8-kb
SALF mRNA was not visible in mRNA from any of the tissues
examined, including placenta, liver, and testis from which SALF can be
amplified by PCR. These results indicate that ALF and TFIIA
/
are
the major transcripts encoding human TFIIA large subunits and that SALF
is relatively rare.

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Fig. 5.
Northern blot analysis of ALF,
TFIIA / , and
TFIIA transcripts. Poly(A) mRNA from
various human tissues was probed with various gene-specific probes (see
"Experimental Procedures" for details). A, ALF;
B, 5'-SALF; C, TFIIA / ; D,
TFIIA ; and E, actin. Tissues are indicated above each
lane, and size markers are shown to the left.
Overexpression of the actin control in skeletal muscle is a
characteristic of this tissue.
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Hybridization with a probe specific for the 5'-end of SALF (5'-SALF)
revealed a 6.5-kb species that was present at the highest levels in
heart, placenta, kidney, prostate, and uterus (Fig. 5B,
lanes 1, 3, 7, 11, and
13) and at lower levels in other tissues. This transcript,
termed RNA6.5, was not detected using the ALF-specific probe (Fig.
5A), indicating that it does not contain a downstream ALF
domain. Thus, RNA6.5 is an independent human transcript that contains
sequences similar or identical to those present at the 5'-end of
SALF.
Together with the cloning data, these experiments reveal the existence
of three related mRNA transcripts: 1) a 1.8-kb testis-specific ALF
mRNA encoding a human TFIIA
/
homolog, 2) a ubiquitously expressed 6.5-kb mRNA (RNA6.5) whose exact function is unknown but
may encode a protein involved in membrane trafficking, and 3) a rare
~3.8-kb SALF mRNA that contains sequences that are present in and
possibly derived from the more abundant ALF and RNA6.5 transcripts (see
"Discussion").
Northern experiments were also performed using probes for human
TFIIA
/
and TFIIA
. Hybridization with TFIIA
/
revealed transcripts at 6.5 and 7.0 kb, as described earlier (4). These species
were present alone or together in all tissues and were enriched in
placenta (10-fold), skeletal muscle (9-fold), and testis (13-fold)
(Fig. 5C, lanes 3, 6, and
12). Hybridization with a TFIIA
-specific probe revealed a
1.0-kb transcript that was present in all tissues and was enriched
32-fold in testis (Fig. 5D, lane 12). In contrast
to the restricted expression of ALF in human tissues, the widespread
distribution of the TFIIA
/
and TFIIA
mRNAs is consistent
with their role as generally required basal transcription factors.
RNA Dot Blot Analysis--
We also examined mRNA from 50 human
adult and fetal tissues for the presence of ALF, SALF, and RNA6.5.
Using an ALF-specific probe, we observed a strong signal in testis that
is due to the 1.8-kb ALF transcript (Fig.
6A, position D1).
In addition, weak signals were observed in approximately 24 of the
remaining tissues, including the small intestine, bladder, uterus, and
prostate (positions E3, C5, C6, and
C7). These weak signals indicate that ALF or SALF is
expressed at low levels in non-testis tissues, and their detection in
this experiment likely reflects the greater sensitivity of the dot
blot. When this blot was stripped and reprobed with the 5'-SALF probe,
signals were detected in all tissues. A short exposure (shown in Fig.
6B) showed highest levels in placenta, uterus, spinal cord,
fetal kidney (positions F4, C6, B7, and G3), and several others and lower levels in the remaining tissues. Because this
probe detected high levels of RNA6.5 (but not SALF) in Northern blot
analysis, we believe the signals in Fig. 6B are primarily due to expression of RNA6.5.

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Fig. 6.
RNA dot blot analysis of ALF,
TFIIA / , and
TFIIA transcripts. A single dot blot
containing poly(A) mRNA from multiple human tissues is probed with
ALF (A), 5'-SALF (B), TFIIA /
(C), TFIIA (D), and a ubiquitin control
(E). The source of the mRNA for each spot is listed in
F, and dashes indicate positions that do not
contain mRNA. The signal at position H4 (E. coli DNA) is typically observed with probes derived from vectors
grown in E. coli.
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Further inspection of the data reveals that the signals detected with
the ALF-specific probe in Fig. 6A are present in a range of
tissues that are nearly identical to those observed in Fig. 6B. This holds true for approximately 20 tissues, including
bladder, uterus, prostate, ovary, placenta (positions C5, C6, C7,
D2, and F4), and others but not for testis
(position D1). Likewise, the absence of signals in Fig.
6A correlates with the absence of signals in Fig.
6B. It is unlikely that this similarity is because of nonspecific hybridization or incomplete removal of the earlier probe
because: 1) the signals in Fig. 6B are much stronger than background, and 2) the experiment in Fig. 6B was performed
after stripping the relatively faint signals in Fig. 6A and
not vice versa. Although the molecular basis for this observation is
not yet clear, the results suggest a relationship between the
expression of RNA6.5 with ALF-containing transcripts (possibly SALF)
present at low levels in non-testis tissues.
Hybridization with human TFIIA
/
- and TFIIA
-specific probes
(Fig. 6, C and D) shows that the corresponding
mRNAs are expressed in all tissues. Quantitation of the results
confirms that ALF (50-fold), TFIIA
/
(4-fold), and TFIIA
(10-fold) are enriched in testis tissue (see "Discussion").
Expression and Reconstitution of ALF/
Complexes--
To prepare
recombinant ALF protein for functional assays, we overexpressed a
479-amino acid histidine-tagged polypeptide that spans residues
Val7 to Trp478. The predicted size of this
polypeptide is 53 kDa, but the mobility on SDS-polyacrylamide gel
electrophoresis is 69 kDa (Fig.
7A, lane 2). This
observation is similar to results showing that the predicted 42-kDa
product of hTFIIA
/
migrates at 55 kDa (3, 4) and may be due to
the effect of charged region III. The mobilities of the purified
recombinant rat TFIIA
/
(p55) and TFIIA
(p12) subunits used in
these experiments are also shown in Fig. 7A (lanes
3 and 4). These polypeptides are at least 98% identical to their human counterparts (GenBank Accession numbers AF000943 and AF000944).

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Fig. 7.
ALF (p69) has functional characteristics
expected of a TFIIA large subunit polypeptide.
A, Coomassie-stained SDS-polyacrylamide gel
electrophoresis shows that the recombinant histidine-tagged ALF protein
migrates at 69 kDa (lane 2) and that the recombinant rat
TFIIA / and TFIIA proteins migrate at 55 and 12 kDa,
respectively (lanes 3 and 4). The band at 25 kDa
(lane 2) is a minor degradation product in this particular
preparation. B, p69 (ALF) can substitute for p55
(TFIIA / ) in stabilizing the interaction between TBP and the
adenovirus major late promoter TATA element ( 40 to 16). Additions
to each reaction are listed above each lane. Antiserum against human
p55 is added to reactions in lanes 8 (2 µl), 9 (4 µl), and 10 (4 µl). C, addition of p69
(ALF) and p12 (TFIIA ) restores activity to transcriptionally
inactive TFIIA-depleted HeLa nuclear extracts. Control (undepleted) and
TFIIA-depleted extracts are indicated by a C and
D, respectively. D, a T7 promoter-driven SALF
construct produces a [35S]-labeled protein of
approximately 170 kDa in in vitro transcription-translation
reactions.
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We then tested the activity of these polypeptides in electrophoretic
mobility shift assays using human TBP. Under the conditions of this
assay, TBP alone is unable to bind DNA (Fig. 7B, lane 1). However, the presence of TFIIA
/
(p55) and TFIIA
(p12)
stabilizes the TBP-DNA interaction via TFIIA
/
/
·TBP·DNA
complex formation (lane 2). Likewise, the recombinant ALF
(p69) polypeptide, in conjunction with the TFIIA
(p12) subunit, is
able to form an ALF/
·TBP·DNA complex (lane 5).
Formation of this complex depends on the presence of both ALF and
TFIIA
(data not shown). Interestingly, although ALF (p69) is 102 amino acids longer than TFIIA
/
(p55) and migrates as a larger
species in SDS-polyacrylamide gel electrophoresis, the
ALF/
·TBP·DNA complex migrates slightly faster than the
TFIIA
/
/
·TBP·DNA complex (lanes 2 and
5). These reactions were run side-by-side on the same gel
using ALF and TFIIA subunits that had been purified and renatured using
the same procedure. In these experiments approximately 5-fold higher
ALF concentrations were used to generate shifted complexes equivalent
to that observed with TFIIA
/
. This fact may reflect differences
in the activities of these two factors or, alternatively, differences
in their ability to be purified and renatured in active form. The
specificity of the ALF/
·TBP·DNA complex is similar to the
TFIIA
/
/
·TBP·DNA complex, as judged by competition with
specific TATA (lanes 3 and 6) and nonspecific Sp1
site (lanes 4 and 7) oligonucleotides. In
addition, both complexes are supershifted to the well when co-incubated
with antiserum against hTFIIA
/
(lanes 8 and
9), indicating that ALF and TFIIA
/
are immunologically
related and are present in their respective complex.
To further support the idea that ALF is a functional polypeptide, we
performed TFIIA-dependent in vitro transcription
assays. For this purpose, we took advantage of the fact that
TFIIA
/
contains an intrinsic seven-histidine region that allows
for the efficient removal of TFIIA from HeLa cell nuclear extracts
using nickel-NTA-agarose (3-5, 7). Depleted extracts are
transcriptionally inactive but can be restored to normal activity by
the addition of TFIIA. As shown in Fig. 7C (lane
1), control (undepleted) extracts produced a
[
-32P]CTP-labeled G-free RNA transcript expressed
under the control of the AdML promoter (pMLC2AT) (52). The
addition of recombinant ALF (p69) and TFIIA
(p12) to these extracts
did not enhance transcription (lane 2). TFIIA-depleted
extracts were transcriptionally inactive and were not affected by the
readdition of either ALF (p69) or TFIIA
(p12) alone (lanes
3-5). However, the addition of both ALF (p69) and TFIIA
(p12)
at concentrations similar to those reported for recombinant
TFIIA
/
and TFIIA
(3, 4, 6, 7) restored transcription to the
level observed with control extracts (lane 6). Based on the
results of the electrophoretic mobility shift and in vitro
transcription assays shown in Fig. 7, B and C, we
conclude that ALF is a functional homolog of TFIIA
/
and that both
ALF and TFIIA
/
require the TFIIA
subunit for activity.
To determine whether a full-length SALF cDNA construct was capable
of directing the translation of an intact protein, we performed in vitro transcription-translation reactions. As shown in
Fig. 7D, rabbit reticulocyte lysates programmed with
pT7T3-SALF produced a [35S]methionine-labeled polypeptide
that migrated at 170 kDa (compared with a predicted size of 132 kDa).
Lysates programmed with pT7T3-SALF truncated at an internal
EcoRI site at nucleotide position 960 (see Fig.
1B) produced a 36-kDa product similar to the predicted size
of 32 kDa (data not shown). These results demonstrate that although
SALF contains a suboptimal ATG initiation codon (AAGATGT) and encodes a large ORF composed of two distinct regions, it can be
translated efficiently in this assay. However, we have not produced
full-length SALF proteins in quantities sufficient to test whether it
is transcriptionally active.
 |
DISCUSSION |
This report describes the isolation and characterization of human
cDNA clones that encode a TFIIA
/
homolog, ALF, and a related factor, SALF, and provides evidence for multiplicity and tissue specificity among the human general transcription factors.
Multiplicity among Human TFIIA-like Factors--
The predicted
amino acid sequence of ALF is organized into four regions (I-IV) that
are characteristic of all TFIIA large subunits (Fig. 3A),
including the conserved N- and C-terminal domains (Fig. 3B).
In addition, the recombinant ALF protein, together with the TFIIA
(p12) subunit, can stabilize TBP-DNA interactions in an electrophoretic
mobility shift assay (Fig. 7B) and can restore RNA
polymerase II-dependent transcription from the AdML
promoter using TFIIA-depleted HeLa cell nuclear extracts (Fig.
7C). These results show that there are at least two genes
for functional TFIIA
/
-like subunits in humans, namely ALF and
TFIIA
/
. Likewise, multiple genes that encode the general
transcription factor TBP have been described in Arabidopsis
(At-1 and At-2) (39), Drosophila (TBP and TRF) (40), and
humans (TBP and TLF) (41). Together, these studies reveal a complexity
among the basal transcription factors that has not been evident from
the biochemical characterization of fractionated nuclear extracts.
As TFIIA is composed of large (
/
) and small (
) subunits, the
question is raised as to whether there are additional human genes for
TFIIA
. The results of computer data base searches did not identify
homologous sequences, and the genomic Southern blotting experiments
suggest that TFIIA
could be a single copy gene (Fig. 4) but will
require genomic characterization to verify. Moreover, the ubiquitous
co-expression of TFIIA
/
and TFIIA
(Figs. 5 and 6) and the
especially high levels of both ALF and TFIIA
mRNAs in testis
(Fig. 5, A and D) suggest that TFIIA
is a
common subunit for both factors. Interestingly, induction of the
Drosophila dTFIIA-S gene (but not dTFIIA-L) is required for
development of photoreceptors (55), implying that the formation or
function of TFIIA-like complexes (such as TFIIA
/
/
and ALF/
)
might be regulated by the availability of the small subunit.
Expression of General Transcription Factors in Testis--
At
least five of the RNA polymerase II general transcription factors are
expressed to higher levels in testis than in other tissues. First, as
reported here, mRNA for both TFIIA
/
and TFIIA
are more
abundant (4-13-fold and 10-32-fold, respectively) in human testis
(Figs. 5 and 6). Second, mRNA for TFIIB is enriched 6-11-fold in
rat testis (56). Third, mRNA for TBP is enriched 50-80-fold in
rodent testis (56, 57) and is estimated to be 1000-fold higher in
haploid round spermatid cells of mice (57). Finally, protein levels of
the TFIIE
subunit, as well as the RNA polymerase II large subunit,
are several-fold higher in rodent testis (56, 57).
In addition, two homologs of general transcription factors are
testis-specific or nearly so. ALF is expressed to 50-fold higher levels
in human testis than in other tissues, as seen in the dot blot analysis
(Fig. 6), and is the first example of a human tissue-specific general
transcription factor. The level of ALF expression might be even higher
if restricted to a particular cell type, as noted for TBP. Likewise,
the Drosophila TBP-related factor TRF is expressed only in
the central nervous system and primary spermatocytes of adult flies
(40, 42). Interestingly, TRF is associated with a distinct set of
neuronal TRF-associated factors and has been localized to a limited
number of sites on salivary gland polytene chromosomes, implying that
it may function at the promoters of relatively few genes (42).
The observations above suggest that general transcription factors and
their homologs have a unique role in testis gene expression (see Refs.
59 and 60 and the references therein). The main function of this organ
is the production of haploid spermatozoa from undifferentiated stem
cells, a process called spermatogenesis (reviewed in Ref. 61). It is
notable that mutations in the Drosophila trf gene result in
immotile sperm and male sterility (40), indicating that TRF might be
selectively involved in the transcription of genes whose products are
required for spermatogenesis. In addition, transcription of some genes
in testis occurs from promoters that are not used in somatic cells,
giving rise to mRNAs with novel 5'-end sequences (59, 60). For
example, the mouse tbp gene initiates from at least six
promoters in testis, five of which are testis-specific, and produces 10 different transcripts (58). These observations suggest an unusual
flexibility in core promoter recognition by the basal factors or a
greater accessibility to core promoter-like sequences in genomic DNA
that may occur during spermatogenesis. Finally, the overall level of
transcription in testis is relatively high, possibly consistent with
the enrichment of the RNA polymerase II machinery in this tissue. As
our understanding of the relationship between testis gene expression
and the general transcription factors is incomplete, ALF may be an
ideal factor on which to focus future studies of this problem.
Functional Significance of ALF--
The results of in
vitro transcription assays show that ALF/
and TFIIA
/
/
complexes can restore RNA polymerase II-dependent transcription in TFIIA-depleted HeLa cell nuclear extracts (Fig. 7C). Although these results are limited to the AdML
promoter, they raise the possibility that ALF may be functionally
identical with TFIIA
/
. If so, the importance of the human ALF
gene may be to transcribe high levels of TFIIA large subunit mRNA
in testis that cannot be obtained by transcription of the TFIIA
/
gene alone. This idea is similar to the suggestion that the increase in
phosphoglycerate kinase PGK-2 gene expression during
spermatogenesis compensates for the loss of PGK-1
transcripts, in this case because of the inactivation of the
PGK-1 gene (62).
An alternative, and more interesting, possibility is that ALF, like
TRF, may be required in vivo for the expression of a subset of class II genes. For instance, ALF/
complexes might be selectively required to stabilize TBP (TFIID)-DNA interactions on the core promoter
sequences of testis-specific genes or to help mediate the function of
testis-specific transcriptional activators. These activities may
further depend on whether ALF is co-expressed in the same cell types as
TFIIA
/
and whether it is post-translationally processed into
ALF
and ALF
subunits analogous to TFIIA
and TFIIA
. A more
detailed biochemical and functional characterization of the recombinant
and endogenous factors will be required to address these issues.
Structure and Functional Significance of SALF--
This paper also
describes the isolation of a 3.8-kb cDNA, SALF, that predicts a
nearly intact ALF protein connected to an N-terminal domain homologous
to Drosophila Stoned B and clathrin APs µ1
(AP47) and µ2 (AP50). The conclusion that this cDNA
represents a naturally occurring human mRNA is based on: 1) the
identification of a partial human EST (I.M.A.G.E. Consortium Clone ID
259637) (Fig. 1A); 2) PCR amplification of products that
span the Stoned B-TFIIA
/
junction from placenta, liver, and
testis cDNA libraries (Fig. 1D); and 3) the isolation of
contiguous upstream Stoned B-like sequences using primers within the
downstream TFIIA
/
-like region (pRACE4) (Fig. 1B). It
is important to note that novel ALF-containing transcripts are not
limited to SALF, as two additional ESTs (I.M.A.G.E. Consortium Clone
IDs 785133 and 1657721) that predict an alternative C terminus were
identified in a data base search and verified by PCR analysis (data not shown).
The structure of SALF raises several issues. First, it is likely
significant that sequences similar or identical to the 5'-end of SALF
are present in an abundant and widely distributed 6.5-kb mRNA
(RNA6.5) (Fig. 5B) and that expression of RNA6.5 and
ALF-containing RNAs overlaps in non-testis tissues (Fig. 6,
A and B). These observations suggest that SALF
might be derived from a gene (or adjacent genes) that contains RNA6.5-
and ALF-like sequences or, alternatively, that it is formed from
post-transcriptional processing of RNA6.5- and ALF-containing
transcript(s). Second, the fact that SALF contains a domain potentially
involved in the clathrin-dependent trafficking of membrane
proteins (49, 50) suggests that, in this context, it might affect the
subcellular distribution or activity of the C-terminal ALF domain. In
any event, a bona fide role for SALF in gene regulation
would probably require the protein to be active even at low levels in
the cell or that expression is higher in other cell types or under
conditions not considered here.
In summary, the identification of ALF (and SALF) shows that multiple
genes encoding TFIIA large subunits are present in human. Furthermore,
the testis-specific expression of ALF, together with the overexpression
of several other general transcription factors in this tissue, points
to a special importance in testis biology and perhaps spermatogenesis.