Characterization of the mRNA Capping Apparatus of Candida albicans*

Beate SchwerDagger , Kevin Lehman§, Nayanendu Saha§, and Stewart Shuman§

From the Dagger  Department of Microbiology and Immunology, Weill Medical College of Cornell University and § Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

Received for publication, July 10, 2000, and in revised form, September 18, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mRNA capping apparatus of the pathogenic fungus Candida albicans consists of three components: a 520- amino acid RNA triphosphatase (CaCet1p), a 449-amino acid RNA guanylyltransferase (Cgt1p), and a 474-amino acid RNA (guanine-N7-)-methyltransferase (Ccm1p). The fungal guanylyltransferase and methyltransferase are structurally similar to their mammalian counterparts, whereas the fungal triphosphatase is mechanistically and structurally unrelated to the triphosphatase of mammals. Hence, the triphosphatase is an attractive antifungal target. Here we identify a biologically active C-terminal domain of CaCet1p from residues 202 to 520. We find that CaCet1p function in vivo requires the segment from residues 202 to 256 immediately flanking the catalytic domain from 257 to 520. Genetic suppression data implicate the essential flanking segment in the binding of CaCet1p to the fungal guanylyltransferase. Deletion analysis of the Candida guanylyltransferase demarcates an N-terminal domain, Cgt1(1-387)p, that suffices for catalytic activity in vitro and for cell growth. An even smaller domain, Cgt1(1-367)p, suffices for binding to the guanylyltransferase docking site on yeast RNA triphosphatase. Deletion analysis of the cap methyltransferase identifies a C-terminal domain, Ccm1(137-474)p, as being sufficient for cap methyltransferase function in vivo and in vitro. Ccm1(137-474)p binds in vitro to synthetic peptides comprising the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II. Binding is enhanced when the C-terminal domain is phosphorylated on both Ser-2 and Ser-5 of the YSPTSPS heptad repeat. We show that the entire three-component Saccharomyces cerevisiae capping apparatus can be replaced by C. albicans enzymes. Isogenic yeast cells expressing "all-Candida" versus "all-mammalian" capping components can be used to screen for cytotoxic agents that specifically target the fungal capping enzymes.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The m7GpppN cap structure of eukaryotic mRNA is formed by three enzymatic reactions as follows. (i) The 5' triphosphate end of the nascent pre-mRNA is hydrolyzed to a diphosphate by RNA 5' triphosphatase, (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase, and (iii) the GpppN cap is methylated by AdoMet:RNA (guanine-N7) methyltransferase (1). RNA capping is essential for cell growth.

The mRNA capping apparatus differs in significant respects in fungi, metazoans, protozoa, and viruses (1). Hence, the cap-forming enzymes are potential targets for antifungal, antiviral, and antiprotozoal drugs that would interfere with capping of pathogen mRNAs but spare the mammalian host capping enzymes. Mammals encode a two-component capping system consisting of a bifunctional triphosphatase-guanylyltransferase polypeptide (named Mce1p in the mouse and Hce1p in humans) and a separate methyltransferase polypeptide (Hcm1p in humans) (2-8). The budding yeast Saccharomyces cerevisiae encodes a three-component system consisting of separate triphosphatase (Cet1p), guanylyltransferase (Ceg1p), and methyltransferase (Abd1p) gene products (9-12). Cet1p and Ceg1p interact in trans to form a bifunctional heteromeric enzyme (13). Although the physical association of the triphosphatase and guanylyltransferase activities is common to budding yeast (in trans) and mammals (in cis), the structures and catalytic mechanisms of the yeast and mammalian RNA triphosphatases are completely different (14-17).

Our mutational analyses of S. cerevisiae Cet1p, Ceg1p, and Abd1p have resulted in the delineation of minimal catalytic domains for each protein and the identification of catalytically important amino acid side chains that comprise the triphosphatase, guanylyltransferase, and methyltransferase active sites (16, 18-24). Genes and/or cDNAs encoding homologues of Cet1p, Ceg1p, and Abd1p have been isolated from other fungal species, including Schizosaccharomyces pombe and Candida albicans (8, 20, 25-27), but the enzymes have not been well characterized biochemically, and functional domains have not been defined.

In considering ways to identify antifungals that target cap formation, one wishes to focus on the capping apparatus from a clinically significant human pathogen such as C. albicans. The triphosphatase (CaCet1p), guanylyltransferase (Cgt1p), and methyltransferase (Ccm1p) components of the Candida capping apparatus have now been identified (8, 25-27). Here, we define a minimal functional domain of the RNA triphosphatase CaCet1p capable of complementing a S. cerevisiae cet1Delta mutant. Analysis of the guanylyltransferase Cgt1p includes the demarcation of functional domain boundaries and studies of the binding of recombinant Cgt1p to its docking site on yeast triphosphatase. We also present studies of the cap methyltransferase Ccm1p including delineation of a minimal functional domain and biochemical characterization of recombinant Ccm1p and truncated version thereof. We show that Ccm1p binds to the phosphorylated C-terminal domain (CTD)1 of RNA polymerase II and that binding is enhanced when the CTD is phosphorylated on both Ser-2 and Ser-5. Finally, we construct yeast strains in which the entire S. cerevisiae capping apparatus is replaced by C. albicans enzymes. The availability of isogenic yeast cells expressing all-Candida versus all-mammalian capping components should facilitate screening in vivo for antifungals that block cap formation.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Expression Vectors for Candida RNA Triphosphatase-- Yeast CEN TRP1 plasmid vectors containing CaCET1, CaCET1(203-520), or CaCET1(217-520) under the control of the yeast TPI1 promoter were described previously (13). New N-terminal deletion mutants CaCET1(223-520), CaCET1(229-520), CaCET1(257-520), and CaCET1(267-520) were constructed by PCR amplification with mutagenic sense-strand primers that introduced an NdeI restriction site and a methionine codon in lieu of the codons for Thr-222, Asn-228, or Lys-266 or an NdeI restriction site at the Met-257 codon. The PCR products were digested with NdeI and BamHI, then inserted into pYN132 (CEN TRP1). The inserts of each deletion plasmid were sequenced to verify that no unwanted coding changes were introduced during amplification and cloning.

Yeast CGT1 Expression Plasmids-- The CGT1 open reading frame encoding C. albicans mRNA guanylyltransferase was amplified from a genomic library (28) by PCR using oligonucleotide primers designed to introduce an NdeI restriction site at the translation start codon and a BamHI site 3' of the stop codon. The PCR product was digested with NdeI and BamHI and inserted into yeast expression vector pYN132. In this vector, expression of the C. albicans guanylyltransferase is under the control of the yeast TPI1 promoter. C-terminal truncation mutants of CGT1 were constructed by PCR amplification with mutagenic antisense-strand primers that introduced stop codons in lieu of the codons for Gln-427, Arg-409, Arg-388, or Lys-368 along with a BamHI site 3' of the new stop codon. The PCR products were digested with NdeI and BamHI, then inserted into pYN132. The inserts of each plasmid were sequenced to verify that no unwanted coding changes were introduced during amplification and cloning.

Expression and Purification of Recombinant Cgt1p-- NdeI-BamHI restriction fragments containing the wild type CGT1 gene or C-terminal truncation mutants were excised from the respective yeast expression vectors and inserted into the bacterial expression plasmid pET16b. The resulting plasmids, pET-Cgt1, pET-Cgt1-CDelta 23, pET-Cgt1-CDelta 41, pET-Cgt1-CDelta 62, and pET-Cgt1-CDelta 82, were introduced into Escherichia coli BL21(DE3). Cultures (500-ml) derived from single transformed colonies were grown at 37 °C in Luria-Bertani medium containing 0.1 mg/ml ampicillin until the A600 reached 0.5. The cultures were placed on ice for 20 min and then adjusted to 0.4 mM isopropyl-1-thio-beta -D-galactopyranoside and 2% (v/v) ethanol. Incubation was continued for 20 h at 17 °C with constant shaking. Cells were harvested by centrifugation, and the pellets were stored at -80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria were resuspended in 25 ml of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% sucrose). Cell lysis was achieved by the addition of 50 µg/ml lysozyme and 0.1% Triton X-100. The lysates were sonicated to reduce viscosity, and insoluble material was removed by centrifugation. The soluble lysates were applied to 1-ml columns of nickel nitrilotriacetic acid-agarose that had been equilibrated with lysis buffer containing 0.1% Triton X-100. The columns were washed with 5 ml of lysis buffer containing 0.1% Triton X-100 and then eluted stepwise with 2-ml aliquots of a buffer solution (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM DTT, 10% glycerol, 0.05% Triton X-100) containing 50, 100, 200, 500, and 1000 mM imidazole. SDS-PAGE analysis showed that the recombinant Cgt1p proteins were recovered in the 100 and 200 mM imidazole eluate fractions. The peak fractions containing the recombinant proteins were pooled, adjusted to 5 mM EDTA, and dialyzed against buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 2 mM DTT, 5 mM EDTA, 10% glycerol, 0.05% Triton X-100. The enzyme preparations were stored at -80 °C.

Yeast CCM1 Expression Plasmids-- The CCM1 open reading frame encoding C. albicans cap methyltransferase was amplified from genomic clone pCCM1-6.6 (8) by PCR using oligonucleotide primers designed to introduce an NdeI restriction site at the translation start codon and a XhoI site 3' of the stop codon. The PCR product was digested with NdeI and XhoI and inserted into yeast expression vector pYN132 (CEN TRP1), placing expression of the C. albicans cap methyltransferase gene under the control of the yeast TPI1 promoter. N-terminal deletion mutants of CCM1 were constructed by PCR amplification with mutagenic sense-strand primers that introduced an NdeI restriction site and a methionine codon in lieu of the codon for Ser-136 or Val-150 or an NdeI restriction site at the Met-175 codon. The PCR products were digested with NdeI and XhoI, then inserted into pYN132. The inserts of each plasmid were sequenced to verify that no unwanted coding changes were introduced during amplification and cloning. KpnI-XhoI fragments containing the CCM1 gene and the N-terminal deletion mutants were then transferred from their respective CEN plasmids to the yeast high copy vector pYX232 (2µ TRP1), with methyltransferase expression under the control of the TPI1 promoter.

Expression and Purification of Recombinant Ccm1p-- NdeI-XhoI restriction fragments containing the wild type CCM1 gene and N-terminal deletion mutants were excised from the respective yeast CEN expression vectors and inserted into the bacterial expression plasmid pET16b. The resulting plasmids, pET-Ccm1, pET-Ccm1Delta 136, pET-Ccm1Delta 150, and pET-Ccm1Delta 174, were introduced into E. coli BL21(DE3). Cultures (1 liter) derived from single transformed colonies were grown at 37 °C in Luria-Bertani medium containing 0.1 mg/ml ampicillin until the A600 reached 0.5. The culture was adjusted to 0.2 mM isopropyl-1-thio-beta -D-galactopyranoside and 2% ethanol, and incubation was continued for 16 h at 18 °C. Cells were harvested by centrifugation, and the pellet was stored at -80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria were resuspended in 50 ml of buffer L (50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 10% sucrose). Cell lysis was achieved by the addition of lysozyme and Triton X-100 to final concentrations of 50 µg/ml and 0.1%, respectively. The lysate was sonicated to reduce viscosity, and insoluble material was removed by centrifugation. The soluble extract was mixed for 1 h with 2 ml of nickel nitrilotriacetic acid-agarose resin that had been equilibrated with buffer L. The suspension was poured into a column and washed with buffer L followed by IMAC buffer (20 mM Tris HCl (pH 7.9), 0.5 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol) containing 30 mM imidazole. The column was then eluted stepwise with IMAC buffer containing 50, 200, and 500 mM imidazole. The polypeptide compositions of the column fractions were monitored by SDS-PAGE. The recombinant Ccm1p proteins were recovered in the 200 mM imidazole eluate. Aliquots of the nickel-agarose fraction (60 µg of protein) were applied to 4.8-ml 15-30% glycerol gradients containing 0.5 M NaCl in 50 mM Tris HCl (pH 8.0), 1 mM EDTA, 2 mM DTT, 0.1% Triton X-100. The gradients were centrifuged for 12 h at 50,000 rpm in a Beckman SW50 rotor. Fractions (0.2 ml) were collected from the bottoms of the tubes. The fractions were stored at -80 °C.

Yeast Strains-- YBS50 (MATa leu2 ade2 trp1 his3 ura3 can1 ceg1::hisG cet1::LEU2 p360-CET1/CEG1) is deleted at the chromosomal loci encoding RNA triphosphatase and guanylyltransferase. YBS40 (MATa leu2 ade2 trp1 his3 ura3 can1 abd1::hisG p360-ABD1) and YBS10 (MATalpha ura3 trp1 lys2 his3 leu2 abd1::LEU2 p360-ABD1) are deleted at the chromosomal locus encoding cap methyltransferase. YBS52 (MATa leu2 ade2 trp1 his3 ura3 can1 ceg1::hisG cet1::LEU2 abd1::KAN p360-CET1/CEG1/ABD1) is deleted at the chromosomal loci encoding all three components of the yeast capping apparatus.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Genetic Interaction between CaCet1p and Fungal Guanylyltransferases-- We reported previously that either full-length CaCET1 or N-terminal deletion alleles CaCET1(179-520), CaCET1(196-520), or CaCET1(203-520) complemented growth of a cet1Delta mutant of S. cerevisiae, whereas the more extensively truncated CaCET1(217-520) gene did not complement (13). CaCET1(203-520) cells displayed a temperature-sensitive growth phenotype that was suppressed by overexpression of CEG1. These experiments delineated a short peptide segment between residues 203 and 216 that is required for CaCet1p function in vivo and also implied a functional interaction between the Candida triphosphatase and the endogenous Saccharomyces guanylyltransferase.

Here we tested complementation of the cet1Delta mutation by a new series of truncated CaCET1 alleles alone versus complementation by truncated CaCET1 alleles plus the wild type Candida guanylyltransferase gene CGT1 present on the same CEN plasmid (Fig. 1). Note that biochemical studies of the purified recombinant proteins encoded by CaCET1 alleles Delta 202, Delta 216, Delta 222, Delta 228, and Delta 256) showed that the N-terminal truncated versions of CaCet1p were fully active in gamma -phosphate hydrolysis in vitro (29).



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Fig. 1.   In vivo deletion analysis of C. albicans RNA triphosphatase. The amino acid sequence of S. cerevisiae (Sce) Cet1p from residues 241-291 is aligned with the homologous segment of C. albicans (Cal) CaCet1p. The margins of the serial N-terminal deletion alleles of CaCET1 are denoted below the aligned sequences. Yeast strain YBS20 (cet1Delta ) was transformed with CEN TRP1 plasmids containing (i) CaCET1 or the indicated deletion mutants driven by the TPI1 promoter, (ii) CaCET1 or the indicated deletion mutants plus CGT1 driven by the GPD1 promoter, or (iii) CaCET1 or the indicated deletion mutants plus CEG1 driven by the GPD1 promoter. Trp+ isolates were streaked on agar plates containing 0.75 mg/ml 5-FOA. Growth was scored after 7 days of incubation at 25 and 30 °C. Lethal alleles (scored as a minus sign) were those that failed to form colonies on 5-FOA at either temperature. For the viable alleles, individual colonies were picked from the 5-FOA plates and patched on YPD agar. Two isolates of each mutant were streaked on YPD agar at 16, 25, 30, 34, and 37 °C. Growth was assessed as follows: ++ indicates wild type colony size at all temperatures; + indicates that the strains formed small colonies at the permissive temperature. Temperature-sensitive (ts) strains failed to form colonies at 37 °C. Cold-sensitive (cs) strains failed to form colonies at 16 °C.

In the present in vivo experiments, CaCET1 expression was driven by the TPI1 promoter, and CGT1 expression was driven by the strong constitutive GPD1 promoter. The temperature-sensitive (ts) phenotype of the CaCET1(203-520) triphosphatase mutant (Delta 202) was completely suppressed by GPD-CGT1. The temperature-sensitive (ts) phenotype was also suppressed when GPD-CEG1 was present in cis on a CaCET1(203-520) plasmid (Fig. 1). The new and instructive findings were that GPD-CGT1 suppressed the lethality of the more extensively truncated Delta 216, Delta 222, and Delta 228 mutations of Candida triphosphatase (Fig. 1). Yeast cells containing GPD-CGT1 plus CaCET1(217-520), CaCET1(223-520), or CaCET1(229-520) formed wild type-sized colonies on rich medium at 16, 25, 30, and 37 °C (not shown). The segment of CaCet1p from residues 203 to 228 corresponds to the guanylyltransferase-binding site of S. cerevisiae Cet1p (30). Apparently, the overexpression of Candida guanylyltransferase can compensate for the loss of key contacts between the triphosphatase and guanylyltransferase. The lethal Delta 216 and Delta 222 mutations of Candida triphosphatase were also suppressed by GPD-CEG1 (Fig. 1).

The lethal CaCET1(257-520) allele (Delta 256) was weakly complemented by GPD-CGT1 at 25 °C. Cells containing CaCET1(257-520) plus GPD-CGT1 formed tiny colonies on rich medium at 25° and were unable to grow at either 15° or 37 °C, i.e. they were cold-sensitive and temperature-sensitive (Fig. 1). The Delta 266 allele was lethal at all temperatures, even when cotransformed with GPD-CGT1.

Genetic and Biochemical Analysis of Candida RNA Guanylyltransferase-- The C. albicans CGT1 gene encodes a 449- amino acid polypeptide with extensive sequence similarity to S. cerevisiae Ceg1p (25). The active-site lysine that becomes covalently bound to GMP during the nucleotidyltransferase reaction is located at position 67 in Cgt1p and position 70 in Ceg1p. Because even small deletions from the N terminus of Ceg1p result in loss of function in vivo and in vitro (19), we presumed that N-terminal deletions of Cgt1p would also be deleterious. Therefore, we constructed a series of C-terminal deletion alleles of CGT1 that were cloned into CEN vectors under the control of the TPI1 promoter and then tested by plasmid shuffle for complementation of the S. cerevisiae ceg1Delta mutant. The CGT1(1-426), CGT1(1-408), and CGT1(1-387) mutants were viable, whereas CGT1(1-367) was lethal (Fig. 2A).



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Fig. 2.   Deletion analysis of Candida guanylyltransferase. A, yeast strain YBS50 (ceg1Delta ) was transformed with CEN TRP1 plasmids containing the indicated genes. Trp+ isolates were streaked on agar containing 0.75 mg/ml 5-FOA. The plate was photographed after incubation for 3 days at 30 °C. B, polypeptide composition. Aliquots (4 µg) of the nickel-agarose preparations of wild type Cgt1p and truncation mutants CDelta 23, CDelta 41, CDelta 62, and CDelta 82 were analyzed by SDS-PAGE. A Coomassie Blue-stained gel is shown. The positions and sizes (in kDa) of marker proteins are indicated on the left. C, guanylyltransferase activity. Reaction mixtures (20 µl) contained 50 mM Tris HCl (pH 8.0), 5 mM DTT, 5 mM MgCl2, 0.17 µM [alpha 32P] GTP, and 0.5 µg of the indicated enzyme preparations. The reaction products were resolved by SDS-PAGE. An autoradiograph of the dried gel is shown. The positions and sizes (in kDa) of prestained marker polypeptides are indicated on the left. WT, wild type.

Full-length Cgt1p and the C-terminal deletion mutants Cgt1(1-426)p (CDelta 23), Cgt1(1-408)p (CDelta 41), and Cgt1(1-387)p (CDelta 62), and Cgt1(1-367)p (CDelta 82) were expressed in bacteria as His10-tagged fusions and purified from soluble bacterial lysates by nickel-agarose column chromatography. The elution profile of full-length Cgt1p is shown in Fig. 3. SDS-PAGE analysis showed that a 55-kDa polypeptide corresponding to Cgt1p was recovered in the 100 and 200 mM imidazole eluate fractions (Fig. 3A). Guanylyltransferase activity was assayed by the formation of a covalent enzyme-[32P]GMP adduct (EpG) when the protein fractions were incubated with [alpha -32P]GTP and magnesium. The activity detected in the soluble lysate (Fig. 3B, lane S) was adsorbed to Ni2+-agarose and eluted in the 100 and 200 mM imidazole fractions in parallel with the recombinant Cgt1p polypeptide (Fig. 3B).



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Fig. 3.   Purification and guanylyltransferase activity of recombinant Cgt1p. A, nickel-agarose chromatography. The polypeptide compositions of the soluble lysate of isopropyl-1-thio-beta -D-galactopyranoside-induced BL21(DE3)/pET-Cgt1 cells (lane S), the nickel-agarose flow-through (lane F), and wash (lane W) fractions, and the 50, 100, 200, and 500 mM imidazole eluate fractions were analyzed by SDS-PAGE. The polypeptides were visualized by staining with Coomassie Blue dye. The positions and sizes (kDa) of coelectrophoresed marker polypeptides are indicated on the left. The recombinant Cgt1p is indicated by an arrow on the right. B, guanylyltransferase activity. Reaction mixtures (20 µl) contained 50 mM Tris HCl (pH 8.0), 5 mM DTT, 5 mM MgCl2, 0.17 µM [alpha -32P] GTP, and 1 µl of the soluble lysate or the indicated nickel-agarose fractions. The reaction products were resolved by SDS-PAGE. An autoradiograph of the dried gel is shown. The positions and sizes (in kDa) of prestained marker polypeptides are indicated on the left. The enzyme-[32P]GMP complex (EpG) is denoted by the arrow on the right.

The SDS-PAGE analysis of the nickel-agarose fractions of the full-length and truncated Cgt1p proteins revealed similar extents of purification and the expected increments in electrophoretic mobility (Fig. 2B). Mutants CDelta 23, CDelta 41, and CDelta 62 retained guanylyltransferase activity, whereas the CDelta 82 protein was unable to form the protein-GMP complex (Fig. 2C). Thus, there was a clear correlation between the in vivo lethality and the loss of catalytic activity elicited by deletion of the Cgt1p segment from positions 367 to 387.

Full-length recombinant Cgt1p was sedimented in a 15-30% glycerol gradient with internal standards catalase, bovine serum albumin, and cytochrome c (Fig. 4A). Note that the catalase and Cgt1p polypeptides migrated identically during SDS-PAGE. The more rapidly sedimenting polypeptide corresponds to catalase, whereas the component sedimenting as a discrete peak between bovine serum albumin and cytochrome c corresponds to Cgt1p. The guanylyltransferase activity profile was coincident with the slower sedimenting Cgt1p protein in fractions 17 to 21 (not shown). Thus, we conclude that Candida guanylyltransferase is a monomeric enzyme.



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Fig. 4.   Sedimentation of Candida guanylyltransferase. Aliquots (30 µg) of the nickel-agarose preparations of wild type Cgt1p (panel A), CDelta 62 (panel B), or CDelta 82 (panel C) were mixed with catalase (25 µg), bovine serum albumin (BSA) (25 µg), and cytochrome c (cyt c, 25 µg) in 0.2 ml of buffer G (50 mM Tris HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.05% Triton X-100). The mixtures were layered onto a 4.8-ml 15-30% glycerol gradient containing buffer G. The gradients were centrifuged in a Beckman SW50 rotor at 50,000 rpm for 16 h at 4 °C. Fractions (0.2 ml) were collected from the bottoms of the tubes. Aliquots (20 µl) of odd-numbered fractions were analyzed by SDS-PAGE along with samples of the input protein mixtures (lane L) for each gradient. Polypeptides were visualized by staining with Coomassie Blue dye.

The catalytically active CDelta 62 protein (clearly resolved from catalase during SDS-PAGE) also sedimented as a discrete monomeric peak between bovine serum albumin and cytochrome c (Fig. 4B). The same was true of the CDelta 82 mutant (Fig. 4C), which suggests that the loss of catalytic activity accompanying the incremental deletion was not caused by gross misfolding and aggregation of the CDelta 82 polypeptide.

Further characterization of the guanylyltransferase reaction was performed using the peak glycerol gradient fraction of wild type Cgt1p. Formation of the covalent Cgt1p-[32P]GMP adduct during a 10-min reaction at 37 °C with 5 µM [alpha 32P]GTP was absolutely dependent on a divalent cation cofactor. Either magnesium or manganese at 2.5 mM could satisfy the cofactor requirement, but calcium, cobalt, copper, and zinc could not (data not shown). Activity was proportional to magnesium concentration up to 2.5 mM and reached a plateaued at 2.5-10 mM MgCl2 (not shown). Cgt1p-[32P]GMP formation in the presence of manganese was optimal at 1.25 mM MnCl2 (not shown). The yield of Cgt1p-[32P]GMP increased with GTP concentration up to 2.5 µM GTP and leveled off at 5 µM GTP (not shown). Kinetic analysis showed that Cgt1p-[32P]GMP formation in the presence of 5 µM GTP increased with time up to 2 min and plateaued at 5 min (data not shown). Approximately 9% of the input Cgt1p polypeptide was labeled with [32P]GMP during the in vitro reaction with 5 µM GTP.

Effects of Cgt1p Deletions on Binding to Cet1(232-265)-- S. cerevisiae RNA triphosphatase (Cet1p) and RNA guanylyltransferase (Ceg1p) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Although it is not known if CaCet1p and Cgt1p exist as a complex in vivo in C. albicans, the findings of Yamada-Okabe et al. (26) that CaCet1p interacts with either Cgt1p or Ceg1p in a two-hybrid reporter assay in S. cerevisiae are at least consistent with the existence of a bifunctional complex in Candida. We recently localized the guanylyltransferase binding function of S. cerevisiae Cet1p to a short segment from residues 232 to 265 (30). This domain is conserved in the C. albicans RNA triphosphatase CaCet1p and is essential for in vivo function when CaCet1p is expressed in S. cerevisiae (Fig. 1).

The experiment in Fig. 5 shows that the recombinant wild type C. albicans guanylyltransferase Cgt1p bound nearly quantitatively to a biotinylated 34-amino acid synthetic peptide Cet1(232-265) coupled to streptavidin-coated beads. The input Cgt1p was eluted with SDS from the peptide-containing beads along with the smaller streptavidin polypeptide (Fig. 5, lane B). Cgt1p did not bind at all to streptavidin beads alone (30). The catalytically active truncated proteins CDelta 62 also bound to the immobilized Cet1(232-265) peptide (Fig. 5). The instructive finding was that the catalytically inactive CDelta 82 protein retained the capacity to recognize the triphosphatase peptide ligand (Fig. 5). We conclude that the binding site for the triphosphatase resides within the N-terminal 367 amino acids of Cgt1p. Further truncation of Cgt1p from the C terminus resulted in extensive proteolysis when the recombinant polypeptide was expressed in bacteria; this problem impeded our efforts to delineate the C-terminal margins of the triphosphatase-binding site on Cgt1p.



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Fig. 5.   Effects of Cgt1p deletions on binding to Cet1(232-265). The sequence of the Cet1(232-265) peptide is shown. An N-terminal biotin anchors the peptide to a streptavidin-coated magnetic bead. Affinity chromatography was performed by mixing 4 µg of Cgt1p, CDelta 62, or CDelta 82 with 0.6 mg of Cet1 peptide beads (estimated to contain 390-540 pmol of peptide) in 50 µl of binding buffer (25 mM Tris HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, 5% glycerol, 0.03% Triton X-100). After incubation for 20-30 min on ice, the beads were concentrated by microcentrifugation and then washed 3 times with 0.5-ml aliquots of binding buffer. After the third wash, the beads were resuspended in 50 µl of binding buffer. Aliquots of the input protein fraction (L) (equivalent to 40% of total material loaded), the free-unbound fraction (F) (40% of the first supernatant), and the bead-bound fraction (B) (40% of the SDS eluate of the beads) were analyzed by SDS-PAGE. A Coomassie Blue-stained gel is shown. The guanylyltransferase and streptavidin polypeptides are indicated on the right. WT, wild type.

Deletion Analysis of Candida Cap Methyltransferase Delineates a Functional Domain-- The CCM1 gene was isolated from a C. albicans library by screening for complementation of the conditional growth defect of S. cerevisiae abd1-ts mutants (8). To test whether CCM1 could fully replace ABD1, the complete CCM1 coding sequence was cloned into a yeast CEN TRP1 plasmid such that its expression was under the control of the constitutive S. cerevisiae TPI1 promoter. The CCM1 plasmid was introduced into a yeast strain in which the chromosomal ABD1 locus was deleted. Growth of the abd1Delta strain is contingent on maintenance of an extrachromosomal ABD1 gene on a CEN URA3 plasmid. Trp+ transformants were plated on medium containing 5-fluoroorotic acid (5-FOA) to select against the URA3 ABD1 plasmid. Control cells transformed with a TRP1 ABD1 plasmid grew on 5-FOA, whereas cells transformed with the TRP1 vector were incapable of growth on 5-FOA. Cells bearing the CCM1 plasmid grew on 5-FOA (Fig. 6A). Thus, the C. albicans cap methyltransferase was functional in lieu of the endogenous S. cerevisiae enzyme.



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Fig. 6.   Characterization of Candida cap methyltransferase. A, complementation of abd1Delta . Yeast strain YBS10 (abd1Delta ) was transformed with CEN TRP1 plasmids containing the indicated genes. Trp+ isolates were streaked on agar containing 0.75 mg/ml 5-FOA. The plate was photographed after incubation for 3 days at 30 °C. B, protein purification. Aliquots (3.5 µg) of the peak glycerol gradient fractions of wild type Ccm1p (WT), Ccm1(137-474)p (Delta 136), Ccm1(151-474)p (Delta 150), and Ccm1(175-474)p (Delta 174) were analyzed by SDS-PAGE. A Coomassie Blue-stained gel is shown. The positions and sizes (in kDa) of marker proteins are indicated on the left. C, methyltransferase activity. Reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 50 µM AdoMet, 20 fmol of 32P cap-labeled poly(A), and recombinant Ccm1p as specified were incubated at 37 °C for 10 min. The reaction products were digested with nuclease P1 and analyzed by polyethyleneimine-cellulose TLC as described (Saha et al. (8)). The extent of cap methylation [m7GpppA/(m7GpppA + GpppA)] was determined by scanning the chromatogram with a phosphorimager. D, overexpression of Ccm1(151-474)p partially restores function in vivo. Strain YBS10 (abd1Delta ) was transformed with 2µ TRP1 plasmids containing the CCM1 or CCM1(151-474) [Delta 150] genes. Trp+ isolates were streaked on agar containing 0.75 mg/ml 5-FOA. 5-FOA-resistant isolates were tested for growth on YPD agar. The YPD plate was photographed after incubation for 4 days at 30 °C.

Several N-terminal deletion mutants of CCM1 were cloned into the CEN TRP1 vector and tested for function in vivo by plasmid shuffle. CCM1(137-474) complemented growth of the abd1Delta strain on 5-FOA. However, the more extensively truncated alleles CCM1(151-474) and CCM1(175-474) were lethal in vivo (Fig. 6A). Based on an alignment of the amino acid sequences of the Ccm1p and Abd1p polypeptides (24), the viable N-terminal Delta 136 deletion of Ccm1p would be analogous to a deletion of 108 amino acids from the N terminus of Abd1p. The lethal N-terminal Delta 150 and Delta 174 deletions of Ccm1p correspond to Delta 122 and Delta 146 deletions of Abd1p. Prior studies showed that deleting 109 amino acids from the N terminus of Abd1p had no effect on yeast cell growth, whereas deletion of 142 or 155 residues was lethal (23). Thus, the N-terminal margins of the functional domains of the Candida and Saccharomyces cap methyltransferases are fairly similar.

Purification and Characterization of Recombinant Ccm1p-- Full-length Ccm1p and the N-terminal deletion mutants Ccm1(137-474)p (Delta 136), Ccm1(151-474)p (Delta 150), and Ccm1(175-474)p (Delta 174) were expressed in bacteria as His10-tagged fusions and purified from soluble bacterial lysates by nickel-agarose chromatography, followed by glycerol gradient sedimentation. SDS-PAGE analysis of the polypeptide compositions of the peak glycerol gradient fractions of Ccm1p, Delta 136, Delta 150, and Delta 174 is shown in Fig. 6B. The apparent sizes of the predominant polypeptides corresponding to Ccm1p and serially truncated versions thereof are in good agreement with the calculated sizes of the His-tagged gene products.

RNA (guanine-N7-)-methyltransferase activity of the Ccm1p preparation was detected by the conversion of 32P cap-labeled poly(A) to methylated cap-labeled poly(A) in the presence of AdoMet. The reaction products were digested to cap dinucleotides with nuclease P1 and then analyzed by polyethyleneimine-cellulose thin layer chromatography, which resolves the GpppA cap from the methylated cap m7GpppA. The radiolabeled product synthesized by Ccm1p comigrated with m7GpppA generated in a parallel reaction mixture containing purified recombinant vaccinia virus cap methyltransferase (not shown). Methylation of capped poly(A) varied linearly with input Ccm1p protein and was nearly quantitative at saturation (Fig. 6C). Cap methylation depended on inclusion of S-adenosylmethionine in the reaction mixture. Half-maximal activity was observed at ~5 µM AdoMet (not shown). S-Adenosylhomocysteine did not support cap methylation and was inhibitory in the presence of AdoMet (not shown).

The specific activity of Delta 120 in cap methylation was similar to that of full-length Ccm1p, whereas Delta 150 was one-fifth as active, and Delta 174 was inactive (Fig. 6C). The failure of Delta 150 to sustain growth of abd1Delta cells when expressed in a single copy despite retention of partial activity in vitro raised the possibility that cell growth requires a threshold level of cap methyltransferase activity. To test this hypothesis, we increased the gene dosage of the Delta 150 mutant by cloning the CCM1(151-474) gene into a high-copy 2µ vector under the control of the constitutive TPI1 promoter. abd1Delta cells bearing the 2µ CCM1(151-474) plasmid did form small colonies on 5-FOA (not shown). When tested for growth on rich medium (YPD) at 30 °C, the 2µ CCM1(151-474) cells formed uniformly small colonies compared with 2µ CCM1 cells (Fig. 6D). 2µ CCM1(151-474) cells also grew slowly at 25 and 37 °C (not shown). These results are consistent with a threshold requirement for cap methyltransferase activity in vivo. Note that the catalytically defective Delta 174 mutant failed to support growth of abd1Delta cells even when introduced on a 2µ plasmid (not shown). We infer that the segment from residues 151 to 174 is essential for cap methylation.

Candida Cap Methyltransferase Binds to the Phosphorylated CTD of RNA Polymerase II-- In vivo, the mRNA capping reactions occur cotranscriptionally, i.e. the substrates for the capping enzymes are nascent RNA chains engaged within RNA polymerase II elongation complexes. Targeting of cap formation to transcripts made by polymerase II is achieved through direct physical interaction of components of the capping apparatus with the phosphorylated CTD of the largest subunit of polymerase II (2-4, 31). The CTD, which is unique to polymerase II, is composed of a tandemly repeated heptad motif (consensus sequence = YSPTSPS). The CTD undergoes a cycle of extensive phosphorylation and dephosphorylation, which is coordinated with the transcription cycle (32). Cyclin-dependent protein kinases are implicated in CTD phosphorylation at positions Ser-2 and Ser-5 of the heptad repeats.

Ho and Shuman (33) employed synthetic CTD phosphopeptides to delineate the requirements for the interaction of capping enzymes with the CTD. They found that mammalian guanylyltransferase binds to 28-mer CTD peptides containing phosphoserine at either position 2 or position 5 of all four heptad repeats but not to unphosphorylated CTD peptides.

Here we have analyzed the binding of Candida cap methyltransferase to CTD Ser-2 and Ser-5 phosphopeptides; we also extended the binding studies to include a synthetic 28-mer CTD phosphopeptide ligand that is phosphorylated on both Ser-2 and Ser-5 of each heptad repeat. An N-terminal biotin moiety was added during chemical synthesis so that the peptides could be linked to streptavidin beads for affinity chromatography purposes (Fig. 7). CTD phosphopeptide-containing beads and control beads containing an unphosphorylated 28-mer CTD peptide were incubated with purified recombinant Ccm1Delta 136 protein. The beads were recovered by centrifugation and held in place with a magnet while the supernatant-containing free methyltransferase was withdrawn. The beads were washed with buffer, and the bead-bound material was eluted from the beads with 1% SDS. The input methyltransferase protein (L) and the free (F) and bead-bound (B) fractions were then analyzed by SDS-PAGE (Fig. 7A). The streptavidin polypeptide was stripped off the beads by 1% SDS and recovered in every bound eluate fraction.



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Fig. 7.   Binding of Candida cap methyltransferase to CTD phosphopeptides. The 28-amino acid CTD peptide ligand consisted of a tandem array of 4 heptad repeats, as shown, with either no modification (no PO4) or with phosphoserine introduced at position 2 (Ser2-PO4), position 5 (Ser5-PO4), or positions 2 and 5 (Ser2-PO4 Ser5-PO4) of each heptad repeat. An N-terminal biotin anchors the CTD peptide to a streptavidin-coated magnetic bead. A, affinity chromatography was performed by mixing 4 µg of Ccm1Delta 136 with 0.5 mg of CTD peptide beads (estimated to contain 375-450 pmol of the unmodified or serine-phosphorylated peptide, as specified) in 50 µl of binding buffer (50 mM Tris HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, 5% glycerol, 0.03% Triton X-100). After incubation for 30 min on ice, the beads were concentrated by microcentrifugation and then washed 3 times with 0.5 ml aliquots of binding buffer. After the third wash, the beads were resuspended in 50 µl of binding buffer. Aliquots of the input protein fraction (L) (equivalent to 40% of total material loaded), the free-unbound fraction (F) (40% of the first supernatant), and the bead-bound fraction (B) (40% of the SDS eluate of the beads) were adjusted to 1% SDS and then analyzed by SDS-PAGE. A Coomassie Blue-stained gel is shown. The positions and sizes (kDa) of coelectrophoresed marker polypeptides are indicated on the left. The Ccm1Delta 136 and streptavidin polypeptides are indicated on the right. B, affinity chromatography was performed by adsorbing 4 µg of Ccm1Delta 136, Ccm1Delta 150, or Ccm1Delta 174 to 0.5 mg of streptavidin beads containing the Ser-2-PO4 Ser-5-PO4 CTD peptide. Aliquots of the input protein fraction, the free-unbound fraction, and the bead-bound fraction were analyzed by SDS-PAGE as described above.

We found that the biologically active Candida methyltransferase domain Ccm1Delta 136 bound to either the Ser-5-PO4 CTD or the Ser-2-PO4 CTD peptide. About half of the input protein was retained on the beads. This binding was specific for CTD-PO4, because the methyltransferase did not bind at all to the beads containing unphosphorylated CTD peptide (Fig. 7A). The salient finding was that the methyltransferase bound more avidly to the CTD when Ser-2 and Ser-5 were phosphorylated than when either Ser2 or Ser5 were phosphorylated singly (Fig. 7A). This experiment shows that the affinity of the cap methyltransferase for the CTD can be modulated by altering the phosphorylation array.

Ccm1Delta 136 and Ccm1Delta 150 bound to the Ser-2-PO4/Ser-5-PO4 CTD, whereas Ccm1Delta 174 did not (Fig. 7B). We conclude from that the Ccm1p segment from amino acids 151 to 174 is required both for catalysis of cap methylation and for interaction of the methyltransferase with the CTD.

Complete Replacement of the Saccharomyces Capping Apparatus by Candida Enzymes-- A plausible strategy for capping-specific antifungal drug discovery is to identify compounds that inhibit the growth of yeast cells containing fungus-encoded capping activities without affecting the growth of otherwise identical yeast cells bearing the mammalian capping enzymes. Ideally, the fungal capping enzymes that sustain growth of the tester yeast cells would be those encoded by a clinically significant fungal pathogen. To achieve this scenario, we sought to construct S. cerevisiae strains in which the entire S. cerevisiae capping apparatus was replaced by enzymes from the pathogenic fungus C. albicans.

A CEN TRP1 expression plasmid (pCan-CAP-1) was constructed that contained three C. albicans genes under the control of S. cerevisiae promoters, as follows: CaCET1(179-520) controlled by the TPI1 promoter; CGT1 driven by the GPD1 promoter; CCM1 controlled by the TPI1 promoter. Another CEN TRP1 expression plasmid (pCan-CAP-2) was constructed that contained CaCET1(179-520) controlled by the TPI1 promoter, CGT1 driven by the GPD1 promoter, and CCM1(137-474) controlled by the TPI1 promoter. pCan-CAP-1 and pCan-CAP-2 were transformed into the S. cerevisiae cet1Delta ceg1Delta abd1Delta triple-deletion strain YBS52 (8), the growth of which is sustained by a CEN URA3 CET1 CEG1 ABD1 plasmid. Plasmid shuffle experiments showed that YBS52 cells transformed with either pCan-CAP-1 and pCan-CAP-2 or with an "all-Saccharomyces" capping enzyme expression plasmid (CEN TRP1 CET1 CEG1 ABD1) grew on 5-FOA, whereas plasmids containing only CET1, CEG1, or ABD1 did not complement growth on 5-FOA (not shown).

5-FOA-resistant isolates were then streaked on YPD plates at 30 °C. Using colony size as a rough estimate of growth, it is surmised that cells containing an all-Candida capping apparatus grew as well as the cells containing an all-Saccharomyces capping apparatus (Fig. 8). Also shown on this plate is the growth of cells containing an all-mammalian capping system encoded by CEN MCE1 plus 2µ HCM1(121-476) plasmids (Fig. 8).



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Fig. 8.   Complete replacement of the Saccharomyces capping apparatus by Candida enzymes. Yeast strain YBS52 (cet1Delta ceg1Delta abd1Delta ) was transformed with TRP1 plasmids as specified below and then selected on 5-FOA. FOA-resistant isolates were streaked on a YPD agar plate. The plate was photographed after incubation for 3 days at 30 °C. ABD1 CET1 CEG1 was transformed with the three S. cerevisiae genes on a single CEN plasmid. CaCET1(179-520), CGT1 CCM1(137-474), and CaCET(179-520) CGT1 CCM1 were transformed with the indicated sets of three C. albicans genes on CEN plasmids pCAN-CAP-2 and pCAN-CAP-1. MCE1 HCM1(121-476) was cotransformed with CEN MCE1 and 2µ HCM1(121-476).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study defines the essential domains of all three components of the C. albicans mRNA capping apparatus: the triphosphatase CaCet1p, the guanylyltransferase Cgt1p, and the methyltransferase Ccm1p. We used in vivo complementation of S. cerevisiae cet1Delta , ceg1Delta , or abd1Delta mutants as a means of demarcating functional domain boundaries for the Candida proteins. Characterization of purified recombinant Candida capping enzymes and truncated versions thereof reveals (i) that for C-terminal deletions of Cgt1p, there is a clear correlation between retention of guanylyltransferase activity in vitro and complementation in vivo, (ii) that serial N-terminal deletions of CaCet1p elicit lethality in vivo before loss of catalytic activity in vitro. We infer that protein segments flanking the catalytic domain of CaCet1p mediate important ancillary functions in vivo. Our studies provide new insights into the influence of CTD phosphorylation on its binding to the fungal cap methylating enzyme. They also highlight how the physical and functional properties of the capping enzymes are conserved among fungi and how the differences between fungal and metazoan capping enzymes, especially the triphosphatase, can be exploited pharmacologically.

Triphosphate-Guanylyltransferase Interactions-- The C-terminal catalytic domain of CaCet1p from residue 257 to 520 suffices for phosphohydrolase activity in vitro (29). N-terminal deletions of 216 to 228 amino acids of CaCet1p eliminate the high affinity guanylyltransferase-binding site (30) and are lethal in vivo. This result is in keeping with the proposal that fungal guanylyltransferase chaperones the triphosphatase to the RNA polymerase II elongation complex (13). The finding that guanylyltransferase overexpression suppressed the growth defects of strains expressing CaCet1p N-terminal deletion mutants Delta 202, Delta 216, Delta 222, and Delta 228 suggests that a second "low affinity" guanylyltransferase-binding site exists distal to amino acid 229. The putative secondary site apparently does not promote sufficient interaction between the truncated Candida triphosphatase and the endogenous pool of Ceg1p to sustain growth. However, increased guanylyltransferase expression can sustain growth by driving guanylyltransferase binding to the low affinity site by mass action. Yeast cell growth apparently requires a threshold level of RNA triphosphatase and guanylyltransferase activities that is severalfold lower than the level in wild type yeast cells (16, 19). Thus, it is probably not necessary to completely restore the wild type level of triphosphatase-guanylyltransferase interaction to sustain cell growth. The efficacy of CGT1 suppression declined when the CaCet1p segment from residue 229 to 256 was removed, to the point that the cells grow very slowly at 25 °C and were both cold-sensitive (cs) and temperature-sensitive (ts) (Fig. 1). This result suggests that the peptide region from 229 to 256, which is not present in the S. cerevisiae triphosphatase (Fig. 1), may comprise part of the proposed low affinity guanylyltransferase-binding site on the Candida triphosphatase.

Cap Methyltransferase Interaction with Phosphorylated CTD-- Previous studies of the binding of the S. cerevisiae cap methyltransferase to the RNA polymerase II CTD employed an immobilized ligand composed of recombinant GST-CTD that was enzymatically phosphorylated in vitro using HeLa extract as the source of CTD kinase. The fusion protein contained 15 copies of the YSPTSPS heptad sequence and an average of 3 serine-phosphates per GST-CTD polypeptide (2). Our present study of the cap methyltransferase-CTD interaction employed a defined 4-heptad CTD ligand in which the number and position of the phosphates were known and subject to manipulation. We have used the synthetic CTD phosphopeptides to establish that (i) the interaction of cap methyltransferase with the phosphorylated CTD described initially for S. cerevisiae is conserved in the pathogenic fungus C. albicans, (ii) Candida cap methyltransferase binds to CTD phosphorylated on either Ser-2 or Ser-5 but binds more avidly when both Ser-2 and Ser-5 are phosphorylated, and (iii) the N-terminal domain of Ccm1p is not required for binding to the phosphorylated CTD.

Ser-5 and Ser-2 are both extensively phosphorylated in vivo, and various CTD kinases differ in their site preference (34-36). Zhou et al. (36) showed recently that the phosphorylation site specificity of the mammalian CTD kinase P-TEFb is altered by its interaction with the human immunodeficiency virus Tat protein. P-TEFb phosphorylates Ser-2 of the CTD heptad in the absence of Tat, but it phosphorylates both Ser-2 and Ser-5 when Tat is present (36). The observation of better binding of the Candida cap methyltransferase when both CTD serines are phosphorylated is, to our knowledge, the first example of a specific "added value" incurred by simultaneous modification of both positions. This raises the prospect that dynamic remodeling of the CTD phosphate array may regulate the efficiency or timing of cotranscriptional cap methylation and the longevity of the association of the cap methyltransferase with the transcription elongation complex.

Tools for Antifungal Drug Discovery-- The availability of isogenic yeast strains containing Candida versus mammalian capping systems provides an attractive means of drug discovery aimed at blocking cap formation in pathogenic fungi. Any compound that is selectively cytotoxic to the all-Candida strain but not to the all-mammalian strain is likely to be a specific inhibitor of fungal capping. Secondary screens for cytotoxicity comparing strains in which only a subset of the Candida capping activities are replaced by a mammalian enzyme could pinpoint which of the gene products is targeted by such a compound. The availability of recombinant versions of all three Candida cap-forming enzymes should facilitate the elucidation of structure activity relationships in vitro for inhibitory compounds.


    ACKNOWLEDGEMENT

We thank San San Yi for expert peptide synthesis.


    FOOTNOTES

* This research was supported by National Institutes of Health Grant GM52470.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 212-639-7145; Fax: 212-717-3623; E-mail: s-shuman@ski.mskcc.org.

Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M006072200


    ABBREVIATIONS

The abbreviations used are: CTD, C-terminal domain; AdoMet, S-adenosylmethionine; PCR, polymerase chain reaction; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; 5-FOA, 5-fluoroorotic acid; YPD medium, yeast extract/peptone/dextrose medium.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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