The Escherichia coli argU10 (Ts) Phenotype Is Caused by a Reduction in the Cellular Level of the argU tRNA for the Rare Codons AGA and AGG

Abstract

The Escherichia coli argU10(Ts) mutation in the argU gene, encoding the minor tRNA Arg species for the rare codons AGA and AGG, causes pleiotropic defects, including growth inhibition at high temperatures, as well as the Pin phenotype at 30°C. In the present study, we first showed that the codon selectivity and the arginineaccepting activity of the argU tRNA are both essential for complementing the temperature-sensitive growth, indicating that this defect is caused at the level of translation. An in vitro analysis of the effects of the argU10(Ts) mutation on tRNA functions revealed that the affinity with elongation factor Tu-GTP of the argU10(Ts) mutant tRNA is impaired at 30 and 43°C, and this defect is more serious at the higher temperature. The arginine acceptance is also impaired significantly but to similar extents at the two temperatures. An in vivo analysis of aminoacylation levels showed that 30% of the argU10(Ts) tRNA molecules in the mutant cells are actually deacylated at 30°C, while most of the argU tRNA molecules in the wild-type cells are aminoacylated. Furthermore, the cellular level of this mutant tRNA is one-tenth that of the wild-type argU tRNA. At 43°C, the cellular level of the argU10(Ts) tRNA is further reduced to a trace amount, while neither the cellular abundance nor the aminoacylation level of the wild-type argU tRNA changes. We concluded that the phenotypic properties of the argU10(Ts) mutant result from these reduced intracellular levels of the tRNA, which are probably caused by the defective interactions with elongation factor Tu and arginyl-tRNA synthetase.

Functional defects in transfer RNAs lead to the accumulation of ribosomal RNA precursors

“…It is possible that the increased accumulation of rRNA precursors, due either to tRNA defects or to antibiotic treatment, has a common basis, which we speculate as the reduced production of one or more factors that have a role in ribosome biogenesis. In an analogous situation, a ts mutation in argU, the gene encoding the rare tRNA Arg4 , was reported to cause DNA replication defects, probably because the mutant strain is unable to sufficiently produce one or more factors that play a role in replication (Henson et al 1979;Garcia et al 1986;Sakamoto et al 2004).…”

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

“…This could affect the formation of the ternary complex (EF-Tu · GTP · aa-tRNA) required for tRNA selection to the A-site codon. 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).…”

“…Observed frameshifting occurred when the arginyl-tRNA Arg mnm 5 UCU levels were decreased only by twofold. We assume that reduction down to "trace" levels of arginylated tRNA Arg mnm 5 UCU , as in the argU10(Ts) mutant (Sakamoto et al 2004), will elicit extensive frameshifting, at least at -GAA-AGA-sites. The peptidyl-tRNA Glu mnm 5 s 2 UUC , which frameshifts at the -GAA-AGA-site, "takes off" from the GAA codon and lands at the AA-A codon.…”

“…The argU mutants described in this paper did not induce any cell division defects at high temperature (data not shown). The argU10(Ts) mutant having only 5% of the wild-type amount of the arginyl-tRNA Arg mnm 5 UCU at permissive temperature has no growth defects then, but stops dividing at high temperature, when the amount of tRNA Arg mnm 5 UCU is reduced to trace level (Sakamoto et al 2004). Among the mutants described in this paper, the most affected mutant [argU2343 (G53A)] has 21% of its arginyltRNA Arg mnm 5 UCU left.…”

“…Note, however, that monitoring TCA-precipitable material does not reveal changes in the frequency of frameshifting. The argU10(Ts) mutant has a severely decreased amount of tRNA Arg mnm 5 UCU , a reduced level of arginylation, and impaired binding of this tRNA to EF-Tu · GTP (Sakamoto et al 2004). Therefore, it was inferred that decoding of the cognate AGA codon in this mutant is inefficient and inhibits the synthesis of specific protein(s) containing AGA codons in their mRNA.…”