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INTRODUCTION |
Elongin B is a subunit of the heterodimeric Elongin BC complex,
which was originally identified as a positive regulator of RNA
polymerase II elongation factor Elongin A (1, 2). Subsequently the
Elongin BC complex was identified as a component of both the multiprotein von Hippel-Lindau tumor suppressor complex (3, 4), where
it appears to function in tumor suppression (5) and regulation of
expression of hypoxia-inducible genes (6-8), and the suppressor of
cytokine signaling-1 complex, where it may function at least in part to
regulate the stability of the suppressor of cytokine signaling-1
protein (9).
Previous studies from our laboratory have shown that Elongin B and C
perform distinct functions in regulation of Elongin A transcriptional
activity (10). Elongin C is capable of binding directly to a site in
the Elongin A elongation activation domain and inducing Elongin A
transcriptional activity in vitro in the absence of Elongin
B (2, 10, 11). Elongin B does not directly affect Elongin A activity in
the absence of Elongin C; instead, Elongin B appears to bind directly
to Elongin C and to promote its interaction with Elongin A.
Molecular cloning of the mammalian Elongin B gene revealed that it
encodes a 118-amino acid protein composed of an ~84 residue NH2-terminal ubiquitin-like domain fused to an ~34
residue COOH-terminal tail (2). The ubiquitin family comprises a
diverse collection of proteins that fall into at least three classes.
One class is composed of ubiquitin, which is conjugated by E2/E3
ubiquitin ligases to a variety of proteins involved in signal
transduction and cell cycle control and targets them for degradation by
the proteosome (12, 13). A second class is composed of a small set of
ribosomal proteins that contain NH2-terminal ubiquitin moieties, which are proteolytically removed following incorporation of
these proteins into ribosomes (14, 15). A third class includes Elongin
B and a growing number of other ubiquitin-like proteins. Among the
members of this class are NEDD8/Rub1p, SUMO, and Rad23, which have
roles in cell cycle control, nucleocytoplasmic transport, and DNA
repair, respectively (16-19).
As part of our effort to understand the structure of the Elongin
BC complex and its mechanism of action in regulation of Elongin A
transcriptional activity, we are carrying out a systematic
structure-function analysis of each of the Elongin subunits. In this
report, we demonstrate that the Elongin B ubiquitin-like domain is
necessary and sufficient for interaction of Elongin B with Elongin C
and for regulation of Elongin A activity. In addition, by site-directed
mutagenesis of the Elongin B ubiquitin-like domain, we identify a short
Elongin B region that is important for its interaction with Elongin C and that is evolutionarily conserved in Elongin B homologs from D. melanogaster and C. elegans.
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EXPERIMENTAL PROCEDURES |
Materials--
Unlabeled ultrapure ribonucleoside
5'-triphosphates were purchased from Pharmacia Biotech Inc.
[
-32P]CTP (>650 Ci/mmol) was obtained from Amersham
Corp. Ni2+-nitrilotriacetic acid-agarose
(Ni2+-agarose)1
was from Invitrogen. Placental ribonuclease inhibitor (RNasin) and
acetylated bovine serum albumin were from Promega. Guanidine hydrochloride (sequanal grade) was purchased from Pierce.
Phenylmethylsulfonyl fluoride and polyvinyl alcohol (average molecular
weight 30,000-70,000) were obtained from Sigma. Phenylmethylsulfonyl
fluoride was dissolved in dimethyl sulfoxide to 1 M.
Polyvinyl alcohol was dissolved in water to 20% (w/v) and centrifuged
or filtered through a 0.2-µm filter prior to use.
DNA Template for Transcription--
pDN-AdML plasmid DNA (20)
was isolated from Escherichia coli using the Triton-lysozyme
method (21). Plasmid DNA was banded twice in CsCl-ethidium bromide
density gradients, precipitated with ethanol, and dissolved in TE
buffer (20 mM Tris-HCl (pH 7.6), and 1 mM
EDTA). A restriction fragment prepared by digestion of pDN-AdML DNA
with EcoRI and NdeI was used as template in
transcription reactions. The fragment was purified from 1.0% low
melting temperature agarose gels using GELase (Epicentre Technologies)
according to the manufacturer's instructions. After phenol-chloroform
extraction and ethanol precipitation, purified DNA fragments were
resuspended in TE buffer.
Preparation of RNA Polymerase II and Initiation Factors--
RNA
polymerase II (22) and TFIIH (rat
, TSK DEAE 5-PW fraction (23))
were purified from rat liver nuclear extracts as described. Recombinant
yeast TBP (24) and rat TFIIB (rat
(25)) were expressed in E. coli and purified as described. Recombinant TFIIE was prepared as
described (26), except that the 56-kDa subunit was expressed in
BL21(DE3)-pLysS. Recombinant TFIIF was purified as described previously
from E. coli strain JM109(DE3) infected with M13pET-Rap30
and M13pET-Rap74 (27).
Assay of Run-off Transcription--
~200 ng of wild type or
mutant Elongin B proteins were mixed with ~40 ng of wild type rat
Elongin A and ~200 ng of rat Elongin C-(
41-50) and diluted ~2
fold to a final volume of 50 µl with 40 mM Hepes-NaOH (pH
7.9), 100 mM KCl, 50 µM ZnSO4,
and 10% (v/v) glycerol. After 90 min on ice, the mixtures were
dialyzed for 2 h at 4 °C against 40 mM Tris-HCl (pH
7.9), 40 mM KCl, 0.1 mM EDTA, and 10% (v/v)
glycerol. Elongin complexes were then assayed in 60-µl reaction
mixtures as follows. Preinitiation complexes were assembled by
preincubation of ~10 ng of the EcoRI to NdeI fragment from pDN-AdML, ~10 ng of recombinant TFIIB, ~10 ng of recombinant TFIIF, ~7 ng of recombinant TFIIE, ~40 ng of rat TFIIH, ~20 ng of TBP, ~0.01 unit of RNA polymerase II, and 8 units of RNasin in 20 mM Hepes-NaOH (pH 7.9), 20 mM
Tris-HCl (pH 7.9), 60 mM KCl, 2 mM DTT, 0.5 mg/ml bovine serum albumin, 2% (w/v) polyvinyl alcohol, and 3% (v/v)
glycerol for 30 min at 28 °C. 10 µl of dialyzed Elongin complexes
were then added to each reaction mixture. Transcription was initiated
by addition of 7 mM MgCl2 and 50 µM ATP, 50 µM GTP, 10 µM CTP,
2 µM UTP, and 10 µCi of [
-32P]CTP.
After incubation of reaction mixtures for 12 min at 28 °C, run-off
transcripts were analyzed by electrophoresis through 6% polyacrylamide
gels containing 7.0 M urea. Transcription was quantitated using a Molecular Dynamics PhosphorImager.
Construction of Elongin B Mutants--
Elongin B mutants were
constructed by oligonucleotide-directed mutagenesis (28) of
M13mpET-Elongin B (2) with the Muta-Gene M13 in vitro
mutagenesis kit (Bio-Rad) and confirmed by dideoxy DNA sequencing with
the fmol DNA Sequencing System (Promega). Mutagenic
oligonucleotides included 15 nucleotides from the parental rat Elongin
B sequence on either side of the site of mutation.
Expression of Elongin B Mutants and Elongin Subunits in Mammalian
Cells--
cDNAs encoding full-length Elongin B and Elongin B
mutants containing NH2-terminal c-Myc epitope tags were
subcloned into the pcDNA3.1 vector (Invitrogen). Full-length
Elongin C containing an NH2-terminal HSV epitope tag was
subcloned into the pCI-neo vector (Promega). 24 h after
transfection of 293T cells, cells were collected and lysed in ice-cold
buffer containing 40 mM Hepes-NaOH (pH 7.9), 150 mM NaCl, 1 mM DTT, 0.5% (v/v) Triton X-100,
10% (v/v) glycerol, 5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml
peptstatin A, and 5 µg/ml aprotinin. Lysates were then centrifuged at
10,000 × g for 20 min at 4 °C.
Immunoprecipitation and Western Blotting--
Anti-c-Myc epitope
antibodies were from Roche Molecular Biochemicals and anti-HSV epitope
antibodies were from Novagen. To immunoprecipitate Elongin B and
associated proteins from 293T cell lysates, the lysates were incubated
with anti-c-Myc antibody for 1 h at 4 °C and then with protein
A/G PLUS-agarose (Santa Cruz Biotechnology) for 1 h at 4 °C.
Protein A/G beads were washed two times in buffer containing 40 mM Hepes-NaOH (pH 7.9), 500 mM NaCl, 1 mM DTT, 0.5% (v/v) Triton X-100, 10% (v/v) glycerol and
once in 40 mM Hepes-NaOH (pH 7.9), 150 mM NaCl,
1 mM DTT, 10% (v/v) glycerol. Immunoprecipitated proteins
or proteins in total cell lysates were analyzed by electrophoresis
through 13.5% SDS-polyacrylamide gels and transferred to
polyvinylidine difluoride membranes (Millipore) and vizualized by
Western blotting using the chemiluminescense reagent (NEN Life Science
Products Inc.).
Expression and Purification of Elongin B Mutants and Elongin
Subunits--
NH2-terminal histidine-tagged, wild type rat
Elongin A was overexpressed in E. coli using a pET16b
expression vector (Novagen) and purified as described (29). Untagged
wild type rat Elongin C and NH2-terminal histidine-tagged
wild type rat Elongin C, rat Elongin C-(
41-50), wild type rat
Elongin B, and rat Elongin B mutants were overexpressed in E. coli using the M13mpET bacteriophage expression system (27) and
purified as described below. Briefly, 100-ml cultures of E. coli strain JM109(DE3) were grown at 37 °C to an
A600 of 0.6 in Luria broth. Cells were infected
with the appropriate M13mpET bacteriophage expression vectors at a multiplicity of infection of 10-20. After an additional 2 h at 37 °C, cells were induced with 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside, and cultures were
incubated an additional 3 h. Cells were harvested by
centrifugation at 2000 × g for 10 min at 4 °C. The
cell pellets were resuspended in 7 ml of 20 mM Tris-HCl (pH
8.0), 1 mg/ml lysozyme, and 10 mM imidazole (pH 8.0) and
incubated for 30 min on ice. After one cycle of freeze-thaw, the
suspensions were centrifuged at 100,000 × g for 35 min. Inclusion bodies were solubilized by resuspension in 7 ml of
ice-cold 6 M guanidine hydrochloride, 40 mM
Tris-HCl (pH 8.0), 0.5 M KCl, 10 mM imidazole
(pH 8.0), and 0.5 mM phenylmethylsulfonyl fluoride, and the
resulting suspensions were clarified by centrifugation at 100,000 × g for 35 min. All histidine-tagged proteins were further
purified by Ni2+-agarose chromatography in guanidine
hydrochloride as described (29).
Expression and Purification of D. melanogaster and C. elegans
Elongin B Proteins--
A TBLASTN search of the GenBank EST data base
identified potential Elongin B homologs from D. melanogaster
and C. elegans. EST yk121b3 encoding the potential C. elegans Elongin B homolog was obtained from Y. Kohara (National
Institute of Genetics, Mishima, Japan). EST LD16189 encoding the
potential Drosophila Elongin B homolog was obtained from
Genome Systems, Inc. The entire open reading frames of the
Drosophila and C. elegans proteins were overexpressed in E. coli with NH2-terminal
histidine tags using the M13mpET bacteriophage expression system
(27).
Assay of Elongin BC Complex Formation--
~2 µg of
histidine-tagged wild type rat Elongin B or rat Elongin B mutants were
mixed with ~2 µg of untagged wild type rat Elongin C and diluted
20-fold with 40 mM Hepes-NaOH (pH 7.9), 100 mM
KCl, 50 µM ZnSO4, and 10% (v/v) glycerol.
After 30 min on ice, the mixtures were dialyzed for 2 h at 4 °C
against 40 mM Hepes-NaOH (pH 7.9), 0.1 mM EDTA,
100 mM KCl, and 10% (v/v) glycerol. Following dialysis,
the mixtures were incubated for 1 h at 4 °C with ~20 µl of
Ni2+-agarose pre-equilibrated in dialysis buffer containing
10 mM imidazole (pH 8.0) and then centrifuged for 20 s
at 2000 rpm in a Micromax microcentrifuge (International Equipment
Co.). Following centrifugation, the supernatants containing unbound
proteins were collected, and the Ni2+-agarose was washed 3 times by resuspension in 500 µl of 40 mM Hepes-NaOH (pH
7.9), 100 mM KCl, 0.1 mM EDTA, 10% (v/v)
glycerol, and 40 mM imidazole (pH 8.0) and centrifugation
for 20 s at 2000 rpm. Finally, bound material was eluted with 300 µl of the same buffer containing 300 mM imidazole (pH
8.0). Aliquots of each fraction were analyzed by 13.5 or 18%
SDS-polyacrylamide gel electrophoresis, and the proteins were
visualized by silver staining.
Assay of Elongin ABC Complex Formation--
~6 µg of
histidine-tagged wild type rat, C. elegans, or D. melanogaster Elongin B or rat Elongin B mutants were mixed with ~45 µg of wild type, histidine-tagged rat Elongin A and ~6 µg of wild type, histidine-tagged rat Elongin C and diluted 5-fold with 40 mM Hepes-NaOH (pH 7.9), 100 mM KCl, 50 µM ZnSO4, and 10% (v/v) glycerol. After 90 min on ice, the mixtures were dialyzed for 2 h at 4 °C against
40 mM Tris-HCl (pH 7.9), 40 mM KCl, 0.1 mM EDTA, and 10% (v/v) glycerol. Following dialysis, the
mixtures were centrifuged at 60,000 × g for 15 min at
4 °C. The resulting supernatants were applied to TSK SP-NPR columns
(35 × 4.6 mm, Toso-Haas) pre-equilibrated in 40 mM
Hepes-NaOH (pH 7.9), 100 mM KCl, 1 mM DTT, and
10% (v/v) glycerol and fractionated using a SMART microchromatography
system (Pharmacia) at 8 °C. The columns were eluted at 0.6 ml/min
with a 9-ml linear gradient from 100 to 900 mM KCl in 40 mM Hepes-NaOH (pH 7.9), 1 mM DTT, and 10% (v/v) glycerol. Aliquots of each column fraction were analyzed by
12.5% SDS-polyacrylamide gel electrophoresis, and the proteins were
visualized by silver staining.
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RESULTS |
Evolutionary Conservation of Elongin B--
We previously
described cloning of rat and human Elongin B cDNAs (2).
Characterization of these cDNAs revealed that Elongin B is composed
of an ~84 amino acid NH2-terminal ubiquitin-like domain
fused to an ~34-amino acid COOH-terminal tail. The
NH2-terminal ubiquitin-like domain can be modeled as
ubiquitin (2), a compact globular structure containing a 3.5 turn
-helix (
1), a short 310 helix, and a 5-stranded mixed
-sheet (30).
As an initial step in our analysis of the structure and function of
Elongin B, we identified and characterized potential D. melanogaster and C. elegans Elongin B homologs.
Comparison of the amino acid sequences of mammalian Elongin B and the
potential Drosophila and C. elegans Elongin B
homologs indicates that they are highly conserved (Fig.
1). According to the BESTFIT program of
GCG, the rat and Drosophila proteins are 55% identical and 78% similar; the rat and C. elegans proteins are 39%
identical and 62% similar; and the Drosophila and C. elegans proteins are 36% identical and 56% similar. A TBLASTN
search of the S. cerevisiae data base revealed no S. cerevisiae open reading frames with significant sequence
similarity to mammalian Elongin B, even though the S. cerevisiae genome includes an open reading frame that encodes a
100-amino acid protein that exhibits significant sequence similarity to
mammalian Elongin C (29, 31).

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Fig. 1.
Evolutionary conservation of Elongin B. The alignment of rat and human Elongin B with D. melanogaster and C. elegans Elongin B homologs was
determined with the MACAW program (31) using the BLOSUM 80 score table.
Positions of identical residues conserved in at least three of the four
Elongin B proteins are indicated in the alignment by dark
shading, and the positions of similar residues conserved in at
least three of the four Elongin B proteins are indicated by light
shading. Positions of predicted Elongin B structural elements are
shown above the alignment, and positions of ubiquitin
structural elements identified in the crystal structure (30) are shown
below the alignment. Ub, ubiquitin; ,
-helix; , -strand.
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To determine whether the potential Drosophila and C. elegans Elongin B homologs possess activities similar to those of
mammalian Elongin B, we investigated their abilities to substitute for
rat Elongin B in reconstitution of the Elongin complex. In previous studies, we showed that the transcriptionally active Elongin ABC complex could be reconstituted by recombining individual Elongin subunits purified from rat liver (1, 32) or by refolding bacterially
expressed Elongin subunits purified from guanidine hydrochloride-solubilized inclusion bodies (10). Formation of the
Elongin ABC complex can be assayed by ion exchange HPLC using TSK
SP-NPR (2, 29). Elongin BC complexes and free Elongin B and C
flow-through TSK SP-NPR at low ionic strength, whereas Elongin ABC
complexes bind tightly to TSK SP-NPR and elute with ~0.3
M KCl.
To investigate the abilities of the potential Drosophila and
C. elegans Elongin B homologs to assemble with mammalian
Elongin A and C into chromatographically isolable ternary complexes,
the Drosophila and C. elegans proteins were
expressed in E. coli, purified from guanidine-solubilized
inclusion bodies, refolded together with bacterially expressed rat
Elongin A and C, and subjected to TSK SP-NPR HPLC. As shown in Fig.
2A, like rat Elongin B, both the potential Drosophila and C. elegans Elongin B
homologs are capable of assembling with rat Elongin A and C to form
ternary complexes that can be isolated by TSK SP-NPR HPLC; neither the Drosophila nor C. elegans proteins bound to TSK
SP-NPR in the absence Elongin A (data not shown).

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Fig. 2.
Characterization of D. melanogaster and C. elegans Elongin B
homologs. Panel A, Drosophila and C. elegans
Elongin B homologs were assayed for their abilities to form Elongin ABC
complexes by TSK SP-NPR HPLC as described under "Experimental
Procedures." L, load; FT, flow-through.
Panel B, the indicated Elongin B molecules were assayed for
Elongin transcriptional activity as described under "Experimental
Procedures."
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To determine whether the potential Drosophila and C. elegans Elongin B homologs function similarly to mammalian Elongin
B in transcription, we assayed them for their abilities to promote activation of Elongin A transcriptional activity by Elongin C. We
previously showed that Elongins B and C play different roles in
activation of Elongin A activity (10). Elongin C functions as the
inducing ligand and activates Elongin A by binding to a site in the
Elongin A elongation activation domain. Although Elongin B is not
essential for activation of Elongin A by Elongin C, it promotes
interaction of Elongin C with Elongin A and, in so doing, increases
both the yield and stability of the functional Elongin complex.
To devise an assay for Elongin transcriptional activity with the
strongest possible dependence on Elongin B, we took advantage of an
Elongin C mutant (Elongin C-(
41-50)) (29) that does not bind stably
to Elongin A in the absence of Elongin B (data not shown) and that does
not detectably activate Elongin A transcriptional activity unless
Elongin B is present (Fig. 2B). In these experiments, Elongin complexes were assembled with rat Elongin A and Elongin C-(
41-50) in the absence of Elongin B or in the presence of either rat Elongin B or the potential Drosophila or C. elegans Elongin B homologs. The Elongin complexes were then
assayed for their abilities to stimulate the rate of accumulation of
run-off transcripts synthesized by RNA polymerase II from the AdML
promoter in a purified basal transcription system reconstituted with
the general initiation factors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. As
shown in Fig. 2B, like rat Elongin B, the potential
Drosophila and C. elegans Elongin B homologs were
capable of strongly promoting activation of Elongin A by Elongin
C-(
41-50). Taken together, the results of both binding and
transcription assays argue that the Drosophila and C. elegans proteins are homologs of mammalian Elongin B.
The Elongin B Ubiquitin-like Domain Is Sufficient for Elongin B
Function in Vitro--
To determine whether the
NH2-terminal Elongin B ubiquitin-like domain, the
COOH-terminal tail, or both are required for Elongin B function, a
series of NH2-terminal, COOH-terminal, and internal deletion mutants of rat Elongin B were constructed (Fig.
3A), expressed in E. coli, purified from guanidine hydrochloride-solubilized inclusion
bodies, and assayed for their abilities to assemble into Elongin BC and
ABC complexes and to promote activation of Elongin A by Elongin
C-(
41-50).

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Fig. 3.
The Elongin B ubiquitin-like domain is
necessary and sufficient for Elongin B activity In
Vitro. Panel A, Elongin B deletion mutants.
Panel B, wild type rat Elongin B and Elongin B mutants were
assayed for their abilities to form Elongin BC complexes by
Ni2+-agarose chromatography as described under
"Experimental Procedures." Panel C, Elongin B deletion
mutants were assayed for their abilities to form Elongin ABC complexes
by TSK SP-NPR HPLC as described under "Experimental Procedures."
L, load; FT, flow-through. Panel D,
Elongin B deletion mutants were assayed for transcriptional activity as
described under "Experimental Procedures".
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To assess the abilities of Elongin B deletion mutants to assemble with
Elongin C into Elongin BC complexes, we assayed rat Elongin B mutants
containing NH2-terminal histidine tags for their abilities
to retain untagged wild type rat Elongin C on Ni2+-agarose.
In these experiments, individual wild type Elongin B or Elongin B
deletion mutants were refolded together with Elongin C and subjected to
Ni2+-agarose chromatography. Unbound and bound protein
fractions were collected, and equivalent amounts of each fraction were
analyzed by SDS-polyacrylamide gel electrophoresis. As shown in Fig.
3B, untagged Elongin C does not bind to
Ni2+-agarose, but is retained on the resin in the presence
of histidine-tagged wild type Elongin B. Elongin C was quantitatively
retained on Ni2+-agarose only by the COOH-terminal deletion
mutant Elongin B-(1-84), which is the only deletion mutant with an
intact ubiquitin-like domain. Elongin C was not retained on
Ni2+-agarose by any Elongin B mutants with deletions of the
NH2-terminal portion of the ubiquitin-like domain, and only
very small amounts of Elongin C were retained on
Ni2+-agarose by Elongin B mutants with deletions of the
COOH-terminal portion of the ubiquitin-like domain. Taken together,
these results argue that the Elongin B ubiquitin-like domain is
necessary and sufficient for stable interaction with Elongin C in
vitro.
To assess the abilities of the Elongin B deletion mutants to support
formation of functional Elongin ABC complexes, we assayed Elongin B
mutants for their abilities (i) to form Elongin ABC complexes isolable
by TSK SP-NPR HPLC and (ii) to promote activation of Elongin A by
Elongin C-(
41-50). In experiments investigating the abilities of
Elongin B deletion mutants to support formation of the Elongin ABC
complex, individual wild type Elongin B and Elongin B deletion mutants
were refolded together with bacterially expressed wild type rat
Elongins A and C and subjected to TSK SP-NPR HPLC. In experiments
investigating the abilities of Elongin B deletion mutants to promote
activation of Elongin A by Elongin C-(
41-50), bacterially expressed
wild type rat Elongin A and Elongin C-(
41-50) were refolded
together in the presence or absence of wild type rat Elongin B or
Elongin B deletion mutants. Elongin complexes were then assayed for
their abilities to stimulate the rate of accumulation of full-length
run-off transcripts synthesized from the AdML promoter by RNA
polymerase II and purified initiation factors TBP, TFIIB, TFIIE, TFIIF,
and TFIIH.
As shown in Fig. 3, C and D, Elongin B-(1-84),
which includes the entire ubiquitin-like domain and binds stably to
Elongin C (Fig. 3B), was also capable of assembling with
Elongin A and C into an isolable Elongin ABC complex and of strongly
promoting activation of Elongin A transcriptional activity by Elongin
C-(
41-50) mutant. In contrast, Elongin B deletion mutants lacking
as few as 10 amino acids from the NH2 terminus of the
ubiquitin-like domain did not form Elongin ABC complexes and did not
promote activation of Elongin A by Elongin C-(
41-50). Elongin
B-(1-74), Elongin B-(1-64), and Elongin B-(
58-66), which have
deletions in the COOH terminus of the ubiquitin-like domain, did not
promote detectable activation of Elongin A by Elongin C-(
41-50),
but did form Elongin ABC complexes. Thus, the entire Elongin B
ubiquitin-like domain is necessary and sufficient for promoting
activation of Elongin A transcriptional activity by Elongin
C-(
41-50). Elongin B residues 58-84 of the ubiquitin-like domain
are not essential for formation of Elongin ABC complexes, although
deletion mutants lacking these residues are severely impaired in their
abilities to form Elongin BC complexes. Elongin B-(
58-66), Elongin
B-(1-64), and Elongin B-(1-74) lack portions of predicted helix 2, the extended surface loop, and/or predicted
-sheet 5, regions of the
protein that would be important for maintaining a ubiquitin-like
tertiary structure (30, 33-35). Our results raise the possibility that contacts between Elongin B and the Elongin AC complex may contribute to
proper folding of the Elongin B ubiquitin-like domain by compensating for loss of the predicted surface loop, portions of helix 2, or predicted
-sheet 5.
Mutagenesis of the Elongin B Ubiquitin-like Domain--
Although
ubiquitin is similar in sequence to Elongin B, ubiquitin neither binds
detectably to Elongin C nor promotes activation of Elongin A
transcriptional activity by Elongin C (2), suggesting that Elongin B
sequences differing from those of ubiquitin are important for Elongin B
function. As discussed above, the Elongin B ubiquitin-like domain can
be modeled as ubiquitin. Notable features of the Elongin B model
include its striking conservation of the ubiquitin hydrophobic core and
conservation of the hydrophobic character of residues corresponding to
ubiquitin surface residues Phe-4 and Leu-71. The Elongin B model also
predicts Elongin B features not shared by ubiquitin; these include two
additional hydrophobic surface residues Phe-15 and Phe-25, a prominent
basic surface patch composed predominantly of residues Arg-8, Arg-9, His-10, and Lys-11, and an extended surface loop that falls between residues 62 and 70 and accommodates a 7-amino acid insertion in the
ubiquitin-like domain.
To explore the relationship between Elongin B and ubiquitin and define
in more detail Elongin B residues critical for its function, we
investigated the activities of a collection of additional Elongin B
mutants that were constructed either by replacing predicted Elongin B
surface residues with those from the corresponding positions of
ubiquitin, mutating the predicted Elongin B surface hydrophobic residues, or mutating Elongin B residues within the predicted basic
patch. These mutants, which contained mutations spanning the entire
ubiquitin-like domain, were tested for their abilities, (i) to assemble
into isolable Elongin BC complexes in cells and (ii) to assemble into
Elongin BC and ABC complexes in vitro and to promote
activation of Elongin A by Elongin C-(
41-50) in vitro. To assay formation of Elongin BC complexes in cells, c-Myc-tagged Elongin B and Elongin B mutants were coexpressed with HSV-tagged Elongin C in 293T cells and tested for their abilities to interact with
one another by coimmunoprecipitation with an anti-c-Myc antibody. Formation of Elongin BC and ABC complexes and activation of Elongin transcription activity in vitro were assayed as described
above. Results of these experiments can be summarized as follows.
(i) Of the Elongin B mutants tested, only three exhibited reduced
ability to form Elongin BC complexes in cells (Fig.
4). These mutants, R29A,
G33D/I34K/L35E/K36G, and EloB(
31-40), also failed to assemble into
isolable Elongin BC and ABC complexes in vitro and to
promote activation of Elongin A by Elongin C-(
41-50) (Fig.
5). R29A, G33D/I34K/L35E/K36G, and
EloB(
31-40) mutations are confined to a short region in the
COOH-terminal portion of predicted helix 1 and in the predicted turn
between helix 1 and
-strand 3. Notably, the R29A and GILK to DKEG
mutations change Elongin B residues to the corresponding residues from
ubiquitin. Additional ubiquitin-like mutants, including F15T, D17E,
K19E/E20P, Y45I/K46F/D47A/D48G/Q49K, D52E, K55R, G76H, and the mutant
in which Ala-81 and Asp-82 were mutated to LRGG, were capable of binding Elongin C in vivo (Fig. 4), suggesting that
differences between Elongin B and ubiquitin in helix 1 and in the turn
immediately following may be sufficient to account for the inability of
ubiquitin to bind Elongin C.

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Fig. 4.
Interaction of Elongin B mutants with Elongin
C in mammalian cells. 293T cells were transiently transfected in
35-mm wells with 1 µg of wild type or mutant c-Myc-Elongin B and 1 µg of HSV-Elongin C expression vectors as described under
"Experimental Procedures." Cell lysates containing approximately
equivalent amounts of wild type Elongin B or Elongin B mutants were
immunoprecipitated with anti-c-Myc antibodies (IP:
myc-EloB), except that ~30% less Elongin B R29A was present in
the cell lysate used for immunoprecipitation of that mutant. The
concentrations of Elongin B proteins in cell lysates were determined by
densitometry of appropriate exposures of Western blots. Elongin B and
Elongin C proteins were detected in both total lysate and
immunoprecipitated fractions by Western blot using anti-c-Myc
(WB: myc-EloB) and anti-HSV (WB: HSV-EloC)
antibodies, respectively.
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Fig. 5.
Analysis of Elongin B mutants with
ubiquitin-like substitutions. Panel A, wild type rat
Elongin B and Elongin B mutants were assayed for their abilities to
form Elongin BC complexes by Ni2+-agarose chromatography as
described under "Experimental Procedures." Panel B,
Elongin B mutants were assayed for their abilities to form Elongin ABC
complexes by TSK SP-NPR HPLC as described under "Experimental
Procedures." L, load; FT, flow-through.
Panel C, Elongin B mutants were assayed for transcriptional
activity as described under "Experimental Procedures."
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(ii) Of the ubiquitin-like mutants that were capable of binding Elongin
C in vivo, none assembled into Elongin BC complexes in
vitro, suggesting that the mutations interfere with proper folding
of Elongin B in vitro (Fig. 5A). Several of these
mutants, however, were able to form Elongin ABC complexes and/or to
promote activation of Elongin A by Elongin C-(
41-50) (Fig. 5,
A and B). The mutant Y45I/K46F/D47A/D48G/Q49K, in
which residues located at the COOH terminus of
-strand 3 and in the
turn immediately following were mutated to the corresponding residues
of ubiquitin, was able to form an Elongin ABC complex and to promote
activation of Elongin A by Elongin C-(
41-50) as well as or better
than wild type Elongin B. Another mutant, constructed by replacing
Elongin B residues Lys-19 and Glu-20 with the predicted corresponding ubiquitin residues Glu-18 and Pro-19, which occupy a position in the
turn between the second
-sheet and the first
-helix, failed to
form isolable Elongin BC or ABC complexes in vitro, but did
promote activation of Elongin A by Elongin C-(
41-50). A third
mutant K11G, formed isolable Elongin ABC complexes in vitro,
but did not promote activation of Elongin A by Elongin C-(
41-50).
(iii) The single Elongin B point mutations R8N, R9G, H10S, and K11N,
which decrease the net positive charge in the predicted basic surface
patch and which alter residues in a predicted turn between
-strands
1 and 2, had no detectable effect on the ability of Elongin B to form
Elongin BC (Fig. 6A) and ABC
complexes in vitro (Fig. 6B) or to promote
activation of Elongin A transcriptional activity A by Elongin
C-(
41-50) (Fig. 6C). Consistent with these observations,
R9G could be immunoprecipitated from cell lysates with Elongin C (Fig.
4). An additional mutant, K11G, could also be immunoprecipitated from
cell lysates with Elongin C (Fig. 4); however, when assayed for
activity in vitro, this mutant was found to assemble into
ABC complexes but not to promote activation of Elongin A by
C-(
41-50) (Fig. 5, B and C). K11G was also
unable to assemble into isolable Elongin BC complexes in
vitro, suggesting that its failure to promote activation of
Elongin A by C-(
41-50) might be due to improper folding of the
protein purified from inclusion bodies.

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Fig. 6.
Analysis of Elongin B mutants with point
mutations in the predicted basic patch. Panel A, wild
type rat Elongin B and Elongin B mutants were assayed for their
abilities to form Elongin BC complexes by Ni2+-agarose
chromatography as described under "Experimental Procedures."
Panel B, Elongin B deletion mutants were assayed for their
abilities to form Elongin ABC complexes by TSK SP-NPR HPLC as described
under "Experimental Procedures." L, load; FT,
flow-through. Panel C, Elongin B mutants were assayed for
transcriptional activity as described under "Experimental
Procedures."
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(iv) Mutations of predicted Elongin B surface hydrophobic residues
Phe-4, Phe-15, Phe-25, and Phe-62, which fall within predicted
-strand 1,
-strand 2, at the NH2 terminus of
predicted helix 1, and in predicted helix 2, respectively, had
different effects on Elongin B function (Fig.
7). Elongin B point mutants F4N, F15N, and F25N could assemble into isolable Elongin BC and ABC complexes in vitro and promote activation of Elongin A by Elongin
C-(
41-50), although Elongin B mutant F15N was less active than wild
type Elongin B in promoting activation of Elongin A. Elongin B point mutant F62N, which contains a mutation at the NH2 terminus
of the predicted Elongin B surface loop not present in ubiquitin, assembled into Elongin BC and ABC complexes, but did not promote activation of Elongin A by Elongin C-(
41-50). Finally, F15T, which
was constructed by replacing Elongin B residue Phe-15 with the
predicted corresponding ubiquitin residue Thr-14, formed isolable Elongin BC complexes in cells but did not assemble into detectable Elongin BC or ABC complexes in vitro and did not promote
activation of Elongin A by Elongin C-(
41-50), suggesting that the
F15T mutation might interfere with proper folding of Elongin B in
vitro.

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Fig. 7.
Analysis of Elongin B mutants with mutations
of predicted surface hydrophobic residues. Panel A,
wild type rat Elongin B and Elongin B mutants were assayed for their
abilities to form Elongin BC complexes by Ni2+-agarose
chromatography as described under "Experimental Procedures."
Panel B, Elongin B deletion mutants were assayed for their
abilities to form Elongin ABC complexes by TSK SP-NPR HPLC as described
under "Experimental Procedures." L, load; FT,
flow-through. Panel C, Elongin B mutants were assayed for
transcriptional activity as described "Experimental
Procedures."
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DISCUSSION |
In this report, we have investigated Elongin B sequences required
for its interaction with Elongin C. Mammalian Elongin B is a 118-amino
acid protein composed of an ~84 amino acid ubiquitin-like domain
fused to an ~34 amino acid COOH-terminal tail (2). The Elongin B
ubiquitin-like domain shares ~30% sequence identity with ubiquitin.
Other ubiquitin-like proteins include NEDD8 and its yeast homologue
Rub1p, SUMO, and Rad23, which have roles in cell cycle control,
nucleocytoplasmic transport, and DNA repair, respectively (16-19).
These ubiquitin-like proteins exhibit a variable degree of similarity
with ubiquitin, ranging from more than 50% sequence identity (NEDD8
and Rub1p) to less than 20% identity (SUMO). Despite the variation in
their primary sequences, ubiquitin, NEDD8, Rub1p, and SUMO have very
similar tertiary structures (33-35). Although a crystal structure of
Elongin B has not yet been reported, Elongin B can be modeled as
ubiquitin (2).
Analysis of the functions of Elongin B mutants has revealed that the
Elongin B ubiquitin-like domain is sufficient for its interaction with
Elongin C in vivo and in vitro and for
reconstitution of the transcriptionally active Elongin complex in
vitro. Furthermore, we observe that neither the predicted surface
hydrophobic residues nor residues that make up the predicted basic
surface patch are critical for reconstitution of functional Elongin
complexes in vitro. We did, however, identify three
mutations, R29A, G33D/I34K/L35E/K36G, and
31-40, which fall within
the ubiquitin-like domain and significantly reduce the interaction of
Elongin B with Elongin C in vivo and in vitro.
Notably, these mutations are confined to a short region in the
COOH-terminal portion of predicted
-helix 1 and in the predicted
turn between helix 1 and
-strand 3. Although we cannot rule out the
possibility that these mutations affect the overall conformation of
Elongin B, our results are consistent with the model that the COOH
terminus of predicted
-helix 1 and the predicted turn between helix
1 and
-strand 3 may form a surface that interacts directly with
Elongin C or may be important for maintaining Elongin B in a
conformation that can interact with Elongin C. Two of the inactivating
mutations, R29A and G33D/I34K/L35E/K36G, change Elongin B residues to
the corresponding residues from ubiquitin. Sequence differences between
Elongin B and ubiquitin in this region may be sufficient to account for
the inability of ubiquitin to bind Elongin C, since eight additional
ubiquitin-like mutations located throughout the ubiquitin-like domain
had no significant effect on the interaction of Elongin B with Elongin
C in cells.
Finally, by characterizing Drosophila and C. elegans Elongin B homologs that efficiently replace mammalian
Elongin B in reconstitution of the transcriptionally active Elongin ABC
complex, we show that both the Elongin B ubiquitin-like domain and
COOH-terminal tail have been highly conserved during evolution.
Although we have not yet identified a function for the Elongin B tail,
it is noteworthy that it includes a PXXP motif that is a
potential target for binding by SH3 domain proteins (36). Efforts to
identify cellular proteins that interact with the Elongin B tail are underway.