PubMed · 4996579
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A Peterkofsky, M Litwack, J Marmor. 1971. Modified bases and transfer RNA function.. https://pubmed.ncbi.nlm.nih.gov/4996579/
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Escherichia coli tRNACys contains an unusual G15.G48 tertiary base pair that is important for recognition and aminoacylation by cysteine tRNA synthetase. This G15.G48 tertiary base pair has a distinctive chemical modification signature that suggests an N2.N3 base pairing. The N2.N3 pairing of a G.G base pair has not been described in any existing RNA structures. Identification of the structural determinant of G15.G48 is of fundamental importance for understanding the formation of an RNA tertiary base pair, as well as the role of RNA tertiary structure in tRNA recognition. We show here that the structural determinant for G15.G48 is an A13.A22 mismatch in the dihydrouridine stem. Introduction of A13.A22 to an unrelated tRNA confers the distinctive chemical modification signature of G15. G48 while substitution of A13.A22 eliminates this signature. The relationship between G15.G48 and A13.A22 enables the unrelated tRNA to be efficiently recognized by cysteine tRNA synthetase. Modeling studies show that A13.A22 has the potential to form a base triple with A46, which is directly connected to G48 in the G15.G48 base pair. The proposed A13.A22.A46 base triple provides a framework for understanding how two RNA structural elements may be related to each other in playing an important role in tRNA aminoacylation.
In spite of variations in the sequences of tRNAs, the genetic code (anticodon trinucleotides) is conserved in evolution. However, non-anticodon nucleotides which are species specific are known to prevent a given tRNA from functioning in all organisms. Conversely, species-specific tRNA contact residues in synthetases should also prevent cross-species acylation in a predictable way. To address this question, we investigated the relatively small tyrosine tRNA synthetase where contacts of Escherichia coli tRNA(Tyr) with the alpha2 dimeric protein have been localized by others to four specific sequence clusters on the three-dimensional structure of the Bacillus stearothermophilus enzyme. We used specific functional tests with a previously not-sequenced and not-characterized Mycobacterium tuberculosis enzyme and showed that it demonstrates species-specific aminoacylation in vivo and in vitro. The specificity observed fits exactly with the presence of the clusters characteristic of those established as important for recognition of E. coli tRNA. Conversely, we noted that a recent analysis of the tyrosine enzyme from the eukaryote pathogen Pneumocystis carinii showed just the opposite species specificity of tRNA recognition. According to our alignments, the sequences of the clusters diverge substantially from those seen with the M. tuberculosis, B. stearothermophilus and other enzymes. Thus, the presence or absence of species-specific residues in tRNA synthetases correlates in both directions with cross-species aminoacylation phenotypes, without reference to the associated tRNA sequences. We suggest that this kind of analysis can identify those synthetase-tRNA covariations which are needed to preserve the genetic code. These co-variations might be exploited to develop novel antibiotics against pathogens such as M. tuberculosis and P. carinii.
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