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© 1999 Oxford University Press

Nucleic Acids Research, 1999, Vol. 27, No. 24 4671–4678

An essential surface motif (WAQKW) of yeast RNA triphosphatase mediates formation of the mRNA capping enzyme complex with RNA guanylyltransferase C. Kiong Ho, Kevin Lehman and Stewart Shuman* Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA Received September 21, 1999; Revised and Accepted October 28, 1999

ABSTRACT

Saccharomyces cerevisiae RNA triphosphatase (Cet1p) and RNA guanylyltransferase (Ceg1p) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Cet1p binding to Ceg1p stimulates the guanylyltransferase activity of Ceg1p. Here we localize the guanylyltransferase-binding and guanylyltransferase-stimulation functions of Cet1p to a 21-amino acid segment from residues 239 to 259. The guanylyltransferase-binding domain is located on the protein surface, as gauged by protease sensitivity, and is conserved in the Candida albicans RNA triphosphatase CaCet1p. Alanine-cluster mutations of a WAQKW motif within this segment abolish guanylyltransferase-binding in vitro and Cet1p function in vivo, but do not affect the triphosphatase activity of Cet1p. Proteolytic footprinting experiments provide physical evidence that Cet1p interacts with the C-terminal domain of Ceg1p. Trypsin-sensitive sites of Ceg1p that are shielded from proteolysis when Ceg1p is bound to Cet1p are located between nucleotidyl transferase motifs V and VI. INTRODUCTION Capping of mRNA entails three enzymatic reactions in which the 5′ triphosphate end of pre-mRNA is hydrolyzed to a 5′ diphosphate by RNA triphosphatase, then capped with GMP by RNA guanylyltransferase, and methylated by RNA (guanine-7) methyltransferase (1). The budding yeast Saccharomyces cerevisiae encodes a three-component capping apparatus consisting of separate triphosphatase (Cet1p), guanylyltransferase (Ceg1p) and methyltransferase (Abd1p) gene products. Each of the cap-forming activities is essential for yeast cell growth (2–7). The yeast RNA triphosphatase Cet1p forms a heteromeric complex with the yeast RNA guanylyltransferase Ceg1p (8–10). The binding of Cet1p to Ceg1p serves two purposes. First, Cet1p– Ceg1p interaction stimulates guanylyltransferase activity by enhancing the affinity of Ceg1p for GTP and increasing the extent of formation of the Ceg1p–GMP reaction intermediate (9). Second, the physical tethering of Cet1p to Ceg1p facilitates recruitment of the triphosphatase to the RNA polymerase II

elongation complex. The yeast guanylyltransferase Ceg1p binds to the phosphorylated C-terminal domain (CTD) of the largest subunit of RNA polymerase II, whereas Cet1p by itself does not bind to the phosphorylated CTD (11–13). The 549-amino acid Cet1p protein consists of three domains: (i) a 230-amino acid N-terminal segment that is dispensable for catalysis in vitro and for Cet1p function in vivo; (ii) a proteasesensitive segment from residues 230 to 275 that is dispensable for catalysis, but essential for Cet1p function in vivo; and (iii) a catalytic domain from residues 275 to 539 (10). A homodimeric quaternary structure for the biologically active fragment Cet1(231–549)p was suggested based on analysis of the purified recombinant enzyme by glycerol gradient sedimentation (10). Cet1(231–549)p binds in vitro to Ceg1p to form a 7.1S triphosphatase–guanylyltransferase complex that is surmised to be a trimer consisting of two molecules of Cet1(231–549)p and one molecule of Ceg1p. The more extensively truncated protein Cet1(276–549)p, which cannot support cell growth, sediments as a monomer and does not interact with Ceg1p (10). These results implicate the segment of Cet1p from residues 230 to 275 in both Cet1p homodimerization and binding to the guanylyltransferase. The interaction of Cet1p with Ceg1p does not require a functional triphosphatase active site in Cet1p, insofar as Cet1p–Ceg1p complex formation is unaffected by mutations in the catalytic domain that abrogate RNA triphosphatase activity (14). Two lines of genetic evidence indicate that the Cet1p–Ceg1p interaction is important in vivo. First, several temperaturesensitive ceg1 mutations are suppressed in an allele-specific manner by overexpression of CET1 (9,13). In turn, the temperature-sensitive cet1-(K250A-W251A) mutation can be suppressed by overexpression of CEG1 (10). This cet1-ts mutation is located with the segment of Cet1p that is suspected to mediate guanylyltransferase-binding. Fifteen other cet1-ts alleles with missense changes mapping elsewhere in the protein were not suppressed by CEG1 overexpression (10). Second, the in vivo function of Cet1(275–549)p, which does not bind to Ceg1p in vitro, is completely restored by fusion of Cet1(275–549)p to the guanylyltransferase domain of the mouse capping enzyme (10). This result shows that the need for Ceg1p-binding by yeast RNA triphosphatase can by bypassed when the triphosphatase catalytic domain is delivered to the RNA polymerase II elongation complex by linkage in cis to the mammalian guanylyltransferase.

*To whom correspondence should be addressed. Tel: +1 212 639 7145; Fax: +1 212 717 3623; Email: [email protected]

4672 Nucleic Acids Research, 1999, Vol. 27, No. 24

Here we use synthetic peptide ligands to localize the guanylyltransferase-binding and guanylyltransferase-stimulation functions of Cet1p. Alanine cluster mutations implicate the Cet1p sequence 247–WAQKW–251 in guanylyltransferasebinding in vitro and in Cet1p function in vivo. The Ceg1pbinding domain of Cet1p is conserved in Candida albicans RNA triphosphatase. Indeed, the Cet1p peptide binds avidly in vitro to the C.albicans RNA guanylyltransferase and binding is abrogated by mutation of the WAQKW motif. MATERIALS AND METHODS Guanylyltransferase expression and purification The S.cerevisiae CEG1 gene was inserted into a customized T7-based expression plasmid (a derivative of pET16b) in such a way as to fuse the 459-amino acid Ceg1p polypeptide in frame to an N-terminal 29-amino acid leader peptide (MGSHHHHHHHHHHSSGHIEGRHSRRASVH) containing 10 consecutive histidine codons (His-tag) and a serine-phosphorylation site (RRASV) for protein kinase A. Recombinant Ceg1p was expressed in Escherichia coli BL21(DE3) and purified from a soluble bacterial lysate by nickel–agarose chromatography as described (10). The C.albicans CGT1 gene was PCR-amplified from a genomic library clone using a sense primer designed to introduce an NdeI site at the translation start codon and an antisense primer that introduced a BamHI site immediately 3′ of the stop codon. The PCR product was digested with NdeI and BamHI and then inserted into yeast expression plasmid pYN132 to yield plasmid pYN-Cgt1. The CGT1 gene was excised from pYN-Cgt1 with NdeI and BamHI and then inserted into the T7-based expression vector pET16b so as to fuse the 449-amino acid Cgt1p polypeptide to a Nterminal 21-amino acid leader peptide containing the His-tag. Recombinant Cgt1p was expressed in E.coli BL21(DE3) and purified from a soluble bacterial lysate by nickel–agarose chromatography. The nickel–agarose preparations of Cet1p and Cgt1p were dialyzed against buffer containing 50 mM Tris– HCl (pH 8.0), 50 mM NaCl, 2 mM DTT, 10% glycerol, 0.05% Triton X-100 and then stored at –80°C. Recombinant mouse guanylyltransferase Mce1(211–597)p was purified as described (15). Protein concentrations were determined using the Bio-Rad dye binding reagent with bovine serum albumin as the standard. Cet1 peptides Peptides composed of Cet1p residues 232–265, 239–265 or 232–259 were synthesized in the Sloan-Kettering Microchemistry Core Laboratory on a Perkin-Elmer Biosystems 431A automated peptide synthesizer using standard Fmoc chemistry. Addition of biotin to the N-terminal amino group of the peptide was performed using reagents purchased from AnaSpec (San Jose, CA) according to the vendor’s instructions. The peptides were purified on a preparative scale by reverse phase HPLC and the purity of the material was confirmed by analytic scale reverse phase HPLC as described (16). The molecular weight of each peptide was analyzed by MALDI-TOF mass spectrometry. The measured masses were in agreement with the calculated theoretical masses within the limits of calibration of the instrument. The lyophilized peptides were dissolved in TE (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) and stored at 4°C.

The UV absorbance profiles of the Cet1 peptides revealed a large peak at

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