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Characterization of mutant N², N²-dimethylguanosine-specific tRNA methyltransferases from Saccharomyces cerevisiae

Title:

Characterization of mutant N², N²-dimethylguanosine-specific tRNA methyltransferases from Saccharomyces cerevisiae

Reuben, Melanie (1997) Characterization of mutant N², N²-dimethylguanosine-specific tRNA methyltransferases from Saccharomyces cerevisiae. Masters thesis, Concordia University.

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Abstract

$N\sp2,N\sp2$-dimethylguanosine-specific tRNA methyltransferase (m$\sbsp{2}{2}$Gtase) is an enzyme which uses S-adenosylmethionine as a substrate to catalyze the transfer of two methyl groups to the 2-amino group of guanosine at position 26 in tRNA. Neither the tertiary structure nor the location of the active sites are known for m$\sbsp{2}{2}$Gtase. In an attempt to characterize this enzyme, the trm1 gene from the Saccharomyces cerevisiae strain SN1015-2a, which lacks functional m$\sbsp{2}{2}$Gtase, was clone and sequenced. Based on the wild-type TRM1 sequence, oligonucleotides were designed and the mutant trm1 gene was isolated from SN1015-2a genomic DNA by polymerase chain reaction (PCR). Sequence comparison to the wildtype TRM1 gene revealed 14 silent point mutations and 4 amino acid substitutions that are common to two PCR products: $\rm Gly\sp3\to Ser,\ Thr\sp⁾\to Ser,\ Ser\spk\to Leu,$ and Gly$\spڿڭto$ Arg. Sub-cloning and site-directed mutagenesis were employed and the single amino acid substitution, Ser$\spk\to$ Leu, was identified as the mutation responsible for inactivating m$\sbsp{2}{2}$Gtase. To elucidate the function of Ser$\spk,$ new mutant enzymes with Thr, Ala and Cys at this position were created. Kinetic studies yielded apparent K$\rm\sb{M}$ and V$\rm\sb{max}$ values that were similar to those of the wild-type m$\sbsp{2}{2}$Gtase demonstrating that Ser at position 467 is not required for enzyme activity.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Reuben, Melanie
Pagination:x, 99 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Theses (M.Sc.)
Program:Dept. of Chemistry and Biochemistry
Date:1997
Thesis Supervisor(s):Joyce, Paul B. M
ID Code:245
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 13:10
Last Modified:08 Dec 2010 10:13
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