Structure of the Saccharomyces cerevisiae Cet1-Ceg1 mRNA capping apparatus

Abstract

The 5' guanine-N7 cap is the first cotranscriptional modification of messenger RNA. In Saccharomyces cerevisiae, the first two steps in capping are catalyzed by the RNA triphosphatase Cet1 and RNA guanylyltransferase Ceg1, which form a complex that is directly recruited to phosphorylated RNA polymerase II (RNAP IIo), primarily via contacts between RNAP IIo and Ceg1. A 3.0 A crystal structure of Cet1-Ceg1 revealed a 176 kDa heterotetrameric complex composed of one Cet1 homodimer that associates with two Ceg1 molecules via interactions between the Ceg1 oligonucleotide binding domain and an extended Cet1 WAQKW amino acid motif. The WAQKW motif is followed by a flexible linker that would allow Ceg1 to achieve conformational changes required for capping while maintaining interactions with both Cet1 and RNAP IIo. The impact of mutations as assessed through genetic analysis in S. cerevisiae is consonant with contacts observed in the Cet1-Ceg1 structure.

Figure 1. Structure of the S. cerevisiae triphosphatase-guanylyltransferase complex

(A) Ribbon representation of the complex between the Cet1 RNA triphosphatase and Ceg1 guanylyltransferase. (B) 90° rotation of the model from panel A. (C) 90° rotation of the model from panel B. Cet1 protomers are color coded magenta and yellow, respectively and Ceg1 protomers are colored blue. Each molecule is labeled. The Ceg1 nucleotidyl transferase (NT) and oligonucleotide binding (OB) domains are denoted. N denotes the respective positions for each Cet1 N-terminus color-coded magenta or yellow. Yellow or magenta circles indicate the six disordered amino acids between the Cet1 N-terminal WAQKW motif and the triphosphatase domain, respectively. Structural representations generated using PYMOL (http://pymol.sourceforge.net/).

Figure 2. Comparison of Cet1 crystallized alone or in complex with Ceg1

(A) Ribbon representation of the Cet1 dimer determined previously highlighting the conserved tryptophan residues in the WAQKW motif (stick representation) near the Cet1 N-terminus (PDB 1D8H) denoted by single letter amino acid code. (B) Ribbon representation of the Cet1 dimer as observed in the Cet1-Ceg1 complex highlighting the swapped configuration of N-terminal WAQKW motifs with residues in stick representation and denoted by single letter amino acid code. One Cet1 protomer is shown in magenta while the other is colored yellow. N and C indicate the amino terminus (amino acid 241) and carboxy terminus (amino acid 549), respectively. Yellow or magenta circles indicate the six disordered amino acids between the Cet1 N-terminal WAQKW motif and the triphosphatase domain, respectively. (C) Ribbon and stick representation of the Cet1 active site as observed in PDB 1D8H. (D) Ribbon and stick representation of the Cet1 active site from a protomer of Cet1 in the present structure.

Figure 3. Two orientations observed for Cet1 with respect to Ceg1 in the Cet1-Ceg1 complex

(A) Ribbon representation of one Cet1-Ceg1 complex with views highlighting the position for Ceg1 in the complex with respect to Cet1. B) Same as (A) but for the other complex between Cet1 and Ceg1 in the heterotetramer. Ceg1 molecules are presented in the same orientation in A and B to highlight the differences in orientations with respect to Cet1. Each molecule is color coded as in Figure 1 with yellow or magenta circles indicating the six disordered amino acids between the Cet1 N-terminal WAQKW motif and the triphosphatase domain, respectively. Nucleotidyl transferase (NT) and oligonucleotide binding (OB) domains are labeled. The distance between Cet1 amino acid 261 and 268 is indicated in each panel. The distance between Cet1 amino acid 510 and Ceg1 amino acid 41 is indicated in each panel.

Figure 4. Comparison between Ceg1 and Cgt1

(A) Ribbon representations of S. cerevisiae Ceg1 (slate) and C. albicans Cgt1 (red). (B) Orthogonal view to A. Ceg1 and Cgt1 were aligned based on their nucleotidyl transferase (NT) domains, highlighting the conformational differences observed for their respective OB domains. The phosphorylated CTD peptide in the Cgt1 structure is colored yellow in stick representation. The Cet1 N-terminal WAQKW motif is colored in magenta in stick representation in the Ceg1 structure. Domains and polypeptides are labeled.

Figure 5. Structural and mutational analysis of the interface between Cet1 and Ceg1

(A) Interactions between the Cet1 WAQKW motif and the Ceg1 OB domain. The OB domain is depicted in ribbon representation with side chains selected for mutational analysis colored yellow in stick representation. The helix-loop insertion in Ceg1 is indicated by a bar and the designation HL. The Cet1 N-terminal element is shown in stick representation in magenta and labeled. Side chains are labeled in black for Ceg1 and magenta for Cet1. B) Similar to (A) but with Ceg1 depicted in surface view to highlight the deep canyon in which the Cet1 polypeptide binds. Selected Ceg1 amino acids (yellow) are labeled. C) and D) Serial dilutions of S. cerevisiae strains bearing indicated CEG1 alleles (top position at OD₆₀₀=0.5 with three serially diluted concentrations along the vertical axis). Top of each panel indicates the CET1 strain utilized in each analysis and individual amino acid substitutions are indicated at the bottom of the panel. Panel (C) shows results of a complementation assay in a strain expressing full length CET1(1–549) and panel (D) shows results of a complementation assay in a strain expressing CET1(241–549). This latter strain contains a fragment of Cet1 that is analogous to the Cet1 domain determined in the structure of the Cet1-Ceg1 complex.

Figure 6. Stereo diagram of the Cet1-Ceg1 complex and structure-based sequence alignment for Ceg1 and Cgt1

(A) Stick representation of the Ceg1 OB domain with side chains targeted by mutagenesis colored yellow and labeled in black. Cet1 amino acids are labeled and colored in magenta. The helix-loop insertion is indicated by a black bar and the designation HL. (B) Sequence alignment for the OB domains from Ceg1 and Cgt1 with secondary structure elements indicated above or below the respective sequence. Residues targeted for mutational analysis are indicated by black triangles. Residues highlighted in red belong to a hydrophobic cluster that is shared between Ceg1 and Cgt1 while residues highlighted in green are located in the unique Ceg1 HL insertion that contacts Cet1. Disordered regions in our structure are indicated by a dashed line.

Figure 7. Model for the organization of the Cet1-Ceg1 capping apparatus

On the left is a schematic of the Cet1-Ceg1 complex with one Ceg1 protomer shown bound to the phosphorylated RNAP IIo CTD via the nucleotidyltransferase domain (NT) in the open configuration ready to bind substrates or release products. Interactions between the Ceg1 OB domain and one Cet1 protomer WAQKW motif is followed by a flexible linker that tethers the Ceg1 OB domain to the Cet1 triphosphatase. On the right is Cet1-Ceg1 complex with Ceg1 in the closed configuration illustrating that Ceg1 can undergo the conformational changes required for capping while maintaining interactions with Cet1 and the phosphorylated RNAP IIo CTD.