Enzymic Aminoacylation of tRNA Acceptor Stem Helixes with Cysteine Is Dependent on a Single Nucleotide

Abstract

The discriminator base U73 at the acceptor terminus of Escherichia coli tRNA(Cys) is a determinant for the specific aminoacylation of this tRNA by the cognate cysteine tRNA synthetase. Substitution of U73 has a major deleterious effect on the catalytic efficiency of aminoacylation. Here, we show that an RNA hairpin minihelix and an RNA hairpin microhelix that recreate, respectively, the 12-base pair acceptor-T psi C stem and the 7-base pair acceptor helix of E. coli tRNA(Cys) were aminoacylated with cysteine. As in tRNA(Cys), alteration of U73 to A73, C73, or G73 in the cysteine mini- and microhelices eliminated aminoacylation. This established that the strong influence of U73 on aminoacylation is fully retained from the full-length tRNA(Cys) to the mini- and microhelixCys. Transfer of U73 to the noncognate minihelixAla conferred cysteine acceptance to the latter, despite the presence of the major determinant for alanine tRNA synthetase. Even minihelixGly, which shares U73 with minihelixCys, was an efficient substrate for aminoacylation with cysteine. Conversely, as long as U73 was present in minihelixCys, introduction of the glycine or alanine determinant could not block charging by cysteine tRNA synthetase. Although the catalytic efficiency of aminoacylation of these small RNA helices with cysteine was reduced by orders of magnitude from that of tRNA(Cys), the single nucleotide U73 determines the ability of these RNA helices to be aminoacylated with cysteine. These results demonstrated a dominant role of U73 for aminoacylation of small RNA helices by cysteine tRNA synthetase.

Recognition of functional groups in an RNA helix by a class I tRNA synthetase

“…A: Sequence and cloverleaf structure of E. coli tRNA Cys , where U73 adjacent to the CCA end is boxed+ B: Sequence of the synthetic wild-type RNA helix, where U73 is boxed+ Variants of the helix containing modifications of U73 were tested for aminoacylation with cysteine+ C: The structures of U (uridine) and its pyrimidine analogs, including C (cytidine), m 5 isoC (5-methyl-isocytidine), dT (deoxy-thymidine), s 4 U (4-thio-uridine), and s 2 U (2-thio-uridine)+ RNA helices containing an analog of U73 that have a k cat /K m value of aminoacylation within 10-fold of that of the wild type are denoted as active (ϩ), whereas the C73 helix with a k cat /K m 10 5 -fold below of that of the wild type is inactive (Ϫ)+ tions+ Based on the Lineweaver-Burk plot (Fig+ 2A), the k cat was 1+81 ϫ 10 Ϫ4 s Ϫ1 , which is reduced from that of the full-length transcript of E. coli tRNA Cys (1+27 s Ϫ1 ) by about 10 4 -fold+ The K m was 160 mM, which is higher than the K m of the full-length tRNA (1+28 mM) by about 10 2 -fold (Table 1)+ Both the k cat and K m values were similar to those of our previous report (Hamann & Hou, 1995)+ Together, the k cat /K m of aminoacylation of the microhelix is reduced from that of the full-length tRNA by six orders of magnitude+…”

“…Kinetics of aminoacylation of cysteine was determined under previously established steady-state conditions (Hamann & Hou, 1995)+ The wild-type transcript was assayed in the range of 0+3-10 mM and with 0+8 nM of E. coli cysteine-tRNA synthetase+ The C73 transcript was assayed in the range of 5-50 mM and with 0+5 mM of the cysteine enzyme+ The wildtype helix and the m 5 isoC73 variant were assayed in the range of 20-300 mM with 3 mM of the cysteine enzyme+ For all of these RNA substrates, the reported K m values were in the same range of substrate concentrations that were tested+ This gave credence to the accuracy of the K m values+ However, the s 4 U73 variant was assayed in the range of 50-400 mM with 4+2 mM of the cysteine enzyme+ As the reported K m (1,300 mM) was outside the range of substrate concentrations, it was an estimation+ All other microhelix variants were assayed at 30 mM with 3 mM of the cysteine enzyme and the initial rates of their aminoacylation reactions were used to estimate k cat /K m + The percentage of charging for each substrate (in Table 1) was determined by aminoacylation with substrate levels of cysteine-tRNA synthetase over a 30-min time course+ The plateau of aminoacylation indicated the amount of the substrate that was functional+ This value was then compared to the standard of 1,600 pmol/OD (based on a 100% functional substrate) to determine the percentage of charging+ In this study, all of the RNA substrates had been gel purified and ethanol precipitated+ The percentage of charging for the transcripts of the wild type and the C73 variant that were synthesized by T7 RNA polymerase was estimated to be 30%, whereas that for minihelices that were chemically synthesized was estimated to be 60-80%+…”

“…Chemical synthesis of RNA containing the m 5 isoC, dU, and dT modifications was achieved at the Nucleic Acid Facility of the University of Pennsylvania+ The dU and dT phosphoramidites were purchased from Glen Research+ The m 5 isoC phosphoramidite was synthesized as described (Strobel et al+, 1994)+ Synthesis of s 4 U and s 2 U phosphoramidites was as described (Kumar & Davis, 1995, 1997)+ All RNAs were examined by HPLC analysis and were purified by electrophoresis through a 12% polyacrylamide/7 M urea gel as described (Hamann & Hou, 1995)+ The composition of the s4U-and s2U-containing RNA helices was also verified by MALDI/MS analysis at the University of Utah+…”

“…RNA helices that recapitulate sequences of the tRNA acceptor stem, including the 39 NCCA nucleotides, can be substrates for aminoacyl-tRNA synthetases (Frugier et al+, 1994;Hamann & Hou, 1995;Martinis & Schimmel, 1995;Quinn et al+, 1995)+ Although the catalytic efficiency of aminoacylation of RNA helices is reduced from that of the full-length parent tRNA, the specificity is maintained+ The specific aminoacylation lies in the ability of aminoacyl-tRNA synthetases to recognize functional groups within the RNA helices+ Analysis of tRNA-synthetase structures has suggested a general principle (Rould et al+, 1989;Ruff et al+, 1991;Arnez & Moras, 1997)+ The class I synthetases, which attach an amino acid initially to the 29-OH of the terminal ribose, approach the acceptor and NCCA end from the minor groove side+ The class II synthetases, which attach an amino acid to the terminal 39-OH, approach from the major groove side (Arnez & Moras, 1997)+ The classspecific approach leads to tRNA-synthetase complexes that are near mirror images of each other and provides a structural rationale for the stereochemistries of aminoacylation+ We report here the identification of a functional group in the acceptor end of Escherichia coli tRNA Cys that is important for the class I cysteine-tRNA synthetase+ This functional group makes one of the largest energetic contributions to aminoacylation+ However, it is located on the major groove side of the acceptor stem+ Kinetic analysis of the contribution of this functional group to aminoacylation suggests new features that are not anticipated from the class-specific approach of synthetases+ The acceptor stem of E. coli tRNA Cys (Fig+ 1A) is a substrate for aminoacylation (Hamann & Hou, 1995)+ For example, an RNA microhelix that contains the acceptor stem, the UCCA end, and the UUCG tetra-loop (Fig+ 1B) is specifically aminoacylated with cysteine+ An RNA minihelix (not shown) that extends the microhelix by including the T⌿C stem is also specifically aminoacylated with cysteine, with a catalytic efficiency (k cat /K m ) of aminoacylation similar to that of the microhelix+ In these RNA helices, the determinant for aminoacylation is U73 (Hamann & Hou, 1995)+ Substitution of U73 with A73, C73, or G73 completely eliminates aminoacylation+ Further, transfer of U73 to RNA helices of a different specificity confers on the latter the ability to accept cysteine+ For example, introduction of U73 to the RNA helix of tRNA Ala enables aminoacylation with cysteine, even though the major determinant for aminoacylation with alanine (G3:U73) is still present in the acceptor stem+ In the transplanted helix, the k cat /K m value of aminoacylation with cysteine is virtually identical to that of the helix of tRNA Cys , whereas k cat /K m of aminoacylation with alanine is reduced by 30-fold (Hamann & Hou, 1995)+ The dominant role of U73 in aminoacylation with cysteine is also observed in the full-length tRNA Cys + In E. coli tRNA Cys , U73 is the most important nucleotide for aminoacylation, and it accounts for 7+1 kcal/mol of the free energy change of activation (Komatsoul...…”

Unusual synthesis by the Escherichia coli CCA-adding enzyme

“…Minihelices were synthesized by the Nucleic Acid Facility of the University of Pennsylvania+ The substrates Cys-35C and Cys-35A were synthesized by the Biotechnology Resources Facility (Howard Hughes Medical Institute Biopolymer Keck Foundation) at the Yale University School of Medicine+ Minihelices of the correct size were separated from impurities by a 15% polyacrylamide gel in 7 M urea, extracted from gel materials, and eluted in 0+125 M ammonium acetate, 0+125 mM EDTA, and 0+025% SDS at room temperature overnight+ The eluted minihelices were further purified by a C18 reversephase Sep-pak column (Waters) and resuspended in an appropriate concentration (Hamann & Hou, 1995)+ Minihelices (8 mM of gel purified materials) were labeled at the 59 end by T4 polynucleotide kinase (5 U, New England Biolabs) and g-32 P-ATP (3,000 Ci/mmol, NEN) in a 10-mL reaction at 37 8C for 15 min+ After heat denaturation of the kinase enzyme, the labeled minihelices were separated from free ATP by a Centrispin-20 column (Princeton Separation) and used directly+…”