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.
Highlights
Introduction of the tsS88L-trmD Mutation to E. coli—The S88L-TrmD mutation was originally isolated from chemical mutagenesis of S. typhimurium (9)
Structural Mapping of ts Mutations of trmD—A genetic study led by Björk and Nilsson (9) isolated a group of ts mutations in S. typhimurium TrmD (StTrmD) that conferred altered thiamine metabolism at a restrictive temperature (P58L/L94F, S88L, G117S, G117N, G117Q, S165L, P184L, G199R, G214D, W217D, and E243K)
The two groups of mutants showed overlapping amino acid substitutions at the protein level and collectively occupied 12 positions in the StTrmD enzyme structure. Because these mutations were isolated before the structure of TrmD was available, nothing was known about their structural context. We mapped these mutations onto the crystal structure of E. coli TrmD (EcTrmD) in complex with AdoHcy (11), which was a logical model based on the over 92% homology in the primary sequence between the S. typhimurium and E. coli enzymes
Summary
Introduction of the tsS88L-trmD Mutation to E. coli—The S88L-TrmD mutation was originally isolated from chemical mutagenesis of S. typhimurium (9). Because the mutation occurred at a highly conserved position in Gram-negative TrmD enzymes (Fig. 2A), we tested its broader significance by introducing it to E. coli This was achieved by replacing the chromosomal wt-trmD locus in E. coli with a plasmid carrying a cassette of the S88LtrmD gene linked to the Kan resistance marker (S88L-trmDKan), using the Red approach (16), such that the reading frame of trmD was preserved. It is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility 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. 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
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
