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Cloning and characterization of two human cDNAs encoding the mRNA capping enzyme
T Tsukamoto1, Y Shibagaki, T Murakoshi
1Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
Biochemical and Biophysical Research Communications
|February 25, 1998
Summary
Human mRNA capping enzymes hCAP1a and hCAP1b were identified, revealing distinct functional domains. hCAP1a possesses both RNA 5'-triphosphatase and mRNA guanylyl-transferase activities, unlike hCAP1b which lacks guanylyl-transferase function.
Area of Science:
- Molecular Biology
- Enzymology
Background:
- Mammalian mRNA capping enzyme is bifunctional (RNA 5 -triphosphatase and mRNA guanylyl-transferase).
- Yeast capping enzyme activities are separated into two subunits (alpha and beta).
Purpose of the Study:
- Compare structural and functional relationships between mammalian and yeast capping enzymes.
- Isolate and characterize human mRNA capping enzymes.
Main Methods:
- Isolation of two human cDNAs (hCAP1a and hCAP1b).
- Sequence comparison with known capping enzymes.
- Purification and activity assays of recombinant hCAP1a.
- Deletion mutant analysis of hCAP1a.
Main Results:
- hCAP1a (597 aa) and hCAP1b (541 aa) identified, differing in C-terminal regions.
- hCAP1a exhibited both RNA 5 -triphosphatase and mRNA guanylyl-transferase activities.
- N-terminal fragment of hCAP1a (213 aa) showed RNA 5 -triphosphatase activity.
- C-terminal fragment of hCAP1a (369 aa) showed mRNA guanylyl-transferase activity.
- hCAP1b showed RNA 5 -triphosphatase activity but lacked guanylyl-transferase activity.
Conclusions:
- hCAP1a is a functional human mRNA capping enzyme with distinct catalytic domains.
- hCAP1b is a variant lacking mRNA guanylyl-transferase activity.
- Structural comparison highlights conserved regions in guanylyl-transferases, with a notable absence in hCAP1b.