A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80.

Abstract

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non‐permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non‐permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.

A mutant cysteinyl-tRNA synthetase affecting timing of chromosomal replication initiation in B. subtilis and conferring resistance to a protein kinase C inhibitor.

“…This might involve, for example, reduced levels of an active (phosphorylated) form of a repressor or increased activity (dephosphorylated) of a positive factor required for initiation. The precise nature and possible role of P70 relative to normal regulation of the timing of initiation is now being investigated, including the relationship between CysRS and P70. This proposal that CysRS is implicated in the control of chromosomal initiation may be extended to a more general hypothesis where different elements of the translation machinery, like aminoacyl-tRNA synthetases or tRNAs (Garcia et al, 1986;Chen et al, 1991; Bouquin,N., Chen,M.X., Kim,S., Bernard,S., Holland,I.B. and Seror,S.J., Mol.…”

Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp

“…2). Plasmid pDV1 (rplL rpoB) suppressed ftsRl thermosensitivity on LBo medium at 42°C ( (9,21), ribosomal proteins (26,35), or elongation factor Tu (42). In some cases, it has been proposed that the filamentation is due to impaired translation of specific mRNA species coding for cell division proteins (9,21).…”

A reduced level of charged tRNAArgmnm5UCU triggers the wild-type peptidyl-tRNA to frameshift

“…The G1-to-A1 change in the acceptor-stem of the same tRNA Arg mnm 5 UCU was demonstrated to impair the EF-Tu · GTP binding (Sakamoto et al 2004). The earlier mentioned G1A mutant in tRNA Arg mnm 5 UCU [argU10(Ts)] and some other tRNA mutants, such as the divE having an A10G change in the major tRNA Ser cmo 5 UGA and feeB having a C77A change in the tRNA Leu U*AG , have one common phenotype-they all induce a cell division defect (Tamura et al 1984;Garcia et al 1986;Chen et al 1991). When the argU10(Ts) mutant is shifted to nonpermissive temperature, it ceases to grow in ∼1 h, although the rate of total protein synthesis, measured as TCA-precipitable material, is reduced only to two-thirds of the wild-type rate and continues for at least 2 h. In contrast, the synthesis of the cI repressor containing five AGA codons read by tRNA Arg mnm 5 UCU is severely reduced.…”