Position of aminoacylation of individual Escherichia coli and yeast tRNAs.

Abstract

Transfer RNAs terminating in 2'-or 3'-deoxyadenosine were prepared from unfractionated E. coil and yeast (Saccharomyces cerevisiae) tRNAs and purified to remove unmodified tRNAs. The modified tRNA species were assayed for aminoacylation with each of the 20 amino acids to determine the initial position of tRNA aminoacylation. The E. coli and yeast aminoacyl-tRNA synthetases specific for arginine, isoleucine, leucine, methionine, phenylalanine, and valine, as well as the E. coil glutamyl-tRNA synthetase, aminoacylated only those cognate tRNAs terminating in 3'-deoxyadenosine (i.e., those having a 2'-OH group). On the other hand, those E. coil and yeast synthetases specific for alanine, glycine, histidine, lysine, proline, serine, and threonine, as well as the yeast synthetase specific for glutamine, utilized exclusively those tRNAs having an available 3'-OH group on the 3'-terminal nucleoside, while the E. coil and yeast synthetases specific for asparagine, cysteine, and tyrosine, and the yeast aspartyl-tRNA synthetase, utilized both of the modified cognate tRNAs. The only observed difference in specificity between the E. coli and yeast systems was for tRNATrP, which was aminoacylated on the 2'-position in E. coli and the 3'-position in yeast. The results indicate that the initial position of aminoacylation is not uniform for all tRNAs, although for individual tRNAs the specificity has been conserved during the evolution from a prokaryotic to eukaryotic organism. Although aminoacyl-tRNA undoubtedly exists in solution as a rapidly equilibrating mixture of the 2'-and 3'-O-aminoacyl species, a description of the initial position of aminoacylation of individual tRNAs by their cognate aminoacyl-tRNA synthetases is of interest as part of a complete description of the precise mechanism of peptide bond formation. Several reports have described the preparation of modified tRNAs terminating in 2'-and 3'-deoxyadenosine and 2'-and 3'-Omethyladenosine and the utilization of those tRNAs in certain of the partial reactions of protein biosynthesis (1-4). Substrate activity of the modified tRNAs in phenylalanine acceptor assays was limited to those species terminating in 3'-deoxyadenosine and 3'-O-methyladenosine; the latter was shown to be aminoacylated poorly relative to unmodified tRNA, also indicating an apparent steric requirement in the aminoacylation reaction. More recently a report has appeared describing the substrate activity of a number of Escherichia coli tRNAs terminating in 2'-and 3'-deoxyadenosine (5) and some of the conclusions were supported by a related study utilizing tRNAs terminating in 2'(3')-amino-2'(3')-deoxyadenosine (6).Abbreviations: tRNA-C-COH, tRNA missing the 3'-terminal adenosine moiety; DBAE-cellulose; N-[N'-(m-dihydroxyborylphenyl)succinamyllaminoethyl-cellulose; Ax unit, the amount of material giving an A of 1.0 at wavelength X when dissolved in 1 ml of solution and measured in a cell with a 1 cm light path; nucleoside Q, 7-(4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanosine...

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

“…Arg-371 and Arg-731 are too far from each other to simultaneously interact with the G3⅐U70 base pair. Our model in mode 2 is compatible with the fact that E. coli AlaRS aminoacylates the 3Ј-OH of A76 (40).…”

Functional group recognition at the aminoacylation and editing sites of E. coli valyl-tRNA synthetase

“…Val terminating with 3Ј-deoxyadenosine can be fully aminoacylated, as expected, because ValRS is known to esterify the 2Ј-hydroxyl group (Hecht and Chinault 1976). This tRNA Val variant, however, is charged at only 17% the rate of wild-type tRNA Val (Fig.…”

“…2). ValRS initially esterifies valine onto the 2Ј-hydroxyl group of the 3Ј-terminal ribose (Hecht and Chinault 1976). As expected, at high concentrations of ValRS (1-2 µM), 3Ј-deoxyadenosine-substituted tRNA…”

An asymmetric underlying rule in the assignment of codons: Possible clue to a quick early evolution of the genetic code via successive binary choices

“…Also, extra care must be taken for those tRNAs whose aminoacylation has been shown to occur on both 29OH and 39OH. This concerns tRNA Tyr and tRNA Cys (Sprinzl and Cramer 1975;Hecht and Chinault 1976). In this respect, it is interesting to note that Tyr and Phe and Cys are considered to be late amino acids (Brooks and Fresco 2002).…”

“…This could be the mark of a recent assignment, or even a codon swapping phenomenon (Szathmary 1991). On the other hand, tRNA Cys , whose primary site of aminoacylation was also measured to be both 29OH and 39OH (Sprinzl and Cramer 1975;Hecht and Chinault 1976), has been shown to be recognized by CysRS (a Class I aaRS) in a purely Class I mode by crystallographic studies of the CysRS-tRNA Cys complex (Hauenstein et al 2004). Also, more recent detailed kinetic studies showed that k cat (29OH) > >k cat (39OH) by a factor of about 20 for CysRS (Shitivelband and Hou 2005).…”