Aminoacylation of Synthetic DNAs Corresponding to Escherichia coli Phenylalanine and Lysine tRNAs

Abstract

Synthetic DNA oligomers (tDNAs) corresponding to Escherichia coli tRNA(Phe) or tRNA(Lys) have been synthesized with either deoxythymidine (dT) or deoxyuridine (dU) substituted in the positions occupied by ribouridine or its derivatives. The tDNAs inhibited the aminoacylation of their respective tRNAs with their cognate amino acids, but not the aminoacylation of tRNA(Leu) with Leu. In the presence of aminoacyl-tRNA synthetase, species of both a tDNA(Phe) synthesized with a 3' terminal riboadenosine and a tDNA(Lys) containing only deoxynucleotides could be aminoacylated with the appropriate amino acids, although the Michaelis constant Km and observed maximal rate Vmax values for aminoacylation were increased by three- to fourfold and decreased by two- to threefold, respectively. The aminoacylation of synthetic tDNAs demonstrates that the ribose backbone of a tRNA is not absolutely required for tRNA aminoacylation.

Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study

“…Nucleotide bases have been recognized as identity elements in interaction of tRNAs with synthetases (Normanly & Abelson, 1989;Söll & RajBhandary, 1995)+ 29-Hydroxyl groups have also been shown to play a role in this process+ These experiments were based on a complete exchange of all 29-deoxy groups in the molecule (Khan & Roe, 1988) or just one of the four nucleotides (Aphasizhev et al+, 1997)+ Additionally, kinetic analysis of single 29-deoxy-modified positions in minihelices or tRNA fragments of tRNA Ala or tRNA Pro showed small but distinct effects in aminoacylation efficiency (Musier-Forsyth & Schimmel, 1992;Yap & Musier-Forsyth, 1995)+ We were interested in an in vitro scanning procedure which should rapidly identify such 29-deoxy-sensitive sites in a complete tRNA rather than testing individual positions in a stepwise fashion+ The E. coli tRNA Asp system was chosen because it offered the opportunity to compare the results with an X-ray structural model still under final refinement (L+ Moulinier & D+ Moras, pers+ comm+)+ Although the simplest method for scanning a tRNA for the importance of 29-hydroxyl groups for aminoacylation should be the random incorporation of 29-deoxynucleotides, the identification of the interfering positions by limited alkaline hydrolysis proved impractical in our system because of high background of cleavage, even though this procedure had been applied to a 15-nt-long tRNA anticodon stem-loop fragment to test ribosomal A site binding (von Ahsen et al+, 1997)+ We therefore chose to couple the 29-deoxy modification with a phosphorothioate modification that generates a signal only at the deoxy positions upon iodine cleavage (Gish & Eckstein, 1988) and thus facilitates the 29-deoxy identification+ As any interference observed with this double modification could result from the deoxy and/or the phosphorothioate substitutions, the effect of the phosphorothioate modification was determined separately by transcribing the E. coli tRNA Asp with dNTPaS or NTPaS+ Both pools were then charged with the cognate synthetase and a tRNAs were ligated using one unmodified half and one half containing the deoxynucleotide at the identified position as described in Materials and Methods+ Each measurement was repeated at least twice+ Error Յ 20%+ (Gish & Eckstein, 1988;Schatz et al+, 1991;Verma & Eckstein, 1998)+ Because the incorporation of the analogues is random, this approach does not rely on any assumption about which positions might be important and thus differs from the previous studies+ In the scanning experiments positions U11, A24, U25, C28, U29, C36, C48, G57, A58, and C67 showed only a deoxy effect+ All others were accompanied by considerable phosphorothioate interference+ At G34, C74, and C75 the deoxy effect on top of the phosphorothioate interference was considered strong enough to be indicative of an additional effect+ The only position where the phosphorothioate effect was stronger than the deoxy effect was U35, the central position of the anticodon+ However, in general, comparison between these two effects is made difficult by the fact that the amount of dNTPaS and NTPaS incorporation is not necessarily the same+ The assignment of deoxy effects was therefore conservative+…”

“…Only the influence of 29-deoxy groups was further characterized kinetically with chemically synthesized halves, ligated enzymatically to full tRNAs containing single 29-deoxy groups at the positions suggested by the interference assay (Table 1)+ Inhibition was observed for all but the dU29-containing tRNA, which showed a slight increase of k cat /K m relative to the allribo ligated tRNA+ This remains unexplained+ The extent of inhibition was most obvious at A76, the 39 terminal nucleotide where the amino acid will be attached+ Considering that the synthetase is a class II type, attaching the amino acid to the 39 hydroxyl, this finding confers significance to the presence of the 29 hydroxy group (Arnez & Moras, 1997)+ A 5-6-fold decrease is seen, except for U25, for positions C74, C36, and G34 in the acceptor stem and in the anticodon loop, both regions interacting with the synthetase (in the yeast system, Ruff et al+ (1991); in the E. coli system, D+ Moras, pers+ comm+)+ An approximate threefold reduction in charging was seen for C67, A58, and A24+ For the other positions the effect was smaller+ In conclusion, it is observed that single 29-deoxy substitutions had, in general, only a minor effect on charging and the effect is well below the losses in aminoacylation efficiency found for base changes of the discriminator nucleotides (Giegé et al+, 1993)+ This observation agrees with earlier results with E. coli tRNA Ala and E. coli tRNA Pro , which, however, had been analyzed using minihelices or tRNAs assembled from fragments (Musier-Forsyth & Schimmel, 1992;Liu & Musier-Forsyth, 1994;Yap & MusierForsyth, 1995)+ In contrast, multiple occurrences of 29-deoxy modifications in at least one half of E. coli tRNA Asp resulted in a dramatic decrease of charging, with the exception of the dA series+ The half harboring the more important interference positions showed the stronger inhibition+ This cumulative effect of 29-deoxy groups has been observed for other tRNAs or tRNA fragments (Khan & Roe, 1988;Perreault et al+, 1989;Musier-Forsyth & Schimmel, 1992;Yap & Musier-Forsyth, 1995; Aphasizhev et al+, 1997)+ An explanation for these observations might be that the presence of several 29-deoxy functions causes a conformational effect incompatible with productive interaction with the enzyme+ A difference in conformation for the yeast tRNA Asp containing any one of the four nucleotides entirely as the 29-deoxy derivative has indeed been verified experimentally (Aphasizhev et al+, 1997)+ The loss of charging activity found by these authors after complete replacement of uridines or guanosines by their 29-deoxy analogues was attributed to a specific effect of U11 and G27 on the basis of the X-ray structure of the complex (Cavarelli et al+, 1993)+ This is an unusually strong effect caused by one position+ It was not found in the present analysis for position 27 and only to a smaller extent at position 11+ A functional difference from the otherwise related yeast system is one explanation+ However, the involvement of several more deoxy positions contributing ...…”

“…The importance of specific 29-hydroxyl groups of an RNA in the interaction with proteins has been investigated for RNA binding to a phage coat protein (Baidya & Uhlenbeck, 1995), for a human double-stranded RNA-binding protein (Bevilacqua & Cech, 1996), for the function of the yeast spliceosome , and HIV regulatory proteins (Iwai et al+, 1992), as well as for recognition of tRNA by aminoacyl-tRNA synthetases+ The first example for the latter was the synthesis of Escherichia coli tRNA Phe and tRNA Lys completely consisting of deoxynucleotides, except for the 39-terminal adenosine (Khan & Roe, 1988)+ These tDNAs required pH 5+5 and the presence of 20% DMSO to restore the acceptor function+ Poor activity was also reported for the deoxy equivalent of E. coli tRNA fMet which was chargeable to 3% compared to the native tRNA (Perreault et al+, 1989)+ RNA-DNA heteroduplexes corresponding to the acceptor stem of tRNA minihelices of E. coli tRNA Ala were also tested for aminoacylation (Musier-Forsyth et al+, 1991)+ The 39-RNA/59-DNA heteroduplex was aminoacylated, but not the 39-DNA/59-RNA heteroduplex+ Studies with oligonucleotide duplexes with single 29-deoxy substitutions, corresponding to the tRNA acceptor stem, identified hydroxyls at G4, U70, C71, and C75 as essential for efficient charging (Musier-Forsyth & Schimmel, 1992)+ The importance of 29-OH groups for the aminoacylation of E. coli tRNA Pro has been investigated with chemically synthesized 59 or 39 oligonucleotides of up to 18 nt in length that were annealed to the corresponding 39-or 59-3/4 tRNA fragment (Liu & Musier-Forsyth, 1994;Yap & Musier-Forsyth, 1995)+ The majority of the single de-oxynucleotide substitutions had only a minor effect on aminoacylation, except U8 which was reduced 22-fold+ These studies indicated that single deoxy substitutions had in most cases only small effects+ Multiple substitutions, however, showed more dramatic effects+ In a recent report yeast tRNA Asp transcripts were enzymatically prepared in which any one of the four nucleotides was replaced by its 29-deoxy derivative (Aphasizhev et al+, 1997)+ Those transcripts containing deoxyuridines or deoxyguanosines were not chargeable+…”

Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain

“…Observations that some tDNAs can be charged with amino acid by aminoacyl-tRNA synthetases (Khan & Roe, 1988;Perreault et al, 1989) and that the tertiary structure of tDNA greatly resembles that of tRNA (Paquette et al, 1990) were equally unexpected. On the basis of these admittedly limited studies, it would seem that both deoxy-and ribonucleotides can adopt a similar conformation (2'-endo, 3'-endo, or variants thereof) depending on the context.…”

Leucyl-tRNA Synthetase from the Hyperthermophilic Bacterium Aquifex aeolicus Recognizes Minihelices

“…DNA analogues serve as active substrates for RNase P or tRNA synthetases (26,27). Moreover, the tDNA minihelix is recognized by a CCA-adding enzyme (28).…”