Elongin from Saccharomyces cerevisiae.

Elongin is a transcription elongation factor that was first identified in mammalian systems and is composed of the three subunits, elongin A, B, and C. Sequence homologues of elongin A and elongin C, but not elongin B, were identified in the yeast genome. Neither yeast elongin A nor C sequence homologues was required for cell viability. The two gene products could be purified from yeast as a complex. A recombinant form of the complex, which could only be produced in bacteria if the gene products were co-expressed, was purified over several chromatographic steps. The complex did not stimulate transcription elongation by yeast RNA polymerase II. Using limited proteolysis, the N-terminal 144 residues of yeast elongin A were shown to be sufficient for interaction with yeast elongin C. The purified complex of yeast elongin C/elongin A(1-143) was analyzed using circular dichroism and nuclear magnetic spectroscopy. These studies revealed that yeast elongin A is unfolded but undergoes a dramatic modification of its structure in the presence of elongin C, and that elongin C forms a stable dimer in the absence of elongin A.

The elongation of mRNA by RNA polymerase II (RNAP II) 1 is regulated at many stages by accessory protein factors (1). These factors fall into three mechanistic classes. The first, typified by TFIIS, re-activates elongation complexes that have paused or arrested at blocks to elongation such as DNA-binding proteins (2)(3)(4)(5)(6). The second class, which includes pTEFb and DSIF, facilitates escape of the elongation complex from the promoter (7)(8)(9). The third class, which includes ELL and elongin, increases the general rate of elongation by suppressing pausing by RNAP II (10 -13).
Elongin was originally identified in rat liver nuclear extracts as a factor that stimulated synthesis of full-length runoff tran-scripts by increasing the rate of RNAP II elongation (10,13). Purified rat elongin is a heterotrimer composed of A, B, and C subunits of 773, 118, and 112 amino acids, respectively (11). Elongin A contains the transcription activity. Elongin C stimulates elongin A activity, and elongin B plays a chaperone-like function that stabilizes the elongin ABC complex (11). Elongins B and C form a complex that is a target for negative regulation by the von Hippel-Lindau tumor suppressor gene product (VHL) (14,15). This gene is mutated in families with VHL disease, a genetic disorder that predisposes individuals to a variety of cancers including hemangioblastomas and renal clear-cell carcinomas (16,17). VHL binds specifically to the elongin BC complex in vivo and in vitro via a motif that is conserved between VHL and elongin A (11, 14 -16). Interestingly, this site is mutated in a subset of naturally occurring VHL mutants, suggesting that the tumor suppressor function of VHL could involve interaction with a subcomplex of elongin.
As well as its involvement in transcription elongation, a role for elongin in the regulation of protein ubiquitination has been inferred from the similarity of elongin to a class of E3 ubiquitin ligases known as SCF complexes (for Skp1-Cullin-F-box). SCFs are heterotetramers that contain a member from each of four protein families, which are conserved from yeast to man. The complex comprises an F-box-containing protein, a Skp1 homologue, the ring finger protein Rbx1, and a cullin (Cdc53 in yeast). Although neither elongin nor elongin complexes have been shown directly to possess E3 ligase activity, elongin bears overall similarity to SCF complexes in many ways. First, mammalian elongins B and C form a complex not only with VHL but also with both cullin 2, a Cdc53 homologue, and Rbx1 (19 -23). Second, elongin C is a sequence homologue of Skp1 (22). Third, elongin B is a ubiquitin-like protein (24). Fourth, elongin A has an F-box (22). Fifth, VHL, and likely the VHL-containing protein complexes, are implicated in protein turnover in vivo; VHL affects the turnover of hypoxia-inducible factor-1 (25). Last, the complex comprising elongins B and C, VHL, cullin 2, and Rbx1 is associated with E3 ubiquitin ligase activity (26,27).
The homologue of elongin C in Saccharomyces cerevisiae, which is named Elc1, stimulates the activity of mammalian elongin A. This protein is termed Elc1 (28). We identified and characterized a yeast protein (Ela1 ϭ elongin A), that forms a complex with Elc1 and displays strong sequence similarity to mammalian elongin A. Ela1 contains two conserved sequence motifs: an elongin C binding site and an F-box. We identified the structural domain of Ela1 that interacts with Elc1. Our results contribute further insight into the genetics and structure of elongin, and identify a core complex amenable to structural analysis by nuclear magnetic resonance (NMR) spectroscopy.

Immunoprecipitation of Elongin Complexes from Yeast-Plasmid
YNL230CY encoding C-terminal V5 epitope-tagged yeast elongin A under control of the GAL1 promoter and with a URA3-selectable marker was purchased from Invitrogen (Carlsbad, CA).
The final PCR product was cloned into vector Topo 2.1 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and then excised with BamHI and HindIII and subcloned into vector YEplac112 placed under control of the GAL1 promoter (30) (TRP1 marker).
For co-immunoprecipitation studies, elongin A was coexpressed with elongin C in strain MDY433 (31). Two liters of culture coexpressing elongin A and elongin C were grown to 5 ϫ 10 7 cells/ml in SC, uraϪ, trpϪ media using 2% galactose as the sole carbon source. The cells were pelleted and washed with one-half volume of ice-cold water and pelleted again. The cell pellet was resuspended with 7 ml of lysis buffer (900 mM (NH 4 ) 2 SO 4 , 3 mM EDTA, 3 mM DTT, 30% glycerol, 150 mM HEPES, pH 7.9) and enough water to bring the final cell suspension volume to 20 ml. 200 l of zymolyase was added, and the extract incubated on ice for 1 h. The cells in the suspension were then lysed with one pass through a French pressure cell at 1,500 p.s.i. 200 l of a protease inhibitor mixture (Sigma P-9599, Sigma) was immediately added. The lysate was then clarified for 30 min at 30,000 rpm in a Beckman 45Ti rotor and the supernatant frozen at Ϫ80°C in aliquots. The cell lysate was thawed and clarified by centrifugation at 13,000 ϫ g at 4°C in a microcentrifuge for 15 min. CaCl 2 was added to the supernatant to a final concentration of 10 mM. 500 l of the supernatant was incubated with 2 g of the appropriate antibody and 30 l of protein A/G-Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h. The beads were then washed four times with 500 l of 20 mM Tris HCI, pH 7.9, 500 mM NaCl, 0.5% Triton, 10 mM CaCl 2 . The final wash was performed with an Immunocatcher spin column (Cytosignal, Irvine, CA). Complexes were then eluted from the beads with SDS sample buffer and analyzed by SDS-PAGE and immunoblotting with the appropriate antibodies. Anti-V5 antibody was purchased from Invitrogen (Carlsbad, CA). Anti-HPC4 antibody was was provided by C. Esmon (Howard Hughes Medical Institute and Oklahoma Medical Research Foundation).
Cloning of Ela1/Elc1 Complexes in a Bacterial Expression Vector-Ela1 and Elc1 and various truncations of Ela1 were expressed in bacteria. The coding sequences were amplified from yeast genomic DNA using Vent polymerase (New England Biolabs) by PCR. For expression of full-length Ela1 and Elc1, the restriction sites NdeI and BamHI were added to the 5Ј and 3Ј ends of the PCR products respectively. The NdeIand BamHI-digested PCR products were inserted between the corresponding sites of the T7 polymerase expression vector pET15b (Novagen, Milwaukee, WI), generating plasmids p15bELA1 and p15bELC1. The recombinant proteins were expressed as fusions to an N-terminal six-histidine tag and a thrombin protease site. To generate a plasmid for coexpression of Ela1 with Elc1, the coding sequence for Ela1 was first inserted between the NdeI and BamHI sites of the T7 polymerase expression vector pET11a, generating plasmid p11aELA1. p11aELA1 was then digested with BamHI and BglII to obtain an 1257-base pair product containing the T7 promoter and the Ela1 protein coding sequence. This fragment was then cloned into the BamHI site of p15bELC1, generating plasmid p15bA/C1. The various truncations of Ela1 were introduced into p15bA/C1 using the Quick-Change mutagenesis kit (Stratagene, La Jolla, CA), generating plasmids as described under "Results." Purification of Recombinant Elongin Complexes-Escherichia coli BL21 (DE3) cells containing the plasmids described above were grown at 30°C to an OD 600 of 0.3-0.4 in 10 liters of medium. For 15 N-labeled proteins, M9 minimal medium containing 15 NH 4 Cl was used; Luria broth was used for unlabeled proteins. Isopropyl-6-D-thiogalactopyranoside was then added to a final concentration of 1.0 mM. The cells were grown for an additional 3.0 h before harvesting and were then resuspended in Buffer A (30 mM HEPES-HCl, pH 7.5, 500 mM NaCl, 10 M ZnSO 4 , 10% glycerol) containing 5 mM imidazole and protease inhibitors (1ϫ PI) (1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mg/ml chymostatin, 1 mg/ml leupeptin, 1 mg/ml pepstatin). The cells were lysed using a French pressure cell, and the supernatant was clarified by centrifugation at 100,000 ϫ g for 40 min at 4°C. All subsequent steps were performed at 4°C. The supernatant solution was loaded onto a 5 ϫ 5-cm DE52 (Whatman, Maidstone, United Kingdom) column, previously equilibrated in Buffer A with 5 mM imidazole. The flow-through was loaded onto a 1 ϫ 6-cm His-bind column (Novagen), washed extensively with Buffer A containing 30 mM imidazole and eluted with Buffer A containing 500 mM imidazole. The eluate was dialyzed immediately against Buffer B (10 mM sodium phosphate, pH 7.5, 5 mM DTT, and 10 M ZnSO 4 ) containing 300 mM NaCl. For the purification of the complex containing full-length Ela1 and Elc1 (Ela1/ Elc1), the dialyzed eluate was applied to a Mono-S HR 5/5 column (Amersham Pharmacia Biotech, Baie D'Urfe, Quebec, Canada), washed with Buffer B containing 500 mM NaCl and eluted with Buffer B containing 1 M NaCl. The eluate was dialyzed immediately against Buffer B with 300 mM NaCl. Purified proteins were stored at Ϫ70°C.
When necessary, the hexahistidine tag was removed by the addition of thrombin in Buffer B according to the pET system technical manual (Novagen). The resulting Elc1 protein contained 3 additional residues at the N terminus (Gly-Ser-His). The digested samples were then concentrated to 2 mM protein and resolved on a Superose 12 HR 10/50 gel filtration column (Amersham Pharmacia Biotech) at 0.2 ml/min. 0.5-ml fractions were collected and analyzed by SDS-PAGE. Fractions containing purified complexes were pooled, concentrated by ultrafiltration (Centricon 10, Amicon), filtered through a 0.22-m Acrodisc (Millex-GP, Millipore, Bedford, MA) and stored at 4°C.
Analysis of Biochemical Activity of Ela1/Elc1-Yeast RNA polymerase II was purified from bakers' yeast as described previously (32), except that 10 M ZnCl 2 was included in all buffers. The polymerase preparations were stored at Ϫ70°C in 20 mM Tris-Cl, pH 7.9, 40 mM ammonium sulfate, 10% glycerol, 10 mM dithiothreitol, 10 mM EDTA, and 10 M ZnCl 2 . The purification of recombinant yeast elongin C/A complex was described under the section "Purification of Recombinant Elongin Complexes." The ability of yeast elongin C/A to stimulate RNA polymerase II was analyzed by an in vitro nonspecific transcript assay. Briefly, transcription by purified RNA polymerase II was carried out in the presence or absence of recombinant yeast elongin C/A complex. Activity was assayed in a 20-l mixture containing 60 mM Tris-OAc, pH 8.0, 5 mM MgOAc, 5% glycerol, 100 mM (NH 4 ) 2 SO 4 , 6 mM spermidine HCl, 0.8 mM each ATP, GTP, and UTP, 0.1 mM CTP, 1 M [␣-32 P]CTP (3000 Ci/mmol, NEN Life Science Products), 1 unit of RNase inhibitor (Roche Molecular Biochemicals, Laval, Quebec, Canada), 50 g/ml native salmon sperm DNA, and 150 ng of RNA polymerase II with increasing amounts of elongin A/C complex or buffer as indicated. Samples were processed after a 25-min incubation at 30°C by spotting on DE81 cellulose, washing away the unincorporated nucleotides and measuring the relative incorporation of [␣-32 P]CTP.
Domain Mapping by Partial Proteolysis-Four micrograms of purified full length Ela1/Elc1 complex were incubated with 4 ng of chymotrypsin (Roche Molecular Biochemicals) at 4°C for up to 8 h in Buffer B containing 300 mM NaCl. The proteolytic products were resolved by denaturing gel electrophoresis and visualized by staining with Coomassie Blue. For preparative purposes, 500 g of purified full-length Ela1/ Elc1 complex were incubated with 500 ng of chymotrypsin at 4°C for 4 h. The reaction was stopped by the addition of trifluoroacetic acid to a final concentration of 0.1%. Proteolytic products were purified by reverse-phase chromatography using a 30-ml linear gradient of 0 -60% acetonitrile in 0.1% trifluoroacetic acid. The absorbance at 220 nm was used to identify peak fractions, which were then precipitated using trichloroacetic acid and analyzed by SDS-PAGE. The molecular weights of the purified products were determined by electrospray ionization mass spectrometry (University of Waterloo).
Separation of Ela1 and Elc1-To isolate Ela1 1-143 and Elc1 individually, the Ela1 1-143 /Elc1 complex was denatured and the individual subunits purified as follows. After elution from the His-bind column, the purified Ela1 1-143 /Elc1 complex was dialyzed against Buffer A containing 5 mM imidazole. Subsequent steps were performed at 25°C. Urea was added to a final concentration of 6.0 M to dissociate the Ela1 1-143 and Elc1 subunits. The denatured complex was then reapplied to a 1 ϫ 6-cm His-bind column (Novagen), previously equilibrated with Buffer A containing 5 mM imidazole and 6.0 M urea. Elc1, which contained a hexahistidine tag, bound to the nickel resin; free, purified Ela1 1-143 flowed through the column. To purify Elc1, the column was washed extensively with Buffer A containing 30 mM imidazole and 6 M urea. Elc1 was eluted with the same solution augmented with 500 mM imidazole.
Renaturation of Ela1 1-143 -All steps were carried out at 4°C. The isolated, denatured Ela1 1-143 was diluted to 0.1 mM and renatured by dialysis against decreasing concentrations of urea in Buffer B containing 500 mM NaCl. The urea concentration was decreased by 0.4 M urea decrements every 6 h. The final dialysate, containing no urea, was clarified by centrifugation at 20,000 ϫ g for 15 min to remove any protein that had precipitated. The supernatant was concentrated to 0.2 mM, filtered through a 0.22-m Acrodisc (Millex-GP, Millipore) and stored at 4°C.
Renaturation of Elc1-Denatured Elc1 was renatured as for Ela1 1-143. After the last dialysis step, the hexahistidine tag was removed by the addition of thrombin (pET System Technical Manual, Novagen). Following digestion for 4 h at 4°C, the protein was loaded onto a 1 ϫ 6-cm His-bind column (Novagen), pre-equilibrated in Buffer B containing 5 mM imidazole and 500 mM NaCl, and washed with the same buffer. Elc1 lacking the hexahistidine tag flowed through the nickel resin while undigested protein remained bound to the resin. The cleaved protein was concentrated to 1.0 -2.0 mM by ultrafiltration (Centricon 10, Amicon), filtered through a 0.22-m Acrodisc (Millex-GP, Millipore) and stored at 4°C.
Renaturation of Ela1 1-143 /Elc1 Complex-To renature the complex of Ela1 1-143 and Elc1, a 2-fold molar excess of Elc1 was added to Ela1 1-143 and the complex renatured as described for free Elc1. Following renaturation, the complex was resolved on a 1 ϫ 10-cm Superose 12 gel filtration column (Amersham Pharmacia Biotech) at 0.2 ml/min. CD Spectroscopy-Circular dichroism experiments were performed on an Aviv-62A DS spectropolarizer using 0.1-cm path length quartz cuvettes. 25 M samples of Ela1 1-143 , Elc1, Ela1/Elc1, or Ela1 1-143 /Elc1 (both native and renatured complexes were used) were prepared in Buffer B containing 500 mM NaCl and lacking DTT. Exact protein concentrations were then determined by amino acid analysis. A wavelength scan measuring observed ellipticities was recorded for each sample at 25°C, employing a 15-s averaging time.
The melting temperatures were determined for each protein by monitoring the observed residue ellipticity at 222 nm from 15°C to 105°C. Data points were collected every 2°C with equilibration and averaging times of 60 and 15 s, respectively.
NMR Spectroscopy-All NMR experiments were acquired on Varian Unity or Unityϩ spectrometers operating at 500-and 600-MHz proton frequencies and equipped with pulse field gradients. 10% D 2 O was added to the samples for a lock signal. All experiments were performed at 25°C. HSQC spectra (33) were acquired with sweep widths of 8000 Hz centered on the hydrogen, oxygen, deuterium peak for the proton dimension, and 2000 Hz centered at 118 ppm relative to external 15 N urea in Me 2 SO. The 1 H chemical shifts were referenced to the hydrogen, oxygen, deuterium peak at 4.772 ppm at 25°C. Data were processed with the NMRPipe/NMRDrawt software package (34) on a Sun SPARC5 workstation. Typically, Lorentzian-to-Gaussian or adjustable sine functions were applied before the transformation. Data were zerofilled in 2 ( 1 H) and 1 ( 15 N) dimensions to give a final matrix of 1024 ϫ 256 real points.

Identification of Yeast Elongin
A and C Homologues-In a search for homologues of mammalian elongin in the yeast S. cerevisiae, we identified two genes with significant sequence similarity to human elongins A and C (35). The yeast elongin C homologue (ELC1) had been identified previously (28). Elc1 is probably the functional homologue of mammalian elongin C because it can activate the transcription activity of mammalian elongin A and can interact with the VHL tumor suppressor protein (28). The open reading frame of the elongin A homologue (YNL230C), which we have termed ELA1, encodes a 379-amino acid protein with a predicted molecular mass of 44 kDa (Fig. 1A). The most significant similarity (31% identity) is exhibited in the region of the mammalian protein most critical for transcriptional activity (36). This portion of the protein also contains an elongin C binding motif (15) and an F-box (37) (Fig.  1B). The ELC1 open reading frame encodes a 99-amino acid protein displaying 41% identity to human elongin C (28).
In an initial characterization of Ela1 and Elc1, yeast strains bearing deletions of ELA1 and ELC1 were generated. Strains bearing deletions of ELA1, ELC1, or both genes had no detectable phenotype. We were also unable to detect a synthetic effect of elongin A or C disruptions in strains bearing mutations in other transcription elongation factors, such as TFIIS, Rpb9, and Spt4 (data not shown).
Ela1 and Elc1 Form a Complex-A structural and biochemical examination of the yeast elongins was also initiated in an attempt to gain insight into their functions. We reasoned that if Ela1 is the bona fide homologue of elongin A, then Ela1 and Elc1 should form a stable heterodimer. To test whether the yeast Elc1 and Ela1 interacted in vivo, we performed co-immunoprecipitation studies on tagged versions of the proteins over- The putative elongin C binding motif is boxed, and the region spanning the F-box is underlined. Only the minimal region of rat elongin A required to stimulate RNAP II is shown (27). expressed in yeast. Yeast cells were transformed with plasmids encoding HPC4-Elc1 and V5-Ela1 under control of a galactoseinducible promoter. Complexes were immunoprecipitated from induced cell lysates with anti-V5 or anti-HPC4 and detected by immunoblotting (see "Materials and Methods"). Fig. 2 illustrates that Ela1 and Elc1 form a stable complex in yeast.
To facilitate the functional studies of the complex, a recombinant Elc1/Ela1 dimer was generated. Neither Elc1 nor Ela1 expressed well in bacterial cells (Ͻ0.2 mg/liter) and both proteins were completely insoluble (data not shown). Co-expression of the two subunits from the same plasmid led to the production of soluble products; in the construct, only Elc1 contained a hexahistidine tag. Both tagged Elc1 and Ela1 expressed to a relatively high level (Ͼ2 mg/liter). These data suggest that there is a co-dependence of the two proteins for proper folding.
To explore if the bacterially expressed proteins formed a stable complex, histidine-tagged Elc1 was purified using a combination of metal-chelate, cation exchange, and gel filtration chromatography and the co-purification of Ela1 monitored. A protein with the same relative mass as Ela1 co-purified with Elc1 over all chromatographic steps. This protein was shown to be Ela1 using mass spectrometry. The co-migration of the Elc1 and Ela1 through all these steps of purification indicated that the two proteins formed a stable complex (termed Ela1/Elc1) and that the ELA1 gene corresponds to a yeast homologue of elongin A (Fig. 3).
Yeast Elongin A/Elongin C Has Undetectable Transcription Activity-Rat elongin is able to stimulate the rate of transcription of rat RNA polymerase II on a double-stranded DNA template. To test if purified yeast Ela1/Elc1 possessed this biochemical activity, we performed nonspecific assays of RNA chain elongation with purified yeast RNA polymerase II. Briefly, RNA polymerase II transcription was initiated on a sheared salmon sperm double-stranded DNA template and the rate of incorporation of ribonucleotides into RNA chains was measured in the presence or absence of Ela1/Elc1. We found that Ela1/Elc1 had no effect on the rate of nucleotide incorporation. A representative experiment is shown in Fig. 4. We monitored activity under a wide range of salts, temperatures, and buffer conditions. Ela1/Elc1 was also unable to stimulate transcription from a mutant RNA polymerase II with dramatically reduced elongation activity (38). This eliminated the possibility that Ela1/Elc1 did not stimulate transcription by yeast RNA polymerase II simply because that enzyme is known to have already a very high intrinsic rate of elongation. 2 The N Terminus of Elongin A Interacts with Elongin C-As the first step in initiating structural studies of yeast elongin, we set out to identify the minimal domains of Ela1 and Elc1 required for formation of a complex. Limited proteolysis was used to dissect the full-length complex into structural domains. Treatment of the Ela1/Elc1 complex with chymotrypsin generated two smaller fragments (Fig. 5A), which persisted throughout the course of the digest, suggesting that they correspond to stable domains. Purification of these fragments by reverse phase high performance liquid chromatography and analysis by mass spectrometry allowed us to identify the fragments as residues 1-174 of Ela1 (Ela1 1-174 ) and the intact Elc1.
Limited proteolysis defined the approximate borders of the smallest stable Ela1/Elc1 heterodimer. Deletion mutagenesis was performed to more accurately define the minimal Ela1/ Elc1 dimer. Elongin A deletion mutants were co-expressed with Elc1, and complex formation was monitored by co-purification. A 143-amino acid region in Ela1 (Ela1 1-143 ) was sufficient for interaction with Elc1 (summarized in Figs. 5B and 6). This fragment of Ela1 contains the short sequence (approximately residues 4 -17) conserved in the VHL protein and previously implicated in elongin C binding (15). Smaller C-terminal deletions of Ela1 1-143 were expressed at much lower levels and did not co-purify with Elc1. These data define a minimal complex as Ela1 1-143 /Elc1.
Stability and Secondary Structure of the Minimal Elongin Complex-To gain more insight into the Ela1 1-143 /Elc1 structure and assembly, we analyzed the far-UV circular dichroism (CD) spectra of native Ela1 1-143 /Elc1. The complex displayed a 2 A. M. Edwards, unpublished data.

FIG. 2. Co-immunoprecipitation of Ela1 and Elc1 from yeast.
Lysates from yeast cells expressing HPC4-Elc1 (HPC4-EloC) and V5-Ela1 (EloA-V5) were immunoprecipitated with anti-HPC4, anti-V5, or anti-HSV (irrelevant antibody). Immunoprecipitated proteins were detected by immunoblotting as indicated. IP denotes immunoprecipitate; WB denotes Western blot. strong minimum at 222 nm, which indicates a significant amount of helical secondary structure in this complex, and had a melting temperature of 55°C.
CD was then exploited to study the structures of the purified subunits and to analyze the co-dependence for proper folding. Since the individual subunits could not be expressed in soluble form, we developed a procedure in which they were purified under denaturing conditions and then renatured. Using a urea denaturation/renaturation protocol, 100% of the input Elc1 and 40% of the input Ela1 1-143 was recovered in soluble form. Solubility was defined as the percentage of the input protein soluble after 100,000 ϫ g centrifugation for 30 min. By renaturing a mixture of Elc1 and Ela1 1-143 , the Ela1 1-143 /Elc1 com- FIG. 5. Identification of the approximate borders of the smallest stable Ela1/Elc1 heterodimer by limited proteolysis. A, purified Ela1/Elc1 complex was digested for the indicated times with chymotrypsin. The proteolytic products were resolved by denaturing gel electrophoresis and stained with Coomassie Blue. Products from the 120-min time point were purified by reverse phase chromatography and the boundaries identified by mass spectrometry (see "Materials and Methods"). B, deletion mutagenesis of the minimal Ela1/Elc1 heterodimer. Several Ela1/Elc1 mutant constructs were co-expressed in E. coli, as shown. Constructs from which Ela1 and Elc1 co-purified are indicated with a checkmark. Ela1 and Elc1 fragments that did not assemble are indicated by an X. The regions of Ela1 corresponding to the elongin C binding motif and the F-box are indicated with an asterisk and an F, respectively. plex was recovered in a 75% yield.
The CD spectra of denatured and renatured Ela1 1-143 /Elc1 were compared, and these spectra compared with those of purified, renatured Ela1 1-143 and Elc1 (Fig. 7A). There were no significant spectral differences between renatured and native Ela1 1-143 /Elc1 (data not shown), and both complexes had identical melting temperatures. By comparison, the CD spectra of both Ela1 1-143 and Elc1 had much weaker signals at 222 nm compared with the complexes, reflecting significantly less helical content in the individual proteins. Temperature denaturation profiles of the individual subunits showed that Elc1 was relatively thermostable, with a thermal denaturation midpoint of 75°C. This suggests that Elc1 has significant regions of three-dimensional structure. By contrast, Ela1 1-143 was unstable, possessing a melting temperature of 37°C (Fig. 7B). Since Ela1 1-143 was significantly more thermolabile in isolation as compared with Elc1 or the Ela1 1-143 /Elc1 complex, the folding of Ela1 is likely modulated significantly by the presence of Elc1.
To test if the Ela1 1-143 /Elc1 complex could be reconstituted from the purified components, we combined equimolar amounts of renatured Ela1 1-143 and Elc1 and compared the CD spectra of the combined proteins with that of the Ela1 1-143 /Elc1 complex. The CD signals for the combined proteins were considerably weaker than for the Ela1 1-143 /Elc1 complex. Formation of the Ela1 1-143 /Elc1 complex, and in particular the folding of Ela1, requires cooperative folding of Ela1 and Elc1. These results strengthen the assertion that Ela1 is a natural partner of Elc1.
NMR Analysis of Elongin A-The structural transitions that occur in Ela1 upon binding to elongin C were studied using NMR. Ela1 1-143 precipitated at relatively low concentrations (Ͻ0.5 mM), and thus we were unable to study that subunit by NMR. We therefore explored the feasibility of studying Ela1 1-143 as a part of a Ela1 1-143 /Elc1 complex, reasoning that Ela1 might be stabilized by binding to its partner Elc1. 15 N HSQC spectra were obtained for native 15 N-labeled Ela1 1-143 / Elc1 (Fig. 8A). Judging from the dispersion of the signals in both nitrogen and proton dimensions and the presence of distinctly downfield-shifted amide resonances, we concluded that Ela1 1-143 /Elc1 contained a significant amount of tertiary structure. The 15 N-1 H HSQC spectra contain a high degree of peak overlap in the center, which is not unusual for large protein systems that are highly helical, but that also severely complicates the structure determination process. Therefore, in an effort to reduce the complexity, we explored a partial labeling strategy in which only Ela1 1-143 was labeled. Since Ela1 expresses well only when co-expressed with Elc1, it was necessary to prepare both labeled and unlabeled Ela1/Elc1 samples, then separate the two components and renature the complex. A 15 N-1 H HSQC spectrum of the complex of 15 N-labeled Ela1 1-143 and unlabeled Elc1 is shown in Fig. 8B. Comparison to Fig. 8A reveals the high level of spectral simplification expected from this selective labeling strategy. Moreover, comparison of the two spectra also permitted the identification of those resonances of Ela1 1-143 /Elc1 that arise from the Ela1 1-143 subunit. The fact that chemical shifts are identical within experimental error for the distinct resonances in Fig. 8B that can be identified in Fig. 6A indicates, again, that denaturation and renaturation of Ela1 1-143 /Elc1 does not alter the three-dimensional structure. These results point to the feasibility of determining the structure of elongin A using a partially labeled sample, and further attest to the relevance of the Ela1/Elc1 interaction. DISCUSSION The yeast elongin C homologue binds both the von Hippel-Lindau tumor suppressor protein and mammalian elongin A and stimulates mammalian elongin A in promoter-independent in vitro transcription assays (28). In this report, we have shown that yeast elongin C can also interact with a non-essential yeast protein that displays strong sequence similarity to mammalian elongin A. We provide several lines of evidence that Ela1 is a yeast homologue of elongin A. First, the sequence includes both an elongin C consensus binding site motif as well as an F-box, each a hallmark of the mammalian elongin A proteins. Second, the proteins form a complex when co-expressed in yeast. Third, when co-expressed in E. coli, Ela1 and Elc1 purified as a complex over several chromatographic steps. In fact, soluble expression of Ela1 or Elc1 in E. coli was dependent upon co-expression of both proteins. Fourth, a subcomplex of Ela1/Elc1, termed Ela1 1-143 /Elc1, was stable to protease digestion and contained both the elongin C binding site and the F-box of Ela1. Fifth, the elongin Ela1 1-143 /Elc1 subcomplex could be denatured and renatured to yield a complex that was structurally indistinguishable from the non-denatured form. Sixth, circular dichroism analysis showed that there was a dramatic increase in the relative amount of secondary structure of upon complex formation.
Although these yeast proteins are structural homologues of the mammalian elongins, we were not able to uncover a connection with transcription elongation; the complex could not stimulate transcription elongation by RNA polymerase II. We were also unable to discover a genetic link between elongin and the other known transcription elongation factors in yeast. If elongin is a transcription factor in yeast, it must have a mechanism that is completely distinct from those that have been studied previously and thus might not display one of the previously known phenotypes. Alternatively, given that the in vitro rate of transcription of yeast RNA polymerase II is very close to the in vivo rate, the role of elongin as a transcription elongation factor in yeast might be insignificant under laboratory conditions. Another possibility is that yeast elongin might play a role completely unrelated to transcription elongation. This last possibility is supported by the observation that a region of mammalian elongin A previously shown to be required for stimulating transcription in vitro (36) is not present in the yeast homologue (see Fig. 1).
If the yeast elongins are not involved in transcription elongation what role might they play? At least one complex of elongins B and C that also contains cullin 2, VHL, and Rbx1 has been shown to be associated with ubiquitin ligase activity in mammals (26,27). Given that homologues of cullin 2 and Rbx1 are present in yeast (18,39), yeast elongins may also be involved in ubiquitination.
Ela1 may also play a role in the regulation of meiotic recombination. Ela1 was isolated recently in a screen designed to identify functions that direct meiotic exchange events away from sister chromatids to homologous chromosomes (40). Meiotic exchange occurs preferentially between homologous chromatids rather than between sister chromatids, as occurs in mitosis. There is likely a pathway that directs the preference of recombination partners, and Ela1 may play a regulatory role in this process, perhaps by targeting a factor for degradation.