Characterization of elongin C functional domains required for interaction with elongin B and activation of elongin A.

The Elongin (SIII) complex stimulates the rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along DNA templates. The Elongin (SIII) complex is composed of a transcriptionally active A subunit, a chaperone-like B subunit, which promotes assembly and enhances stability of the Elongin (SIII) complex, and a regulatory C subunit, which (i) functions as a potent activator of Elongin A transcriptional activity, (ii) interacts specifically with Elongin B to form an isolable Elongin BC complex, and (iii) is bound and negatively regulated in vitro by the product of the von Hippel-Lindau tumor suppressor gene. As part of our effort to understand how Elongin C regulates the activity of the Elongin (SIII) complex, we are characterizing Elongin C functional domains. In this report, we identify Elongin C mutants that fall into multiple functional classes based on their abilities to bind Elongin B and to bind and activate Elongin A under our assay conditions. Characterization of these mutants suggests that Elongin C is composed of multiple overlapping regions that mediate functional interactions with Elongin A and B.

The Elongin (SIII) complex stimulates the rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along DNA templates. The Elongin (SIII) complex is composed of a transcriptionally active A subunit, a chaperone-like B subunit, which promotes assembly and enhances stability of the Elongin (SIII) complex, and a regulatory C subunit,

which (i) functions as a potent activator of Elongin A transcriptional activity, (ii) interacts specifically with Elongin B to form an isolable Elongin BC complex, and (iii) is bound and negatively regulated in vitro by the product of the von Hippel-Lindau tumor suppressor gene. As part of our effort to understand how Elongin C regulates the activity of the Elongin (SIII) complex, we are characterizing Elongin C functional domains. In this report, we identify Elongin C mutants that fall into multiple functional classes based on their abilities to bind Elongin B and to bind and activate Elongin A under our assay conditions. Characterization of these mutants suggests that Elongin C is composed of multiple overlapping regions that mediate functional interactions with Elongin A and B.
Eukaryotic messenger RNA synthesis is an elaborate biochemical process catalyzed by multisubunit RNA polymerase II and governed by the concerted action of a set of general transcription factors that control the activity of polymerase during the initiation and elongation stages of transcription (1)(2)(3)(4). At least six general initiation factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) have been identified in eukaryotic cells and found to promote selective binding of RNA polymerase II to promoters and to support a basal level of transcription (1). In addition to the general initiation factors, five general elongation factors (P-TEFb, SII, TFIIF, ELL, and Elongin (SIII) 1 ) have been defined biochemically and found to increase the efficiency of elongation by RNA polymerase II. P-TEFb catalyzes the conversion of early, termination-prone elongation complexes into productive elongation complexes (5,6). SII prevents RNA polymerase II from terminating transcription prematurely by promoting passage of polymerase through a vari-ety of transcriptional impediments, including DNA sequences that act as intrinsic arrest sites and DNA-bound proteins and drugs (7). The remaining elongation factors, TFIIF (8), ELL (9), and Elongin (SIII) (10,11) all act to increase the overall rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along the DNA. In addition, two of these elongation factors, ELL and Elongin (SIII), may play roles in the development of certain types of cancers. The gene encoding ELL is a frequent target for t (11;19) chromosomal translocations in acute myeloid leukemias (12,13). Elongin (SIII) is a potential target for regulation by the product of the von Hippel-Lindau (VHL) 2 tumor suppressor gene, which is mutated in the majority of clear-cell renal carcinomas and in families with VHL disease, a rare genetic disorder that predisposes individuals to a variety of cancers including clear-cell renal carcinoma, hemangioblastomas and hemangiomas, and pheochromocytoma (14,15).
Elongin (SIII) was originally purified from mammalian cells as a heterotrimer composed of A, B, and C subunits of 773, 118, and 112 amino acids, respectively (10, 16 -18). Elongin A is the transcriptionally active component of the Elongin (SIII) complex. Whereas Elongin A is capable of weakly stimulating the rate of elongation by RNA polymerase II in the absence of Elongin B and C, neither Elongin B nor Elongin C affects the activity of polymerase in the absence of Elongin A (16 -18). Biochemical studies have shown that Elongin B and C are positive regulators of Elongin A activity and function by different mechanisms (16 -18). Elongin C is capable of interacting directly with Elongin A in the absence of Elongin B to form an AC complex with increased specific activity, suggesting that Elongin C functions as a direct activator of Elongin A. Elongin B, a member of the ubiquitin homology gene family, does not appear to interact directly with Elongin A. Evidence suggests that Elongin B plays a chaperone-like role in assembly of the Elongin (SIII) complex by binding to Elongin C and facilitating its interaction with Elongin A.
As part of our effort to understand the structure, mechanism of action, and regulation of the Elongin (SIII) complex, we are carrying out a systematic structure-function analysis of the each of the Elongin subunits. Here we describe studies leading to the identification of Elongin C regions important for binding to Elongin B and for binding and activation of Elongin A.  1 RNA polymerase II elongation factor Elongin is referred to in this paper as Elongin (SIII) because it was originally designated ''SIII'' (10). obtained from American Allied Biochemicals or Promega. [␣-32 P]CTP (Ͼ650 Ci/mmol) was from Amersham Corp. Proteinase K and isopropyl ␤-D-thiogalactoside (IPTG) were purchased from Sigma. Bovine serum albumin (Pentex fraction V, reagent grade) was obtained from ICN Immunobiologicals. Guanidine hydrochloride (sequanal grade) was from Pierce. Phenylmethylsulfonyl fluoride (PMSF) was from Sigma and was dissolved in dimethyl sulfoxide to 1 M. Polyvinyl alcohol (average molecular weight 30,000 -70,000) was from Sigma and was dissolved in water to 20% (w/v) and centrifuged or filtered through a 0.2-m filter prior to use.

Materials-Unlabeled
DNA Template for Transcription-pDN-AdML (19) plasmid DNA was isolated from Escherichia coli using the Triton-lysozyme method (20). 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, 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 a 1.0% low melting temperature agarose gel using GELase (Epicentre Technologies) according to the manufacturer's instructions. After phenolchloroform extraction and ethanol precipitation, purified DNA fragments were resuspended in TE buffer.
Assay of Runoff Transcription-All reaction mixtures were 60 l. Construction of Elongin C Mutants-Elongin C mutants were constructed by oligonucleotide-directed mutagenesis (27) of M13mpET-Elongin C (16) 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 C sequence (16) on either side of the deletion point. Mutagenic oligonucleotides for alanine scanning mutagenesis included 12 nucleotides from the parental rat Elongin C on either side of the alanine substitution point.
Expression and Purification of Elongin Subunits-Histidine-tagged Elongin A was overexpressed in E. coli using a pET16b expression vector (Novagen). The pET16b-Elongin A construct for expression of histidine-tagged Elongin A was prepared by insertion of a polymerase chain reaction-generated fragment containing the entire rat Elongin A open reading frame (18) into the NdeI and BamHI sites of the pET-16b vector. A 1-liter culture of E. coli strain BL21(DE3) transformed with pET16b-Elongin A was grown to an A 600 of 0.6 in Luria broth (LB) (20) containing 100 g/ml carbenicillin at 37°C. Following induction with 0.5 mM IPTG, the culture was incubated for an additional 3 h at 37°C. Cells were harvested by centrifugation at 2000 ϫ g for 10 min at 4°C. The cell pellet was resuspended in 50 ml of ice-cold 20 mM Tris-HCl (pH 8.0), 10 mM imidazole (pH 8.0), and 1 mg/ml lysozyme and incubated on ice for 30 min. After two cycles of freeze-thaw, the suspension was centrifuged at 100,000 ϫ g for 35 min. Inclusion bodies were solubilized by resuspension in 50 ml of ice-cold 6 M guanidine hydrochloride, 40 mM Tris-HCl (pH 8.0), 10 mM imidazole (pH 8.0), 0.5 mM PMSF, and 0.5 M KCl, and the resulting suspension was clarified by centrifugation at 100,000 ϫ g for 35 min. Recombinant Elongin A was purified from the supernatants by Ni 2ϩ -nitrilotriacetic acid-agarose (Invitrogen) affinity chromatography. Ni 2ϩ chromatography was performed at 4°C. 10 ml of supernatant were applied to a 1-ml Ni 2ϩ -column preequilibriated with 6 M guanidine hydrochloride, 20 mM Tris-HCl (pH 7.9), 10 mM imidazole (pH 8.0), 0.5 M KCl, and 0.5 mM PMSF. The column was washed with 10 ml of 5.7 M guanidine hydrochloride, 40 mM Tris-HCl (pH 7.9), 40 mM imidazole (pH 8.0), and 0.5 mM PMSF, and recombinant Elongin A was eluted with 4.2 M guanidine hydrochloride, 40 mM Tris-HCl (pH 7.9), 300 mM imidazole (pH 8.0), and 0.5 mM PMSF.
Overexpression of Elongin B and wild type and mutant Elongin C was accomplished using the M13mpET bacteriophage expression system (16,17). A 100-ml culture of E. coli strain JM109(DE3) (Promega) was grown to an A 600 of 0.6 in LB medium at 37°C. Cells were infected with M13pET bacteriophage carrying the wild type rat Elongin B or wild type or mutant rat Elongin C cDNAs at a multiplicity of infection of 10 -20. After an additional 2 h at 37°C, cells were induced with 0.5 mM IPTG, and cultures were incubated an additional 3 h. Cells were harvested by centrifugation at 2000 ϫ g for 10 min at 4°C. The cell pellet was was resuspended in 7 ml of 20 mM Tris-HCl (pH 8.0), 10 mM imidazole (pH 8.0), and 1 mg/ml lysozyme and incubated on ice for 30 min. After two cycles of freeze-thaw, the suspension was centrifuged at 100,000 ϫ 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), 10 mM imidazole (pH 8.0), 0.5 mM PMSF, and 0.5 M KCl, and the resulting suspension was clarified by centrifugation at 100,000 ϫ g for 35 min. Wild type Elongin B and wild type and mutant Elongin C were purified using Ni 2ϩ -column chromatography as described above.

Identification of an Elongin C Region Important for Binding to Elongin B-Mammalian
Elongin C is a 112-amino acid protein with a calculated molecular mass of 12,473 Da (16). In previous studies, we have shown that transcriptionally active Elongin (SIII) and Elongin subassemblies can be reconstituted by renaturation of combinations of denatured native Elongin subunits purified from rat liver (10,16) or bacterially expressed Elongin subunits purified from guanidine hydrochloride-solubilized inclusion bodies (14, 16 -18). To investigate the requirements for interaction of Elongin C with Elongin B, a systematic series of N-terminal, C-terminal, and internal Elongin C deletion mutants were constructed (Fig. 1), expressed in E. coli, purified from inclusion bodies, and assayed for their abilities to form chromatographically isolable Elongin BC complexes. In these experiments, individual Elongin C mutants were refolded together with wild type Elongin B and subjected to TSK DEAE-NPR HPLC. Consistent with our previous results (14), the wild type Elongin BC complex elutes from TSK DEAE-NPR as a discrete species with chromatographic properties distinct from those of both wild type Elongin B, which flows through TSK DEAE-NPR at low ionic strength, and wild type Elongin C, which binds tighter to this resin than the Elongin BC complex ( Fig. 2A) and elutes over a broad range of ionic strength. Thus, coelution of Elongin B and C from TSK DEAE-NPR is diagnostic of an interaction between the two proteins.
As shown in Fig. 2B, deletion of as many as 28 amino acids from the C terminus of Elongin C does not prevent formation of isolable Elongin BC complexes. Likewise, deletion of as many as 18 amino acids from the N terminus of Elongin C does not prevent formation of isolable Elongin BC complexes. Deletion of 22 amino acids from the N terminus of Elongin C, however, abolished formation of isolable Elongin BC complexes, suggesting that Elongin C residues between 18 and 22 are critical for interaction of Elongin B and C. Consistent with this possibility, an Elongin C internal deletion mutant lacking residues 21-30 did not form an isolable Elongin BC complex (Fig. 2B); this mutant was the only Elongin C internal deletion mutant that failed to bind to Elongin B, suggesting that the Elongin C region between residues 18 and 30 either contains the Elongin B binding site or is crucial for proper folding of the protein.
Notably, although the Elongin C internal deletion mutant lacking residues 61-70 was capable of retaining wild type Elongin B on the TSK DEAE-NPR column, the resulting Elongin BC complex eluted from the column over a broad range of ionic strength. At the present time, we do not know why this mutant Elongin BC complex elutes aberrantly from TSK DEAE-NPR. One possible explanation is that deletion of residues 61-70 alters the conformation of Elongin C without preventing it from binding Elongin B.
To characterize further the Elongin C sequences most critical for binding to Elongin B, we analyzed a set of clustered alanine scanning mutants in which Elongin C residues between 19 and 30 were mutated three at a time to alanines. As shown in Fig.   2C, although Elongin C alanine scanning mutants C-(Ala19 -21) and C-(Ala22-24) were capable of assembling into isolable Elongin BC complexes, C-(Ala25-27) and C-(Ala28 -30) were not.
Characterization of Elongin C Regions Important for Activation of Elongin A-In a previous study, we observed that preassembled Elongin BC complexes are capable of activating Elongin A (14). To investigate the requirements for activation of Elongin A by Elongin C, the isolated wild type and mutant Elongin BC complexes shown in Fig. 2, A and B, were assayed for their abilities to stimulate the rate of accumulation of runoff transcripts synthesized by RNA polymerase II from the AdML promoter in a reconstituted transcription system containing purified Elongin A and the general initiation factors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. As shown in Fig. 3, the entire C-terminal half of Elongin C is critical for activation of Elongin A by preassembled Elongin BC complexes. Whereas Elongin BC complexes containing Elongin C deletion mutants lacking up to 18 amino acids from their N termini or sequences between amino acids 41 and 60 were capable of activating Elongin A, Elongin BC complexes containing Elongin C deletion mutants lacking as few as 14 amino acids from their C termini were inactive. Furthermore, Elongin BC complexes containing all Elongin C internal deletion mutants lacking sequences Cterminal to amino acid 61 were inactive, and an Elongin BC complex containing the internal deletion mutant C-(⌬31-40), which lacks sequences immediately C-terminal to the region important for Elongin B binding, was also inactive. Finally, Elongin BC complexes containing the alanine scanning mutant C-(Ala19 -21) were inactive, although BC complexes containing C-(Ala22-24) was active (Fig. 3C).
Although Elongin B facilitates assembly and enhances stability of the Elongin (SIII) complex, it is not essential for activation of Elongin A by wild type Elongin C (18). As de- scribed above, most of the Elongin C mutants containing mutations in the region between amino acids 19 and 30 were unable to form isolable Elongin BC complexes. To investigate the ability of these Elongin C mutants to activate Elongin A, they were assayed for their abilities to stimulate the rate of accumulation of runoff transcripts synthesized by RNA polymerase II from the AdML promoter in the presence of Elongin A and the general initiation factors, but in the absence of Elongin B. As shown in Fig. 4, although wild type Elongin C strongly activated Elongin A, none of the Elongin C mutants that failed to assemble into Elongin BC complexes was capable of activating Elongin A.
To investigate whether Elongin C deletion mutants that fail to activate Elongin A are defective in their abilities to assemble into Elongin ABC complexes, Elongin C deletion mutants were assayed for their abilities to form chromatographically isolable Elongin ABC complexes. In these experiments, individual Elongin C deletion mutants were refolded together with wild type Elongin A and B and subjected to TSK SP-NPR HPLC. As described previously, Elongin A and Elongin AC and ABC complexes bind tightly to TSK SP-NPR and can all be eluted with ϳ0.3 M KCl, whereas Elongin B and C flow through this resin at low ionic strength (17,18). As shown in Fig. 5, the N-terminal Elongin C deletion mutants C-(15-112) and C- (19 -112), which form isolable Elongin BC complexes that activate Elongin A, were capable of assembling into isolable Elongin ABC complexes, whereas the remaining N-terminal and Cterminal Elongin C deletion mutants, which either fail to form Elongin BC complexes or form inactive BC complexes, were unable to form isolable Elongin ABC complexes. In contrast, with the exception of Elongin C internal deletion mutant C-(⌬21-30), which lacks residues 21-30 and does not form an isolable Elongin BC complex, each of the Elongin C internal deletion mutants was capable of forming an isolable Elongin ABC complex (Fig. 6A). In these experiments, the yield of Elongin B and C in purified Elongin ABC complexes containing Elongin C internal deletion mutants was routinely less than their yield in purified Elongin ABC complexes containing wild type Elongin C, suggesting that Elongin ABC complexes containing the internal deletion mutants assemble less efficiently or are less stable than wild type Elongin ABC.
Runoff transcription assays were used to compare the activities of the isolated wild type and mutant Elongin ABC complexes shown in Fig. 6A. The concentration of the wild type Elongin ABC complex in the reaction shown in lane 2 of Fig. 6B was sufficient to saturate the assay, and the concentrations of mutant Elongin ABC complexes were adjusted so that all reactions shown in Fig. 6B contained equivalent levels of Elongin A. Highlighting the importance of sequences at the C terminus of Elongin C for activation of Elongin A, Elongin ABC complexes containing C-(⌬91-100) did not stimulate the rate of elongation by RNA polymerase II. Likewise, Elongin ABC complexes containing C-(⌬61-70), which forms Elongin BC complexes with aberrant chromatographic properties, were inactive. Interestingly, although Elongin BC complexes containing Elongin C internal deletion mutants C-(⌬71-80) and C-(⌬81-90) were unable to activate Elongin A, Elongin ABC complexes containing these same Elongin C mutants were capable of stimulating the rate of elongation by RNA polymerase II, suggesting that the Elongin C region between amino acids 71 and 90 is not critical for activation of Elongin A in pre-assembled Elongin ABC complexes. In these experiments, it is notewothy that the activity of mutant Elongin ABC complexes does not, in all cases, correlate with the amount of Elongin B and C present, since Elongin ABC complexes containing Elongin C internal deletion mutant C-(⌬71-80) are considerably more active than those containing C-(⌬81-90), even though Elongin ABC complexes containing C-(⌬81-90) contain more Elongin B and C. Thus, the results of these experiments demonstrate that assembly of Elongin C into the complete Elongin ABC complex is not sufficient for activation of Elongin A, and they indicate the importance of the C terminus of Elongin C for activation of Elongin A. Further evidence supporting the importance of the C terminus of Elongin C in activation of Elongin A came from analysis of a set of clustered alanine scanning mutants in which Elongin C residues between 89 and 112 were mutated three at a time to alanines. As predicted, all C-terminal alanine scanning mutants were capable of binding to Elongin B to form  panel (B)), wild type Elongin C (middle panel (C)), or a mixture of wild type Elongin B and C were refolded and subjected to DEAE-NPR HPLC as described under "Experimental Procedures." Aliquots of the indicated column fractions were analyzed by SDS-PAGE, and proteins were visualized by silver staining. B, N-terminal, C-terminal, and internal Elongin C deletion mutants were assayed for their abilities to form Elongin BC complexes as described under "Experimental Procedures." C, Elongin C alanine scanning mutants were assayed for their abilities to form Elongin BC complexes as described under "Experimental Procedures." C-(Ala19 -21) co-electrophoreses with wild type Elongin B during SDS-PAGE. L, load; FT, flow-through. chromatographically isolable Elongin BC complexes (data not shown). In addition, although Elongin BC complexes containing three of these mutants, C-(Ala89 -91), C-(Ala98 -99), and C-(Ala108 -109), were nearly as active as wild type BC complexes, the activity of BC complexes containing C-(Ala92-94), C-(Ala95-97), C-(Ala101-103), C-(Ala104 -105), and C-(Ala110 -112) was significantly impaired (Fig. 7). DISCUSSION In this report, we have investigated the structure and function of Elongin C, a 112-amino acid subunit of the Elongin (SIII) complex (16,18). Elongin (SIII) was initially purified from mammalian cells as a multimeric complex composed of A, B, and C subunits (10). Biochemical studies have shown that Elongin A is the transcriptionally active subunit of Elongin (SIII) and that Elongin B and C regulate its activity by different mechanisms (16 -18). By virtue of its ability to bind directly to Elongin A in the absence of Elongin B to form an AC complex with increased specific activity, Elongin C appears to function as a bona fide activator of Elongin A. Elongin B does not appear to interact with Elongin A in the absence of Elongin C. Elongin B appears to play a chaperone-like role in formation in the Elongin (SIII) complex by binding directly to Elongin C and facilitating its interaction with Elongin A.
As part of our effort to understand how Elongin C regulates the activity of the Elongin (SIII) complex, we have constructed and analyzed a systematic series of Elongin C mutants for their abilities to bind Elongin B and to bind and activate Elongin A under our assay conditions. Elongin C mutations were found to fall into several classes based on their effects on Elongin C activities (Fig. 8). First, the only Elongin C mutations that had dramatic effects on Elongin B binding fell within a short Elongin C region between amino acids 19 and 30, consistent with the possibility that sequences within this region are directly involved in interactions with Elongin B. Deletion mutations in this region, however, were also found to affect the ability of Elongin C to assemble into isolable Elongin ABC complexes and to activate Elongin A transcriptional activity. Thus, it is also possible that some mutations in this region disrupt the overall tertiary structure of Elongin C. Distinguishing between these possibilities must await more detailed structural studies of Elongin C and Elongin C-containing complexes.
Second, the only Elongin C mutations that had dramatic effects on formation of isolable Elongin ABC complexes without affecting formation of Elongin BC complexes were mutations in the extreme C terminus of Elongin C. This result, together with our finding that all Elongin C internal deletion mutants, except C-(⌬21-30), were capable of forming isolable Elongin ABC complexes, suggests that the C terminus of Elongin C plays a crucial role in assembly of the Elongin ABC complex, possibly through direct interactions with Elongin A.
Third, Elongin C mutations that affect formation of Elongin ABC complexes are only a subset of those mutations that affect activation of Elongin A, indicating that assembly of Elongin C into ABC complexes is not sufficient for activation of Elongin A. Interestingly, the size of the Elongin C region sensitive to mutations that affect activation of Elongin A was dependent on the assay used to measure activation. In one assay, which measured the ability of Elongin BC complexes to stimulate the rate of elongation by RNA polymerase II in the presence of Elongin A, Elongin C mutations that fell within the entire C-terminal half of the protein (residues 61-112) drastically reduced Elongin C activity. In contrast, in a second assay, which measured the ability of preassembled Elongin ABC complexes to stimulate the rate of elongation by RNA polymerase II, Elongin C mutations that fell between residues 71 and 90 had a significantly reduced effect on Elongin C activity, indicating that sequences within this Elongin C region are not essential for activation of Elongin A.
Finally, our previous analysis of the predicted open reading frame of the Elongin C cDNA revealed two notable features that suggested the existence of potentially important functional domains (16). First, a FASTA search of the Swiss-Prot data base revealed that Elongin C amino acids 13-75 resemble a portion of the RNA binding domain of E. coli termination protein . This region of is believed to be involved in allosteric coupling of the RNA binding and ATPase activities (29). In light of our evidence that large portions of the -like region can be deleted without affecting Elongin C activities, it is likely that the statistically significant sequence similarity between FIG. 5. Assay of formation of Elongin ABC complexes containing N-terminal and C-terminal Elongin C mutants. Mixtures containing wild type Elongin A and B and wild type or mutant Elongin C were refolded and subjected to SP-NPR HPLC as described under "Experimental Procedures." Aliquots of peak fractions from isolation of Elongin ABC complexes were analyzed by SDS-PAGE, and proteins were visualized by silver staining. A, Elongin A; B, Elongin B; C, wild type or mutant Elongin C.
FIG. 6. Assay of formation and activity of Elongin ABC complexes containing Elongin C internal deletion mutants. A, formation of Elongin ABC complexes was assayed as described in the legend to Fig. 5. B, runoff transcription assays were performed as described under "Experimental Procedures" according to the protocol diagrammed at the bottom of the two proteins does not reflect a similarity of function. Second, the extreme C terminus of Elongin C is predicted by both the Chou and Fasman (30) and Garnier et al. (31) algorithms to form a short, hydrophobic ␣-helix that has the potential to form a coiled-coil protein-protein interaction domain (32,33). A similar short C-terminal ␣-helix with potential to form a coiled-coil has been shown to play a central role in protein-protein interactions involved in microtubule bundling (34). Our finding that mutations in the C-terminal tail of Elongin C do not affect its ability to bind Elongin B, but have dramatic effects on its ability to enter into Elongin ABC complexes and activate Elongin A, leave open the possibility that the potential hydrophobic ␣-helix at the C terminus of Elongin C could mediate proteinprotein interactions with Elongin A.