No abstract

The genetic code is based on aminoacylation reactions where specific amino acids are attached to tRNAs bearing anticodon trinucleotides. However, the anticodonindependent specific aminoacylation of RNA minihelix substrates by bacterial and yeast tRNA synthetases suggested an operational RNA code for amino acids whereby specific RNA sequences/structures in tRNA acceptor stems correspond to specific amino acids. Because of the possible significance of the operational RNA code for the development of the genetic code, we investigated aminoacylation of synthetic RNA minihelices with a human enzyme to understand the sequences needed for that aminoacylation compared with those needed for a microbial system. We show here that the species-specific aminoacylation of glycine tRNAs is recapitulated by a speciesspecific aminoacylation of minihelices. Although the mammalian and Escherichia coli minihelices differ at 6 of 12 base pairs, two of the three nucleotides essential for aminoacylation by the E. coli enzyme are conserved in the mammalian minihelix. The two conserved nucleotides were shown to be also important for aminoacylation of the mammalian minihelix by the human enzyme. A simple interchange of the differing nucleotide enabled the human enzyme to now charge the bacterial substrate and not the mammalian minihelix. Conversely, this interchange made the bacterial enzyme specific for the mammalian substrate. Thus, the positional locations (if not the actual nucleotides) for the operational RNA code for glycine appear conserved from bacteria to mammals.The 20 synthetases are divided into two classes (I and II) of 10 enzymes each (1, 2). Enzymes of each class are approximately comprised of two major domains, where each of the two tRNA domains interacts with a distinct domain of the cognate tRNA synthetase (3-5). The tRNA acceptor-TTC helix ("minihelix" domain) interacts with the class-defining catalytic domain (and insertions into that domain), and segments outside of the acceptor-TTC stem-loop, such as the anticodon, interact with a second, highly variable synthetase domain that is joined to the catalytic core. While many synthetases make contact with their tRNA anticodons, specific mutations in the acceptor stems of their tRNAs severely reduce aminoacylation efficiency (6-8) and, even for these "anticodon" examples, RNA oligonucleotides with sequences based on acceptor stems alone are also aminoacylated with their cognate amino acids (9-11). The operational RNA code based on acceptor stems may have predated the genetic code and was possibly incorporated into or combined with the genetic code when the two domains of tRNAs were assembled into a single molecule (4).Escherichia coli glycyl-tRNA synthetase has an a212 quaternary structure, with a 303-amino acid a chain and 689-amino acid 13 subunit (12, 13). In contrast, the human enzyme is an a2 dimer of 739-amino acid polypeptides (14, 15), similarThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be her...

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.