2004 Fiscal Year Final Research Report Summary
Involvement of mRNA capping enzyme in transcription initiation
| Project/Area Number |
14580630
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Structural biochemistry
|
|---|
| Research Institution | Kitasato University |
|---|
Principal Investigator |
SHIBAGAKI Yoshio Kitasato University, School of pharmaceutical Sciences, Lecturer, 薬学部, 講師 (90235565)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
HISATAKE Koji Saitama Medical School, Assistant Professor, 医学部, 助教授 (70271236)
MIZUMOTO Kiyohisa Kitasato University, School of pharmaceutical Sciences, Professor, 薬学部, 教授 (80092344)
|
|---|
| Project Period (FY) |
2002 – 2004
|
| Keywords | Capping enzyme / C-terminal domain / RNA polymerase II / in vitro transcription / Transcription factor / Protein phosphorylation |
|---|
| Research Abstract |
The mRNA capping occurs soon after transcription initiation and before other processing events. The several mRNA processing factors are coupled to transcription through binding to the C-terminal domain of RNA polymerase II (CTD). mRNA-capping enzyme directly binds to the phosphorylated CTD, and catalyzes the co-transcriptional cap formation. However, the precise mechanism of the involvement of capping enzyme in pol II transcription has been unclear.
(1) To examine the involvement of capping enzymes in pol II transcription, we developed in vitro transcription/capping system using immobilized DNA template. Immobilized templates were incubated with HeLa nuclear extract to form initiation complex (PICs) on promoter region. After washing to remove unbound proteins, PICs were subjected to elongation reaction in the presence of 3'-O-methly CTP in order to stall elongation complex at the first C residue. Using this system, we found that capping occurs in 18-19 nt RNA length in the case of adenovirus major late promoter.
(2) To investigate the functional interaction of capping enzyme and pol II, we subjected highly purified calf thymus pol II to capping assay with various kinds of CTD kinases. Capped RNAs were separated by Boronate-affinity PAGE (BAGE) to evaluate capping efficiency. We found that non-phosphorylated pol II stimulated capping activity about 2.5-fold. Furthermore, CAK phosphorylated pol II increased in capping activity about 1.8-fold compared with non-phosphorylated pol II. These results suggest that the CAK phosphorylation of pol II CTD is important for activation of capping enzyme and other portion of pol II also may involve in this activation.
|
-
-
-
-
-
-
[Journal Article] The role of mRNA capping enzyme in pol II transcription initiation2003
Author(s)
Shibagaki, Y., Nojima, T., Matsui, H., Hisatake, K., Fukuda, A., Mizumoto, K.
-
Journal Title
The New Frontier of RNA Science RNA 2003 Kyoto Abstract
Pages: 175
Description
「研究成果報告書概要(欧文)」より
-
-
[Journal Article] Involvement of mRNA capping enzyme in pol II initiation complex2002
Author(s)
Shibagaki, Y., Hisatake, K., Fukuda, A., Fukamachi, N., Tsukamoto, T., Mizumoto, K.
-
Journal Title
Description
「研究成果報告書概要(欧文)」より
