| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
|
|---|
| Research Abstract |
The mRNA cap structure is synthesized by a series of reactions catalyzed by mRNA capping enzyme (RNA 5'-triphosphatase and mRNA guanylyltransferase) and mRNA (guanine-7-)methyltransferase (mRNA cap methyltransferase). Here we isolated Saccharomyces cerevisiae RNA 5'-triphosphatase (capping enzyme beta subunit) gene (CET1), two human capping enzyme cDNAs (hCAP1a, b), and three human mRNA (guanine-7-)methyltransferase cDNAs (hCMT1a, b, c). CET1 encodes 549 amino acids with a calculated Mr of 61, 849 and bears no significant sequence similarities to RNA 5'-triphosphatase of human capping enzyme (hCAP1) including the tyrosine specific protein phosphatase (PTP) active site motif. Gene disruption experiment showed that CET1 is essential for yeast cell growth. hCAP1a and hCAP1b encode 597 and 541 amino acids, respectively, and are different only at the region coding for the C-terminal portion of the enzyme. The regions conserved among mRNA guanylytransferases are observed in hCAP1 except that
… More
one conserved region was absent in the hCAP1b protein. Deletion mutant analysis of hCAP1a showed that the N-terminal 213 amino acid fragment containing PTP motif catalyzed the RNA 5'-trihosphatase activity and the C-terminal 369 amino acid fragment contained the mRNA guanylyltransferase activity. hCAP1b showed RNA 5'-triphosphatase activity, but neither enzyme-GMP covalent complex formation or cap structure formation was detected. hCMT1a and hCMT1b encode 476 and 504 amino acids, respectively, and differ only at the region coding for the C-terminal portion of the enzyme after amino acid residue 465. RT-PCR showed that all 3 types of mRNAs were expressed in every tissue examined. Comparison of the deduced amino acid sequences with those of other viral and cellular enzymes showed the regions which are highly conserved among mRNA (guanine-7-)methyltransferases. Interactions of hCAP1a, hCMT1a and RNA polymerase II were analyzed using recombinant proeins expressed in bacteria and location indispensable for their interactions were clearly shown. Less
|
