histidyl-tRNA Synthetase Gene Research Articles

Mutant tRNA His ineffective in repression and lacking two pseudouridine modifications

TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons1–4. Histidyl-tRNAHis has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNAHis as the wild type4 and in the one example sequenced, contains tRNAHIS with a structure identical to that of the wild type8. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNAHISin vitro2. The amounts of charged and uncharged tRNAHis present in vivo during physiological derepression of the wild type and in the six classes of regulatory mutants, have been determined9. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNAHisin vivo and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNAHis. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNAHis) and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNAHis which these mutants contain.

Histidine regulation in Salmonella typhimurium: XI. The percentage of transfer RNA His charged in vivo and its relation to the repression of the histidine operon

The cellular concentration of charged transfer RNAHis is shown to be directly correlated with the repression of the histidine operon in Salmonella typhimurium. This conclusion is based on determinations of the amounts of charged and total tRNAHis, tRNAVal and tRNAArg present in vivo in exponentially growing cells of the wild type and of the six classes of regulatory mutants of the histidine biosynthetic operon. The derepression observed in the regulatory mutants hisS (histidyl-tRNA synthetase gene), hisR (tRNAHis structural gene), and hisU and hisW (genes presumably involved in tRNA maturation) may be explained by the lesser amount of charged tRNAHis which these mutants contain. However, mutants in the hisT gene (coding for a dispensable tRNA modification enzyme: uridine to pseudouridine) contain wild-type levels of charged and total tRNAHis, yet are derepressed. HisT tRNAHis then, is a tRNA functional in the charging reaction and protein synthesis, but defective in repression. Mutants in hisO (the operator gene), as expected, contain wild-type levels of charged tRNAHis. Exponentially growing cells of wild type bacteria have about 77% of their tRNAHis charged and 23% uncharged. Adding external histidine increases the percentage of charged tRNAHis to 88%, which is consistent with the known increase in the pool of histidine in the cell and the Km of the histidyl-tRNA synthetase for histidine. Bacteria derepressed physiologically, due to a partial defect in histidine biosynthesis, contain 12% charged and 88% uncharged tRNAHis. Addition of histidine or triazolealanine (a histidine analogue effective in causing repression) to this strain restores charging to the wild-type level. It is shown that a modification of the Folk & Berg (1970) method for the determination of the tRNA charging in vivo gives precise and accurate results.

Histidine regulatory mutants in Salmonella typhimurium: II. Histidine regulatory mutants having altered histidyl-tRNA synthetase

1. (1) The hisS gene of Salmonella typhimurium is the structural gene for histidyl-tRNA synthetase. Mutants in the hisS gene have an altered histidyl-tRNA synthetase and constitute one of the four classes of histidine regulatory mutants. 2. (2) Mutant hisS1520 has a synthetase with a 50-fold decreased affinity for histidine. This mutant has a markedly decreased growth rate on minimal medium and is de-repressed for the biosynthetic enzymes of the histidine operon. Both of these properties appear to reflect the decreased amount of charged histidine-specific tRNA due to the mutant synthetase. The amount of histidyl-tRNA can be increased in this mutant by swelling its internal pool of histidine, and this swelling results in restoration of the normal growth rate and repression of the operon. These results support the idea that histidyl-tRNA is more directly connected with the repression of the histidine operon than histidine itself. 3. (3) Levels of the histidyl-tRNA synthetase are unaffected by de-repression of the histidine operon.