1990 Fiscal Year Final Research Report Summary
Studies on the Mechanism of Formation of mRNA 5'-Terminal Cap Structure and its Function
| Project/Area Number |
63480498
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
代謝生物化学
|
|---|
| Research Institution | The University of Tokyo |
|---|
Principal Investigator |
MIZUMOTO Kiyohisa The Institute of Med. Sci., Associate Prof., 医科学研究所, 助教授 (80092344)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TANAKA Michiko The Inst. of Med. Sci., Research Assistant, 医科学研究所, 教務職員 (60114550)
SUGANO Junjo The Inst. of Med. Sci., Research Associate, 医科学研究所, 助手 (80192756)
|
|---|
| Project Period (FY) |
1988 – 1990
|
| Keywords | Cap Structure / mRNA Capping Enzyme / mRNA Guanylyltransferase / Enzyme-GMP Covalent Intermediate / Active Center / Yeast / Transcription Initiation Complex / Sendai Virus |
|---|
| Research Abstract |
At least four enzymatic activities are required for the synthesis of the 5'-terminal cap structure in eukaryotic mRNA. mRNA capping enzyme which catalyzes the formation of a blocked structure, GpppN, consists of two enzyme activities, the mRNA guanylyltransferase (GTase) and the RNA 5'-triphosphatase (Tpase). To elucidate the molecular mechanism of mRNA capping, we studied the structure and the function of capping enzyme from various sources. The relationship between capping and transcription was also studied.
1. The animal capping enzyme consists of a single polypeptide chain with an Mr of approximately 70,000, while the yeast enzyme is composed of two separate chains, alpha (52 KDa, GTase) and beta (80 KDa, TPase). Cap formation catalyzed by yeast alpha subunit proceeds through an enzyme-GMP covalent reaction intermediate. The GMP in the intermediate was found to be bound to a lysine residue of the enzyme.
2. The gene encoding the alpha subunit (CEG1) was isolated by screening a yeast genomic expression library with antibodies against yeast capping enzyme. The gene is present as one copy in chromosome 7 and is essential for the growth of yeast.
3. CEG1 was expressed in E. Coli and the recombinant alpha chain was highly purified. It catalyzed cap formation as well as the enzyme-GMP complex formation in the absence of the beta chain.
4. The amino acid sequence of the alpha chain active site, including the GMP binding site was determined using the recombinant enzyme.
5. From studies using an in vitro transcription system derived from HeLa cells, we demonstrated that capping enzyme is specifically associated with the initiation complex of RNA polymerase II.
6. To investigate the mechanism of mRNA capping in Sendai virus (a negative strand RNA virus), we developed an efficient and faithful in vitro transcription system with purified virion. We found a unique mechanism of cap formation (GDP transfer mechanism) which is different from the cellular one (GMP transfer mechanism).
|
