Genomic Analysis of Human and Mouse Guanine-7-Methyltransferase with Active Site Characterization

The 5' end of eukaryotic and viral mRNAs contain a "cap" structure with the sequence m7G(5')pppN(5'). The methylation of the 7-position on the guanine cap is very important to proper mRNA processing and initiation of translation. The enzyme responsible for this methylation, RNA guanine-7-methyltransferase, has been cloned and studied from a number of different species, including human, X. laevis, yeast, and C. elegans. The sequences for mouse guanine-7-methyltransferase cDNA and protein have been deduced based upon identity of mouse ESTs to the cDNA of the human enzyme. The deduced mouse cDNA encodes an ORF of 465 amino acids and is 76.4% identical to the human enzyme, or 86.5% within the C-terminal domain. Active site characterization of mouse and human guanine-7-methyltransferase indicates a cysteine residue is important to proper enzyme activity. Enzyme activity was completely eliminated when N-ethylmaleimide (NEM) was added to the assay mixture. When the product of the reaction, S-adenosyl-L-homocysteine (SAH), was added at a concentration of 40uM the mouse enzyme retained 60% activity while enzyme isolated from Human Osteosarcoma (HOS) cells retained 100% of the original activity. SAH demonstrated no protective effects on the cloned human enzyme. Factors that affect binding of RNA to the active site were also investigated. UV-cross-linking of RNA to the active site of the mouse enzyme was inhibited 35% by NEM. Cap analog, GpppG, at a concentration of 1mM, inhibited cross-linking, but the similar nucleotide GMP, at a concentration of 1mM, did not inhibit cross-linking. These analyses have given a clearer understanding of this very important enzyme.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Biochemistry

Department dc:contributor.department

Biochemistry

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2001

Author dc:creator

• Bautz, David James

Chair dc:contributor.committeechair

• Sitz, Thomas O.

Committee members dc:contributor.committeemember

• Gillaspy, Glenda E.
• Luckhart, Shirley

• Genomics
• Cap Structure
• NEM
• Methylation

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

etd-06012001-143530

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/33369