An unusual RNA tertiary interaction has a role for the specific aminoacylation of a transfer RNA.

Abstract

The nucleotides in a tRNA that specifically interact with the cognate aminoacyl-tRNA synthetase have been found largely located in the helical stems, the anticodon, or the discriminator base, where they vary from one tRNA to another. The conserved and semiconserved nucleotides that are responsible for the tRNA tertiary structure have been shown to have little role in synthetase recognition. Here we report that aminoacylation of Escherchia coli tRNACYS depends on the anticodon, the discriminator base, and a tertiary interaction between the semiconserved nucleotides at positions 15 and 48. While all other tRNAs contain a purine at position 15 and a complementary pyrimidine at position 48 that establish the tertiary interaction known as the Levitt pair, E. coli tRNACYS has guanosine -15 and -48. Replacement of guanosine -15 or -48 with cytidine virtually eliminates aminoacylation. Structural analyses with chemical probes suggest that guanosine -15 and -48 interact through hydrogen bonds between the exocyclic N-2 and ring N-3 to stabilize the joining of the two long helical stems of the tRNA. This tertiary interaction is different from the traditional base pairing scheme in the Levitt pair, where hydrogen bonds would form between N-1 and 0-6. Our results provide evidence for a role of RNA tertiary structure in synthetase recognition.All transfer RNAs (tRNAs) fold into a cloverleaf structure that consists of four double-helical stems and four singlestranded regions known as the dihydrouridine (D), anticodon, extra (or variable), and T'IC loops, where T is pseudouridine. Within this cloverleaf, a set of conserved and semiconserved nucleotides establish a network of tertiary interactions that fold the cloverleaf into an "L"-shaped tertiary structure. In this structure (Fig. 1), the amino acid acceptor stem stacks directly on the TTC stem to form one arm of the L structure, while the D stem stacks on the anticodon stem to form the other arm of the L. The two arms are thenjoined by tertiary interactions between the D and the TPC loops and between the D and variable loops so that the 3' CCA sequence and the anticodon are placed at the opposite ends of the L structure (1, 2). The two ends are separated by about 75 A. Various locations scattered along the inside ofthe L tertiary structure have recently been implicated as sites for tRNA recognition by aminoacyl-tRNA synthetases (3-6), which are the group of enzymes that catalyze the specific attachment of an amino acid to the CCA end.The most frequently observed interactions between a tRNA and its cognate synthetase are with the anticodon, the acceptor helix, and the discriminator base (nucleotide 73) adjacent to the CCA end (3-6). In Escherichia coli, the alanyl-and histidinyl-tRNA synthetases primarily recognize determinants in the acceptor helix, while the methionyl-, isoleucyl-, and valyl-tRNA synthetases depend largely on the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertis...

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

“…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 ...…”

“…Aminoacylation reactions were carried out in 50 mM HEPES, pH 7+5, 30 mM KCl, 20 mM MgCl 2 , 2+5 mM ATP, and 77 mM L-U-14 C-aspartic acid (213 mCi/mmol, Amersham, UK) at 37 8C with tRNA transcripts or ligated tRNAs between 0+16 and 6+4 mM and a fixed synthetase concentration of 0+0062 units in a total volume of 60 ml as described in Sampson and Uhlenbeck (1989)+ Kinetic parameters, as the average of at least two independent experiments, were derived from LineweaverBurk or Michaelis-Menten plots using KaleidaGraph software+ Deviation was 620%+ K cat /K m values for ligated tRNAs were determined either from Lineweaver-Burk plots or from plots of rate-versus-tRNA concentration (Segel, 1975;Hou et al+, 1993)+…”

An engineered class I transfer RNA with a class II tertiary fold

“…The severe impairment of tRNA QSer_C9/A13 in glutaminylation suggests an important role for a presumed 9-13-22 base triple in recognition of tRNA QSer by GlnRS+ In tRNA Ser , this base triple is composed of the three purines G9-G13-A22, whereas in the class I tRNA Gln a The five most common nucleotide combinations for each structural element are shown+ Data are compiled for all nonmitochondrial elongator tRNAs+ The total number of sequences analyzed is 1,385 for class I and 353 for class II+ b Secondary structures for two class I tRNAs are ambiguous such that the assignment of nucleotide 9 is not included+ Class II selenocysteine tRNAs possess additional nucleotides between the acceptor and D-stems; for these species, the identity of the nucleotide directly 39 to position 8 is compiled+ c This number includes six selenocysteine tRNAs and five tRNAs of other specificity (Fig+ 5)+ d The nomenclature for D-loop structure follows Giege et al+ (1993), in which a and b represent the number of nucleotides present 59 and 39 to the conserved guanosines at positions 18 and 19+ Nucleotides 13 and 22 are considered to be part of the D-stem, regardless of whether they form a Watson-Crick pair+ the triple at the analogous position is A45-A13-A22 (Fig+ 1)+ To evaluate whether purines are needed at these positions to maintain structure, we again examined the tRNA sequence database (Sprinzl et al+, 1998; Table 2)+ This analysis shows that of 353 class II sequences, cytidine is present at position 9 in only five tRNAs and the C9-A13-A22 triple in tRNA QSer_C9/A13 is present in only three+ There are also only eleven examples of uracil at position 9 in class II tRNAs+ These sequences were examined to assess whether a pyrimidine at position 9 in class II tRNAs might require a compensating structural feature elsewhere in the molecule (Fig+ 5)+ The alignments show that nearly all such class II tRNAs possess at least one unpaired nucleotide 39 to the variable stem, adjacent to the augmented D-stem (nucleotide C48 is paired with G15 in the Levitt interaction; Fig+ 1)+ Interestingly, this nucleotide is conserved as a uracil+ The only exceptions to this are several of the very unusual selenocysteine species' tRNA Sec , which possess unpaired nucleotides 59 to the The correlation of Py9 with unpaired nucleotides adjacent to C48 provides further insight into the requirements for folding in class II tRNAs+ It appears that a purine at position 9 may be crucial for adequate stacking on the base at position 21, which is also very highly conserved as a purine (Fig+ 1, Table 2)+ Thus, a pyrimidine at position 9 may destabilize the structure owing to a decreased favorable free energy of base stacking+ Alternatively, it is possible that Py9 may form fewer or weaker hydrogen bonds within a 9-13-22 base triple than could be formed by a purine+ Because tRNA QSer_C9/A13 retains a large D-loop, it is not immediately evident how additional nucleotides, located between U47q and C48 (Fig+ 1), might help reconstitute the structure+ One plausible hypothesis is that these class II tRNAs possess a class I-like 9-12-23 triple instead of the class II 9-13-22 triple+ Further rearrangements would then be required, to permit one or more of the unpaired nucleotides to stack inside the core region, stabilizing the fold+ Model building and energy minimization should be helpful in evaluating the possibilities+ Such studies might be helpful in providing a hypothesis for why the unpaired base is conserved as a uracil+…”

“…In tRNA QSer_C9/A13 , a pyrimidine at position 9 without the additional unpaired variable loop nucleotide produces a very low proportion of functional transcripts after refolding in the presence of Mg 2ϩ (Table 1)+ The stability of the molecule (Fig+ 3) suggests that it may fold into an alternative stable conformation+ No global alteration to the fold is suggested, however, from a secondary structure prediction algorithm (data not shown), so that the differences from native tRNA may be fairly localized+ GlnRS interacts directly with the 59 side of the D-stem and adjacent D-loop nucleotides but not with discriminating groups on the bases of C9 or A13+ Thus, the very strong effect on k cat is likely manifested via an indirect effect on the enzyme-RNA backbone contacts, ultimately resulting in mispositioning or destabilizing the tRNA acceptor end in the active site+ The k cat effect is unusual because the mutation of nucleotides involved in tRNA architecture often results instead in significant increases in K m (Perret et al+, 1992;Puglisi et al+, 1993;Rogers & Soll, 1993;Frugier et al+, 1994)+ Strong effects on k cat are normally associated with the mutation of identity nucleotides in direct contact with the enzyme (Giege et al+, 1993), although an extreme disruption of the yeast tRNA Asp D-loop/T-loop contacts did substantially lower k cat as well (Puglisi et al+, 1993)+…”

“…The tertiary core regions of a number of class I and class II tRNAs are also implicated as specificity determinants for aminoacyl-tRNA synthetases+ Direct contact with the extra arm of tRNA Ser (Asahara et al+, 1994;Biou et al+, 1994) provides recognition in this class II tRNA+ In class I tRNAs, the variable pocket nucleotides 16,17,20,59, and 60 at the junction of the D and T-loops form a recognition site for E. coli tRNA Arg and yeast tRNA Phe (McClain & Foss, 1988a;Sampson et al+, 1989)+ Important identity elements are also known in other parts of the core region, for the class I species E. coli tRNA Phe , tRNA Glu , tRNA Ile , and tRNA Cys (McClain & Foss, 1988b;Peterson & Uhlenbeck, 1992;Hou et al+, 1993;Nureki et al+, 1994;Sekine et al+, 1996)+ One identity switch involving introduction of a long variable arm into a class I tRNA has been reported (Achsel & Gross, 1993)+ In this study, identity determinants of human tRNA Ser were inserted into human tRNA Val , and the requirements for serylation and maturation evaluated+ In addition to the large variable arm, 13 further nucleotides were necessary to confer efficient serylation capacity on the chimeric molecule+ Ten of these nucleotides, located in the D and T stems, appear important for maintaining the tertiary fold as assessed by efficiency of pre-tRNA processing in in vitro extracts+ Valylation was abolished upon introduction of the variable arm, and its possible reconstitution with further nucleotide substitutions was not studied+ Thus, this work establishes a precedent for introduction of a naturally-occurring large variable loop into a class I tRNA, but does not separate nucleotides required for stable folding from those specifying aminoacylation identity (identity elements of human tRNA Val are unknown)+ An important test of our understanding of the nucleotides specifying class I versus class II tRNA folds is the interconversion of these frameworks while retaining function+ To address this we have chosen the E. coli glutaminyl-tRNA synthetase (GlnRS)-tRNA Gln complex as a model system+ The structure of this synthetasetRNA complex is determined at a resolution of 2+5 Å (Rould et al+, 1989(Rould et al+, , 1991, with all nucleotides of the tRNA core region well resolved+ Identity determinants for tRNA Gln reside primarily in the acceptor end and anticodon loop (Jahn et al+, 1991;Hayase et al+, 1992;Ibba et al+, 1996;Sherman et al+, 1996), and interactions of GlnRS with the globular hinge occur only with the D-stem, D-stem/anticodon stem junction, and 59 D-loop nucleotides+ Integrity of the D-stem, as assessed by the introductio...…”