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. 1994 Nov 25;22(23):5031–5037. doi: 10.1093/nar/22.23.5031

Histidylation by yeast HisRS of tRNA or tRNA-like structure relies on residues -1 and 73 but is dependent on the RNA context.

J Rudinger 1, C Florentz 1, R Giegé 1
PMCID: PMC523774  PMID: 7800496

Abstract

Residue G-1 and discriminator base C73 are the major histidine identity elements in prokaryotes. Here we evaluate the importance of these two nucleotides in yeast histidine aminoacylation identity. Deletion of G-1 in yeast tRNA(His) transcript leads to a drastic loss of histidylation specificity (about 500-fold). Mutation of discriminator base A73, common to all yeast tRNA(His) species, into G73 has a more moderate but still significant effect with a 22-fold decrease in histidylation specificity. Changes at position 36 in the anticodon loop has negligible effect on histidylation. The role of residues -1 and 73 for specific aminoacylation by yeast HisRS was further investigated by studying the histidylation capacities of seven minihelices derived from the Turnip Yellow Mosaic Virus tRNA-like structure. Changes in the nature of nucleotides -1 and 73 modulate this activity but do not suppress it. The optimal mini-substrate for HisRS presents a G.A mismatch at the position equivalent to residues G-1.A73 in yeast tRNA(His), confirms the importance of this structural feature in yeast histidine identity. The fact that the minisubstrates contain a pseudoknot in which position -1 is mimicked by an internal nucleotide from the pseudoknot highlights further the necessity of a stacking interaction of this position over the amino acid accepting branch of the tRNA during the aminoacylation process. Individual transplantation of G-1 or A73 into yeast tRNA(Asp) transcript improves the histidylation efficiency of the engineered tRNA(Asp). However, a tRNA(Asp) transcript presenting simultaneously both residues G-1 and A73 becomes a less good substrate for HisRS, suggesting the importance of the structural context and/or the presence of antideterminants for an optimal expression of these two identity elements.

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Selected References

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