title: The temperature sensitivity of a mutation in the essential tRNA modification enzyme tRNA methyltransferase D (TrmD).
abstract: Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m(1)G37 on the 3' side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: Isao
authors.initials: I
authors.lastname: Masuda
authors.forename: Reiko
authors.initials: R
authors.lastname: Sakaguchi
authors.forename: Cuiping
authors.initials: C
authors.lastname: Liu
authors.forename: Howard
authors.initials: H
authors.lastname: Gamper
authors.forename: Ya-Ming
authors.initials: YM
authors.lastname: Hou
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D011489:Protein Denaturation; D012343:RNA, Transfer / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D012343:RNA, Transfer; C489297:TrmD protein, E coli; D012359:tRNA Methyltransferases
keywords: Bacterial Metabolism; Methyl Transfer; Protein Folding; RNA Methyltransferase; RNA Modification; Transfer RNA (tRNA); Trefoil Knot Fold; TrmD; m1G37-tRNA Modification