Sandbox ceg1p Steven Paris

Function of Ceg1p and the Cet1-Ceg1 mRNA Capping Complex

Ceg1p is an mRNA guanylyltransferase in Saccharomyces cerevisiae that forms a heterotetramer with the RNA triphosphatase Cet1p which is called the mRNA capping apparatus. This complex is recruited by RNA polymerase II (RNAP-II) and performs the first two steps in the 5’-guanisine mRNA capping mechanism. Cet1p hydrolyzes a phosphate from the 5’-triphosphate end of the pre-mRNA.^[1] The Ceg1p guanylyltransferase then adds a guanosine monophosphate to the 5' end of the mRNA, creating a 5'-5' triphosphate linkage. In the final step, an RNA methyltransferase comes and adds a methyl group to the N7 atom of the guanine, completing the 5'-guanine cap.^[2] This 5’ capping is necessary for cells to live in yeast among other organisms. The interactions between Cet1p and Ceg1p are extremely important for inducing capping activity. These interactions both stimulate Ceg1p GMP transfer and help localize the complex in the nucleus where RNAP-II can recruit it.^[3] The capping apparatus is specially recruited to the Rpb1p subunit of RNAP-II. It binds to the serine-5-phosphorylated-carboxy terminal domain (CTD) of Rpb1p. The capping mechanism occurs while transcription is still occurring, beginning when around 20-25 nucleotides are successfully transcribed by RNAP-II.^[2] Another role that the Cet1-Ceg1 complex has is the suppression of RNAP-II transcription. This transcriptional control involves the capping complex keeping RNAP-II from reinitiating transcription.^[4] The capping complex has also been suggested to have regulatory roles in other cellular functions such as cell proliferation and RNA interference.^[2]

Structure of Ceg1p and Interactions with Cet1p in the Capping Apparatus

The is composed of a and two , one on each end of the homodimer. The capping apparatus may also have only one Ceg1p monomer attached, forming a heterotrimer complex. A contains two major domains: a nucleotydil transferase (NT) domain and an oligonucleotide binding (OB) domain. The OB domain interacts with Cet1p, while the NT domain interacts with RNAP-II. For the Cet1p, only amino acids 241-549 of each monomer are required for the complex to properly cap mRNA.^[1] The Ceg1p monomers interact with the Cet1p homodimer though the of Cet1p. The motif is followed by a flexible linker.^[1][5] The motif extends from the Cet1p monomer furthest from Ceg1p to form effectively bridge it to the homodimer. This interaction allows the Ceg1p to maintain a conformation that allows it to bind to both the Cet1p homodimer, and RNAP-II during the capping process. The OB and NT domains open and close so that GMP can be properly transferred to the 5' end of the mRNA. Many components of the Cet1-Ceg1 capping apparatus are conserved across evolution. Some highly conserved components include: the NT and OB domains of Ceg1p, the WAQKW motif of Cet1p and the capping apparatus recruitment by RNAP-II.^[1] In Candida albicans, the guanylyltransferase-binding domain was conserved from S. cerevisiae.^[5] This is one example of the high conservation observed for this apparatus.

Cet1-Ceg1 Interaction with RNAP-II

When 22-25 nucleotides have been transcribed by RNAP-II, the Cet1-Ceg1 apparatus is recruited to the Rpb1p subunit's CTD to perform 5' capping on the mRNA.^[2] Ceg1p comes in direct contact with the CTD so that 5' capping can occur. This interaction with the CTD occurs in the NT domain of Ceg1p. This interaction is highly conserved across both yeast and mammals.^[1][3] Cet1p, while still in complex with Ceg1p, Cet1p does not directly directly bind to RNAP-II.^[6] When Ceg1p binds to the CTD, it gains an unfavorable conformation that doesn't allow 5' capping to occur. It is the combination of Cet1p's flexible linker and the general flexibility of the CTD that allow for Ceg1p to transfer the GMP cap to the pre-mRNA. This flexibility on both sides of the Ceg1p monomer allow both the NT and OB domains to open and close into the conformations needed the GMP transfer mechanism.^[1][6]

About This Structure

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