Alternative design of a tRNA core for aminoacylation1

doi:10.1006/jmbi.2000.4169 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 303, 503±514

Alternative Design of a tRNA Core for Aminoacylation Thomas Christian, Richard S. A. Lipman, Caryn Evilia and Ya-Ming Hou* Department of Biochemistry and Molecular Pharmacology Thomas Jefferson University 233 South 10th Street, BLSB 222, Philadelphia PA 19107, USA

The core of Escherichia coli tRNACys is important for aminoacylation of the tRNA by cysteine-tRNA synthetase. This core differs from the common tRNA core by having a G15:G48, rather than a G15:C48 base-pair. Substitution of G15:G48 with G15:C48 decreases the catalytic ef®ciency of aminoacylation by two orders of magnitude. This indicates that the design of the core is not compatible with G15:C48. However, the core of E. coli tRNAGln, which contains G15:C48, is functional for cysteine-tRNA synthetase. Here, guided by the core of E. coli tRNAGln, we sought to test and identify alternative functional design of the tRNACys core that contains G15:C48. Although analysis of the crystal structure of tRNACys and tRNAGln implicated long-range tertiary base-pairs above and below G15:G48 as important for a functional core, we showed that this was not the case. The replacement of tertiary interactions involving 9, 21, and 59 in tRNACys with those in tRNAGln did not construct a functional core that contained G15:C48. In contrast, substitution of nucleotides in the variable loop adjacent to 48 of the 15:48 base-pair created functional cores. Modeling studies of a functional core suggests that the re-constructed core arose from enhanced stacking interactions that compensated for the disruption caused by the G15:C48 base-pair. The repacked tRNA core displayed features that were distinct from those of the wild-type and provided evidence that stacking interactions are alternative means than long-range tertiary base-pairs to a functional core for aminoacylation. # 2000 Academic Press

*Corresponding author

Keywords: aminoacyl-tRNA synthetases; RNA tertiary base-pairs; RNA stacking interactions; the Levitt base-pair

Introduction RNA molecules can form complex and highly speci®c tertiary structures that are the basis for a wide range of important RNA-RNA and RNA-protein interactions. These structures are primarily de®ned by two elements. One is a network of long-range tertiary hydrogen bonds (H bonds) that are established between bases or between bases and the sugar-phosphate backbone. The other is a set of stacking interactions where bases lie ¯at upon one another. A speci®c RNA function arises from a speci®c interplay of these two elements. However, the question of whether the same function can derive from an alternative interplay has not been addressed. The ability to address this question should shed important light on RNA structures and functions. E-mail address of the corresponding author: [email protected] 0022-2836/00/040503±12 $35.00/0

The core region of transfer RNAs (tRNAs) provides a model system to study the interplay of different RNA structural elements. As shown by crystal structures of natural tRNAs,1 ± 9 the core consists of tertiary H bonding and stacking interactions that are formed by nucleotides of the dihydrouridine (D) stem-loop, the variable (V) loop, and the T C stem-loop. As such, the core connects two functional domains of the L-shaped tRNA structure, one contains the amino acid acceptor site, while the other contains the anticodon triplet. The integrity of the core can be evaluated by its ability to join properly the two domains for recognition by a speci®c aminoacyl-tRNA synthetase. This recognition is the basis for aminoacylation of tRNA as a means to relate amino acids with tRNA anticodon triplets of the genetic code. The core of Escherichia coli tRNACys is attractive for two reasons. First, the crystal structure of this tRNA complexed with the Thermophilus aquaticus elongation factor Tu (EF-Tu) has been solved and # 2000 Academic Press

504 Ê .5 This tRNA, in a ligand-free state, re®ned at 2.6 A has also been modeled based on extensive biochemical studies.10 Although the crystal structure and the model differ in some details in the core, both show features unique to this tRNA that are absent from the common core found in yeast tRNAPhe.3,6 Second, the core of E. coli tRNACys is extremely sensitive to alterations, as it contains an unusual 15:48 base-pair for joining the D and V loops. Instead of the more common G15:C48, this core has G15:G48 (Figure 1)11 and substitution of G15:C48 into tRNACys decreases the catalytic ef®ciency (kcat/Km) of aminoacylation by nearly 100fold.12 In contrast, substitution of G15:C48 in E. coli tRNAAla has little effect on aminoacylation.13 The unusual G15:G48 in the crystal structure of E. coli tRNACys is formed by unusual H bond pairings, where O6 of G15 is paired with N1 and N2 of G48.5 This base-pair is distinct from the G15:C48 base-pair in the crystal structures of other tRNAs, where N1 and N2 of G15 (but not O6) are paired with O2 and N3 of G48, respectively. Above and below G15:G48 are several layers of base-pairs and base-triples which, although also formed with unusual H bond pairings, provide stacking interactions to stabilize G15:G48. The importance of G15:G48 in the cysteine core is likely to modulate the presentation of the cysteine-speci®c U73 and the GCA anticodon to cysteine-tRNA synthetase (Figure 1).14 We and others have shown that, outside of the core, U73 in the acceptor stem and the GCA anticodon are major determinants for aminoacylation.15 ± 17

An Alternative tRNA Core for Aminoacylation

The inability of the cysteine core to accommodate G15:C48 raised the question as to whether an alternative core containing G15:C48 could be identi®ed. If so, the comparison of the alternative core with the wild-type core might shed light onto principles that govern the interplay of structural elements in the cysteine core. To identify an alternative cysteine core, we used the core of E. coli tRNAGln as a framework. We showed recently that although the core of E. coli tRNAGln contains G15:C48, it is functional for cysteine-tRNA synthetase.18 Speci®cally, a mutant of E. coli tRNAGln that maintained the wild-type core but harbored U73 and the GCA anticodon for cysteine is aminoacylated with cysteine. The kcat/Km of aminoacylation of the mutant tRNAGln with cysteine was 0.23 relative to that of the wild-type tRNACys (Figure 1). This is a signi®cant improvement from that of the G15:C48 variant of tRNACys, which retains U73 and the GCA anticodon. The mutant tRNAGln thus provided an example of a tRNA core that accommodates G15:C48 for cysteine-tRNA synthetase. The glutamine core was particularly attractive because the crystal structure of E. coli tRNAGln complexed to its cognate synthetase is available.7 In contrast, although the core of Baccilus subtilis tRNACys also contains G15:C48 and is functional for E. coli cysteine-tRNA synthetase19 (Figure 1), there is no structural information of this core. Analysis of the structure of tRNAGln has revealed features that are distinct from those of tRNACys. In particular, nucleotides at positions 9,

Figure 1. Sequence and cloverleaf secondary structure of E. coli tRNACys, E. coli tRNAGln, and B. subtilis tRNACys. Nucleotides at 9, 15, 21, 48, and 59 are circled to indicate their differences in the core. A mutant of tRNACys that contains G15:C48 has a relative aminoacylation activity 0.01 to that of the wild-type (k ˆ 0.01). In contrast, a mutant of tRNAGln that contains G15:C48 is ef®ciently aminoacylated with cysteine with a relative activity 0.23 to that of tRNACys. The mutant tRNAGln harbors mutations outside of the core, including the U1G, C34G, U35C, G36A, A72C, and G73U substitutions. The B. subtilis tRNACys is ef®ciently aminoacylated by E. coli cysteine-tRNA synthetase (k ˆ 1.6). It contains a functional core, consisting of G15:C48 and U46.

An Alternative tRNA Core for Aminoacylation

21, and 59 that formed long-range tertiary interactions above and below 15:48 are considered important. These long-range tertiary interactions are formed between the D and T C loops. In this study, we tested several long-range tertiary interactions but showed that none could rescue the G15:C48 mutation. Instead, it is the mutations in the V-loop that created an alternative functional core. Structural modeling suggested that while the G15:C48 mutation destabilized the cysteine core, nucleotides immediately adjacent to 48 in the V-loop could be recruited to enhance stacking interactions as an alternative solution to a functional core.

Results Experimental design Inspection of E. coli tRNACys and E. coli tRNAGln showed major differences in the core (Figure 1). Most notably, while tRNACys has G15:G48 and a rare U21, tRNAGln has G15:C48 and the more common A21. The mutant of tRNAGln that was ef®ciently aminoacylated with cysteine contained mutations outside of the core. These mutations were at the distal ends of the tRNA molecule and were not expected to alter the core. For example, mutations U1G, A72C, and G73U were clustered at the acceptor end to place U73 adjacent to a G1:C72

505 base-pair. Recent studies have shown that U73 is most ef®ciently recognized by cysteine-tRNA synthetase when it is in the context of G1:C72 (Lipman and Hou, unpublished results). A second set of mutations occured at the anticodon end, which altered the glutamine anticodon triplet CUG to the cysteine triplet GCA. In the context of all of the mutations, E. coli tRNAGln was aminoacylated with cysteine with a kcat/Km only fourfold below that of tRNACys (0.23). This ef®ciency suggested that the core of tRNAGln was functional for cysteine-tRNA synthetase. In contrast, substitution of G48 with C48 in the core of tRNACys reduced kcat/Km by 100-fold, even with U73 and the GCA anticodon present. Thus, with respect to cysteine-tRNA synthetase, G15:C48 was functional in the glutamine core but was deleterious in the cysteine core. We examined the structure of the core in tRNACys and in tRNAGln to identify tertiary features that differentiated the two (Figure 2).5,7 We rationalized that features unique to tRNACys might have arisen from RNA tertiary packing to accommodate the unusual G15:G48, while those unique to tRNAGln might shed light on solutions to accommodate G15:C48. Two features were noted. First, tRNACys contains a C16:C59 base-pair, which stacks on top of G15:G48. The formation of the 16:59 base-pair might be necessary to stabilize the bulkier G15:G48. In contrast, tRNAGln lacks the 16:59 base-pair, as C16 in this tRNA is ¯ipped out

Figure 2. Comparison of the structure of the core of E. coli tRNACys (left) with that of E. coli tRNAGln (right). The Figure was constructed by superimposing the two tRNAs based on sugar-phosphate backbone atoms at nucleotide positions 15:48, 8:14, 9, 13, and 22, followed by translating the molecules apart while maintaining the same orientation. Structural elements that formed base-pairs or base-triples are color coded.

506

An Alternative tRNA Core for Aminoacylation

mation. This analysis suggested that installment of A59, A21, and C9 into the cysteine core harboring G15:C48 could mimic the glutamine core. The second strategy was to focus on the V-loop that contains nucleotide 48. Although nucleotides in the V-loop are not directly related to 15:48 through long-range tertiary base-pairs, they are immediately adjacent to 48 and are expected to be sensitive to the alteration of G48 to C48 in the cysteine core. Thus, variations in the V-loop might induce re-arrangement of the core to accommodate G15:C48.

to contact glutamine-tRNA synthetase while A59 provides stacking interactions with G15:C48. Second, although both cores stack two layers of base-triples below 15:48, compositions of these triples are different. Speci®cally, tRNACys has the A46:[U8:A14] triple below G15:G48, which is unique to this tRNA and is absent from all other tRNA crystal structures. The recruitment of A46 to pair with U8:A14 might compensate for the ¯ipped-out U21. In contrast, tRNAGln has A21, which stacks in the core and forms the A21:[U8:A14] base-triple that is also found in many other tRNAs. In the next layer of triple, tRNACys has A9:[A13:A22] where A9 is tilted relative to A13 and A22. The 9:[13:22] triple is rare and is only found in one other example, which is the G9:[G13:A22] triple in T. thermoophilus tRNASer.2 In contrast, tRNAGln forms the A45:[A13:A22] triple that is similar in geometry to the 46:[13:22] triple commonly found in tRNAPhe and tRNAAsp.3,6,20 Based on the design of the glutamine core, we developed two strategies to modify the cysteine core. The ®rst was to focus on long-range tertiary base-pairs that were predicted by crystal structures as important for the 15:48 base-pair. These longrange pairs ¯ank the 15:48 base-pair and are formed between the D and T C loops. Speci®cally, the unusual G15:G48 in the cysteine core appeared to be accommodated by a combination of the C16:C59 base-pair, the U21 nucleotide, and the A9:[A13:A22] triple. The glutamine core instead has A59 in the T C loop, A21 in the D loop, and C9 in between the acceptor and D stems. As A59 does not form a base-pair with C16, introduction of A59 to the cysteine core might disrupt the 16:59 interaction. As A21 stacks in the core, introduction of A21 to the cysteine core might prevent the aberrant recruitment of A46 to form the A46:[U8:A14] base-triple. Additionally, as C9 in tRNAGln is positioned to form a 9:[12:23] triple (C9:[C12:G23]), introduction of C9 to the cysteine core might suppress the unusual A9:[A13:A22] triple from for-

Investigation of long-range tertiary base-pairs in the D and T C-loops To the G15:C48 variant of tRNACys, we created mutations A59, A21, and C9 in single or multiple positions and determined if substitutions improved aminoacylation with cysteine. Each mutant was generated from transcription in vitro by T7 RNA polymerase, puri®ed, and re-natured by a heatcool process in the presence of 10 mM Mg2‡. The purity of each tRNA was estimated from the plateau level of aminoacylation assuming homogeneity as 1600 pmole/OD. Based on the plateau, the concentration of each tRNA was then adjusted. Aminoacylation of the wild-type transcript showed a Km of 1.28 mM and a kcat of 1.27 secÿ1 (Table 1). These were similar to the Km of 0.8 mM and kcat of 1.0 secÿ1 of the native tRNACys puri®ed to homogeneity from E. coli (Table 1). This similarity suggested that the tRNA transcript, although lacking natural base modi®cations, is a functional substrate for cysteine-tRNA synthetase. Compared to the wild-type transcript (Cys01, Table 1), the G15:C48 variant of tRNACys (the Cys06 mutant) suffered from a decrease of 100-fold in kcat/Km. This decrease resulted from nearly a tenfold increase in Km and a tenfold decrease in kcat. To measure Km and kcat accurately of the G15:C48 variant, we used elevated concentrations

Table 1. Kinetic parameters of tRNA mutations in the D and T C-loops Wild-type/ mutants a

Native Cys01 (w.t.)b Cys06 Cys13 Cys115 Cys116 Cys117 Cys118 Cys119

15:48

9

21

59

kcat (secÿ1)

Km (mM)

kcat/Km (106 secÿ1 Mÿ1)

k

G:G G:G G:C G:C G:C G:C G:C G:C G:C

A C C C

U A A A A

C A A A

1.00 1.27 0.15 0.15 0.02 0.05 0.14 0.13 0.04

0.84 1.28 15.7 55.7 5.86 2.65 7.00 13.38 2.03

1.19 0.99 0.01 0.0027 0.0034 0.018 0.020 0.0097 0.02

1.19 1.00 0.01 0.0027 0.0034 0.018 0.020 0.01 0.02

All other CysXX tRNAs are variants of the wild-type T7 transcript. Nucleotides of mutants that differ from those of the wild-type at 15:48, 9, 21, and 59 are indicated whereas those that maintain the same as those of the wild-type are indicated by ``-``. Each kinetic parameter Km and kcat was the average of at least two determinations based on the Michaelis-Menten equation and had a standard error of deviation no more than 50 %. The letter k indicates kcat/Km relative to that of Cys01. a Native refers to tRNACys puri®ed from E. coli and contained natural modi®cations. Nucleotides of the wild-type at 15:48, 9, 21, and 59 are shown. b Cys01 (w.t.) tRNA refers to the wild-type T7 transcript of tRNACys.

507

An Alternative tRNA Core for Aminoacylation

of cysteine-tRNA synthetase to assay the tRNA substrate over a range of tRNA concentrations. However, even with elevated cysteine-tRNA synthetase, the concentration of the enzyme was at least 20-fold below the lowest concentration of the tRNA substrate. Under this condition, the enzyme functioned as a catalyst and satis®ed the MichaelisMenten steady-state kinetic mechanism. The principle of using elevated levels of cysteine-tRNA synthetase applied to all other substrates that were defective for aminoacylation. Table 1 shows analysis of tRNA mutations that were introduced to the cysteine core. These mutations were in the D and T C-loops and were designed to recapitulate features of the glutamine core. To the G15:C48 mutant, introduction of C9 alone (Cys115) did not improve the kcat/Km value. Neither did the introduction of A21 alone (Cys13) or A59 alone (Cys117). In fact, the A21 and C9 mutations even decreased kcat/Km further. Also, introduction of double mutations failed to improve kcat/Km. For example, the double mutant containing C9A21 and the one containing A21A59 (Cys116 and Cys118, respectively) maintained a kcat/Km identical to that of the G15:C48 variant. Most importantly, introduction of all three mutations (C9, A21, and A59) also failed to improve kcat/Km. The triple mutant (Cys119) showed a kcat/Km that was only twofold better than the value of the G15:C48 variant. This was still a 50-fold decrease in the catalytic ef®ciency of aminoacylation from that of the wild-type. Thus, although structural analysis implicated A59, A21, and C9 as important for re-designing the cysteine core to be compatible with G15:C48, kinetic analysis did not support the importance. Installation of A59, A21, and C9, either alone or in combination, did not rescue the deleterious G15:C48 mutation in the cysteine core. Examination of the mutants that were tested showed that a combination of defective Km and kcat prevented the improvement of kcat/Km. For example, compared to the parameters of the G15:C48 variant, none of the mutations improved the kcat. In particu-

lar, the C9 mutation (either singly as in Cys115 or in combination as in Cys116 and Cys119) appeared to decrease kcat further. Although the A59 mutation decreased Km by twofold and thus improved kcat/ Km (as in Cys117), this improvement was eliminated in the presence of A21 (as in Cys118) or was compromised by the decrease in kcat in the presence of A21 and C9 (as in Cys119). The single A21 mutation (as in Cys13) increased Km by three- to fourfold and this deleterious effect was also previously observed in the wild-type tRNA.21 However, when A21 was combined with C9 (as in Cys116 or Cys119), the effect on Km was eliminated while the overall kcat/Km was dominated by the decrease in kcat. This analysis indicated that mutations to introduce A59, A21, and C9 affected each other but none of the tested combinations succeeded in restoring the kcat/Km value of the wild-type. Investigation of nucleotides in the V-loop We next investigated nucleotides in the V-loop. To examine the effect of the V-loop alone, separate from that of the long-range tertiary interactions between the D and T C-loops, we replaced the Vloop of tRNACys with that of tRNAGln but kept the wild-type nucleotides at 59, 21, and 9 in the cysteine core. The ®rst mutant (Cys127), which contained G15:C48 and the entire V-loop of tRNAGln, showed a signi®cant improvement of kcat/Km over that of the G15:C48 variant. The improvement suggested that the V-loop would be the basis for exploring further mutations in the cysteine core to identify features that were important to accommodate G15:C48. Table 2 summarizes mutations created in the V-loop and their effect on kinetics of aminoacylation. The Cys127 mutant had a kcat/Km of aminoacylation 26-fold better than that of the G15:C48 variant. This improved kcat/Km to within fourfold of that of the wild-type. Compared to parameters of the G15:C48 variant (Cys06), the improved kcat/Km primarily arose from an enhanced kcat. This enhancement on kcat was unattainable by mutations in the

Table 2. Kinetic parameters of tRNA mutations in the variable loop. Wild-type/ mutants

15:48

44

45

46

Cys01 (w.t.) Cys06 Cys127 Cys128 Cys129 Cys130 Cys131 Cys132

G:G G:C G:C G:C G:C G:C G:C G:C

C C C C C A C C

U U A A U U U A

A A U U U U A C

47

U

U U

48

kcat (secÿ1)

Km (mM)

kcat/Km (106 secÿ1 Mÿ1)

k

G C C C C C C C

1.27 0.15 2.06 2.44 1.51 1.89 0.16 0.18

1.28 15.7 8.01 12.7 11.2 3.47 10.04 3.55

0.99 0.01 0.26 0.19 0.14 0.53 0.02 0.05

1.00 0.01 0.26 0.19 0.14 0.53 0.02 0.05

Sequences of the core at 15:48 and in the V-loop are shown. Cys01 (w.t.) tRNA refers to the wild-type T7 transcript of tRNACys. All other CysXX tRNAs are variants of the wild-type T7 transcript. Each kinetic parameter Km and kcat was the average of at least two determinations based on the Michaelis-Menten equation and had a standard error of deviation no more than 50%. The letter k indicates kcat/Km relative to that of Cys01.

508 D and T C-loops (Table 1) and suggested that further mutations that would improve Km could restore the wild-type activity. We next tested if alteration of the size of the V-loop would improve Km. The V-loop of tRNAGln is larger than that of tRNACys by having an extra U47. Although U47 is directly linked to position 48, we showed that it had little role in aminoacylation. The Cys128 mutant, which maintained the V-loop of tRNAGln but lacked U47, was still active and had a relative kcat/Km (0.19) similar to that of Cys127 (0.26). Conversely, the Cys131 mutant, which maintained the V-loop of tRNACys but had an insertion of U47, remained inactive (relative kcat/Km ˆ 0.02). Thus, deletion of U47 from the V-loop of tRNAGln or insertion of U47 to the V-loop of tRNACys did not affect the activity of aminoacylation. We tested two additional variations in the Vloop to focus on positions 44-46. The Cys129 mutant, which replaced the CAUC sequence in Cys128 with CUUC, did not improve the kinetics of aminoacylation further (relative kcat/Km ˆ 0.14). However, the Cys130 mutant, which harbored the AUUC sequence, improved the relative kcat/Km to 0.53. The improvement came from a reduction of Km, while retaining the kcat of the other mutants with substitutions in the V-loop. Overall, the kcat, Km, and kcat/Km values of Cys130 were within twofold of those of the wild-type. We concluded that variations in the V-loop alone, independent of the D and T C-loops, were suf®cient to rescue the defect of G15:C48 in tRNACys. U46 was an important element in aminoacylation. The Cys129 mutant with a CUUC sequence in the V-loop was active for aminoacylation, whereas the Cys06 mutant with a CUAC sequence was inactive. This suggested that the single replacement of A46 with U46 was responsible for the enhanced activity of Cys129. To further test the signi®cance of U46, we created the mutant Cys132, which contained C46 in the V-loop CACUC sequence. This mutant was to be compared with Cys127, which contained the V-loop CAUUC sequence. Aminoacylation analysis showed that Cys132 was inactive (relative kcat/Km ˆ 0.05), whereas Cys127 was active (relative kcat/Km ˆ 0.26). These results emphasized that it is the identity of U46, but not A46 or C46, which is important for aminoacylation. Analysis of alternative cores by the chemical probe dimethyl sulfate We used the chemical probe dimethyl sulfate (DMS) to determine if the wild-type cysteine core could be distinguished from functional cores that contained G15:C48. The ability to differentiate the wild-type from the variants would provide evidence that they were structurally distinct and that the variants contained an ``alternative'' core. DMS probed the accessibility of N7 of guanine by introducing a methyl group that rendered the base susceptible to cleavage by aniline.22 We previously

An Alternative tRNA Core for Aminoacylation

showed that G15 of the wild-type core (containing G15:G48) was highly sensitive to DMS and that this sensitivity was unique to tRNACys.12,21 In all other tRNAs that have been tested, G15 was protected by stacking interactions and was not sensitive to DMS.23 ± 26 In the G15:C48 mutant of tRNACys (Cys06), however, the sensitivity of G15 to DMS was signi®cantly reduced and this reduction indicated a structural re-arrangement that inactivated the core.12,21,27 We determined the sensitivity of G15 to DMS for the wild-type and several mutants (Figure 3). The tRNA substrates were prepared by T7 transcription, labeled at the 30 end, folded in 10 mM MgCl2, and reacted with DMS under conditions of less than one modi®cation per tRNA molecule. The statistical DMS levels were to prevent multiple modi®cations, which might induce tRNA conformational changes. The DMS modi®cation destabilized the modi®ed base and allowed aniline scission to generate a cleavage product that could be identi®ed by comparison to a size ladder. We generated the size ladder by digesting a full-length tRNA with the nuclease T1, which cleaved at the phosphodiester linkage 30 to G. Because the aniline scission was at the linkage 50 to the site of modi®cation, the scission product (labeled at the 30 end) would be longer than the corresponding T1cleavage product by one nucleotide. For each tRNA, two controls were performed in parallel to identify speci®c signals. One control contained no DMS to identify signals from non-speci®c scission of the tRNA backbone. The other control was the DMS reaction with the tRNA in semi-denaturing conditions (in 1 mM EDTA) to evaluate the reactivity of DMS. By eliminating non-speci®c signals, the intensity of a true signal at G15 was quanti®ed and normalized to that at G34, which is an accessible position at the anticodon.27 The normalization was necessary to minimize sample variations and to provide a de®nitive ``signal'' of modi®cation. Figure 3 shows results of DMS modi®cation and gives the relative signal of each mutant to that of the wild-type. The wild-type tRNACys displayed a DMS signal at G15 as 1.61, which was similar to that previously published.10 The G15:C48 variant displayed a signal of 0.35, which was 0.22 relative to that of the wild-type. Based on our de®nition of tRNA functionality,10 the G15:C48 variant was a non-functional mutant. Speci®cally, we had used the relative DMS signal at G15 as a structural parameter to correlate with the relative kcat/Km as a kinetic parameter. Analysis of these parameters of several mutants of E. coli tRNACys had established a structure-function correlation that separated the active mutants from the inactive. Figure 4 shows that the active mutants clustered in one group and displayed a relative structural parameter r > 0.75 and kinetic parameter k > 0.3, respectively. The inactive mutants clustered separately and displayed a relative structural parameter r < 0.55 and kinetic parameter k < 0.2. Thus, because the

An Alternative tRNA Core for Aminoacylation

509

Figure 3. DMS modi®cation of the wild-type tRNA, the functional Cys130, and the non-functional Cys131 and Cys06. Each tRNA is probed under conditions of the control (C), the native (N), and the semi-denaturing (S) (see Materials and Methods). Bands of modi®cation are identi®ed by comparison to a ladder generated by T1 digestion of the wild-type (the T1 lane) and to another ladder generated by glycine hydrolysis (not shown). The wild-type core differed from the Cys130 core at G15 but shared common features in the rest of the tRNA molecule. G15 in the nonfunctional Cys131 and Cys06 did not react to DMS in semi-denaturing conditions, suggesting that these mutants might have collapsed structures that were resistant to DMS modi®cation. The positions of T1 cleavage at G15, G24, G27, and G34 are shown by arrows. For each tRNA, the ratio of the signal at G15 over that at G34 is measured. The wild-type ratio is normalized as 1.0 (in parentheses) and those of the other mutants relative to that of the wild-type are indicated.

G15:C48 variant had a structural parameter r ˆ 0.22 and a kinetic parameter k ˆ 0.01, it was de®ned as inactive. By de®ning mutants with both structural and kinetic parameters, we could identify those that had recapitulated the structurefunction relationships of the wild-type. The operational de®nition described here had satis®ed all previously analyzed mutations in the core.10,27,28 The most active mutant in Table 2, Cys130, behaved differently than anticipated by the operational de®nition (Figure 4). The r value of Cys130 was 0.28, which indicated a reduced accessibility of G15 to DMS. However, the reduced accessibility did not compromise the k value, which was within twofold of that of the wild-type. Thus, while the core of Cys130 is distinct from that of the wildtype by a different r value, it is nonetheless a functional core. Additional evidence for the core of Cys130 as an alternative to the wild-type core was noted for G18 and G19 in the D loop. These two nucleotides in known tRNA structures formed tertiary H bonds with U55 and U56 respectively and thus were not expected to be accessible to DMS. The wild-type core supported this prediction, where G18 and G19 were inaccessible to DMS. In contrast, the Cys130

core showed accessibility of G18 and G19 to DMS. However, outside of the core, such as the region between G24 and G34, Cys130 displayed a DMS pattern similar to that of the wild-type. This suggested that the structural effect generated by mutations in Cys130 was con®ned to the core. The distinct features of the Cys130 core, such as the reduced accessibility at G15 and the enhanced accessibility at G18 and G19, were similarly observed in the core of the other functional mutant, Cys127, and in the core of tRNAGln (not shown). This suggested that the active mutants contained a core that had become more akin to the glutamine core. The inactive mutant Cys131, which had the insertion of U47 in the V-loop of tRNACys, showed a r value of 0.32 and a k value of 0.02. Although the r parameter of Cys131 was similar to that of the active Cys130, the structure of the core was different. Most evident was the smear banding in Cys131 between G15 and G24, in contrast to the clear banding in the functional mutant and the wild-type. Also noted was the lack of a signal at G27 in contrast to the presence of a signal in the functional mutant and the wild-type. These differ-

510

Figure 4. Correlation of the relative structural parameter with the relative kinetic parameter of several core mutants of E. coli tRNACys (adapted from Figure 5(a) of Reference (10)). This correlation had operationally de®ned functional mutants as those of the relative structural parameter >0.75 and the relative kinetic parameter >0.3, whereas the non-functional mutants as those of the relative structural parameter < 0.6 and the relative kinetic parameter < 0.2. However, this de®nition did not apply to the Cys130 mutant (indicated as such), which had a structural parameter of 0.22 but a kinetic parameter of 0.53.

ences indicate that the inactive Cys131 suffered from additional changes in the core.

Discussion Alternative cysteine cores that contained G15:C48 shared U46 in common We have created alternative functional cores of E. coli tRNACys that contained G15:C48. The core in the functional Cys130 mutant, for example, displayed a pattern of chemical modi®cation quite distinct from that of the wild-type. This provides evidence for an alternative core that confers aminoacylation by cysteine-tRNA synthetase. The mutations in Cys130 and in all other functional mutants are in the V-loop but not in the D or T Cloops. This suggests that nucleotides in the V-loop can respond to the G15:C48 mutation, perhaps better than those in the D or T C-loops that form long-range tertiary H bonds. It is possible that mutations in the D and T C-loops, although designed based on analysis of crystal structures, might severely destabilize the core. Crystal structural analysis of these mutations would be necessary to gain a better insight into their effect on the core. Comparison of functional and non-functional sequences in the V-loop emphasizes the signi®cance of U46 (Table 2). At positions 44-46, the func-

An Alternative tRNA Core for Aminoacylation

tional sequences are CAU, CUU, and AUU, which share U46 in common. The non-functional sequences are CUA and CUC, which differ from the functional CUU by having A46 and C46, respectively. The single change from A46 to U46, for example, is suf®cient to elevate activity from 0.01 to 0.14. This accounts for more than tenfold improvement. Other adjustment in the V-loop, such as the replacement of C44 with A44, then brings the activity to within twofold of that of the wild-type. The ability of U46 to in¯uence kinetic behaviors suggests that it has a role in the structure of the alternative core, either involved in stacking or in tertiary hydrogen interactions. This is unexpected, as uridine bases in RNA loops usually are not critical for RNA structures due to their propensity to ¯ip out from loops. For example, U46 and U47 in tRNAGln are ¯ipped out and have little role in the structure/function of the tRNA V-loop.7 The signi®cance of U46 is also supported by analysis of B. subtilis tRNACys. Although this tRNA contains a G15:C48, it is a functional substrate for E. coli cysteine-tRNA synthetase (relative kcat/Km ˆ 1.6, Figure 1). The V-loop of this tRNA has the UAUC sequence, where U46 is directly linked to C48. This provides a second example of correlation between the presence of U46 and the activity of a core containing G15:C48. B. subtilis tRNACys differs from E. coli tRNACys or tRNAGln by having A9, A21, and A59, suggesting the lack of a correlation between these nucleotides and the aminoacylation activity. Structural modeling of alternative cores To gain insights into the signi®cance of U46 in the alternative core, we performed structural modeling of the most active mutant Cys130. In one modeling study, we used coordinates of the crystal structure of the wild-type tRNACys complexed with EF-Tu as a framework.5 Because the tRNA V-loop does not contact EF-Tu in the crystal structure, we deleted EF-Tu to examine the structure of the core. In a second modeling study, we used coordinates of a modeled structure of the ligandfree tRNA as a framework.10 Within constraints of each structure, we replaced residues in the V-loop of the wild-type with those of Cys130 using the Biopolymer module of Insight II (Molecular Simulations Inc, v98). We then used the Discover module (Insight II) to adjust the structure of the V-loop to satisfy constraints of bond angle and bond length and to achieve energy minimization to arrive at a viable structure. The model built from coordinates of the crystal structure showed a well-stacked U46 in the core. The stacking suggests that U46 participates in the structure of the core and provides a basis to understand how U46 in¯uences kinetic behaviors of tRNA variants. In contrast, the model built from the ligand-free tRNA showed that U46 is not stacked in the core and is less ordered. Figure 5 is

An Alternative tRNA Core for Aminoacylation

511

Figure 5. Structural modeling of the core of the (a) wild-type, (b) the G15:C48 variant, and (c) the Cys130 functional mutant. The sequence in the V-loop of each tRNA is shown below and mutations that differ from the corresponding position in the wild-type are underlined. The structural elements of 15:48, 46:[8:14], 45, and 26:44 are color coded.

based on the model derived from the crystal structure and presents a view that looks down from the top of the core through the helix of the anticodon stem. The wild-type core contains G15:G48 and A46:[U8:A14], a splayed-out U45, and a C44 that interacts with A26 through O2 and N6 (Figure 5(a)). In this core, G48 uses N1 and N2 to form H bonds with O6 of G15. The purine ring of G48 stacks directly on top of A46, which may be important to stabilize the unusual 15:48 base-pair and 46:[8:14] triple. In the G15:C48 variant (Figure 5(b)), however, C48 appears to destabilize the core by two means. First, because C48 cannot provide N1 or N2 as H bond donors, it cannot form the 15:48 base-pair as in the wild-type. The lack of the 15:48 base-pair is consistent with previous chemical modi®cation of the G15:C48 variant.12,27,28 Second, because the pyrimidine ring of C48 does not stack with A46, C48 can not maintain the 48:46 stacking observed in the wild-type, and as such it is likely to damage the integrity of the core. The core of the functional Cys130 mutant (Figure 5(c)), which has AUUC in the V-loop, is distinct from the core of the G15:C48 variant by two new features. First, it has recruited U46 into the core to stack with C48. This feature is also observed in the modeled structure of the other two active mutants, Cys127 and Cys128 (not shown). In all three modeled structures, 15:48 remain unpaired. Thus, the defect of the unpaired 15:48 appears compensated by restoration of the 48:46 stacking interaction. Second, Cys130 differs from the wild-type and all other functional mutants by having A44 instead of C44 (Table 2). A44 is in a position to form a pair of symmetric N1:N6 H bonds with A26. As such, it improves the stability of the 26:44 interaction from one H bond in the wild-type to two in Cys130. The enhanced 26:44

interaction provides a rationale for why Cys130 is the most active mutant; it can contribute to general stacking interactions in the core, and can stabilize the core by strengthening the junction of the D and anticodon stems. Analysis of tRNA crystal structures has identi®ed two types of example where uridine stacks in the core. In the ®rst, the stacked uridine is not constrained by H bond interactions. For example, U60 in the T C-loop of yeast tRNAAsp stacks on U59, which in turn stacks on A15 of the 15:48 base-pair (A15:U48).29 Neither U60 nor U59 is in a base-pair and thus their stacking in the core is not expected. In the second, the stacked uridine is recruited to the core by pairing with nucleotides in the D or V-loop to form a base-pair or base-triple. For example, D20a of T. thermophilus tRNASer is stacked in the core,1 rather than being ¯ipped out as in other tRNA structures. The stacking is achieved by formation of a D20a:[G15:C48] basetriple that helps to stabilize the core. Also, U47q in the long V-loop of T. thermophilus tRNASer is stacked,1 due to its base-pairing with A45 to enhance stacking in the V-loop and general hydrophobic interactions with nucleotides in the D-loop. The molecular details of how U46 stacks in the alternative core and whether it forms H bonds with other nucleotides in the core remain to be de®ned. In principle, a stacked U46 could be distinguished from an unstacked U46 by its protection from chemical probes. However, this is dif®cult to test for two technical limitations. First, the core at positions 44-48 is in general dif®cult to access by chemical probes, in part due to the highly compact local structure.10 Second, the only available probe for uridine is CMCT (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate), which is quite large and does not effectively probe the tRNA core.10,22 Thus, further insights on the

512 stacking of U46 will depend on structural analysis of one of the functional mutants, such as Cys130. Importance of local stacking interactions in the core Our modeling study suggests the importance of local stacking interactions in the core of tRNACys. This importance is also demonstrated in the analysis of the crystal structure of a mutant tRNAGln that has an enhanced af®nity to glutamine-tRNA synthetase.30 The mutant tRNA contains the AGGU sequence in the V-loop, rather than the CAUUC sequence in the wild-type, and this difference improves the tRNA af®nity with glutaminetRNA synthetase by 30-fold. The mutant has a much better stacked core, where all four bases of the V-loop are inserted into the core. In particular, the base of G45 ®lls a gap in the wild-type core and thus greatly increases hydrophobic stacking interactions in the mutant structure. Also, A44 of the mutant forms a much more stable 26:44 basepair than C44 of the wild-type. As such, the more stable 26:44 pair helps to stack the core better, generating a much more global effect on the tRNA structure. The importance of the 46:48 stacking interactions may be a common feature for tRNA cores that contain G15:G48. There are only seven cytoplasmic tRNAs that carry G15:G48 in a recent analysis of tRNA database.18 These tRNAs range from E. coli to zea mays and include speci®city for cysteine, isoleucine, threonine, phenylalanine, histidine, and methionine. Inspection of these tRNAs shows that all contain a purine adjacent to G48. This purine is directly linked to G48 or is separated from G48 by a U47. In both cases, we assume that the purine can be stacked with G48, either directly or by ¯ipping out the intervening U47. Analysis of tRNA crystal structures has shown that uridine ¯anked between two purines is consistently splayed out.3,6,31 This emphasizes the overwhelming preference for a purine adjacent to G48 and suggests the importance of purine: purine stacking interactions to stabilize G48. The study of G15:G48 provides an example of an approach to identify compensatory mutations that rescued a defect in a tRNA structure. Results of this study and those of earlier clearly show that G15:G48 in the E. coli cysteine core is not the direct determinant for recognition by cysteine-tRNA synthetase. Alternative cores containing G15:C48 are possible, so long as local structural elements are compatible with each other to generate a permissible structure for aminoacylation. The core is therefore an indirect determinant that in¯uences the overall presentation of a tRNA to its synthetase. The main question of interest is whether a permissible core provides a better structure for aminoacylation or it provides a lower energy barrier for tRNA conformational transition required for aminoacylation. The answer to this question cannot be directly derived from the present study.

An Alternative tRNA Core for Aminoacylation

All tRNA variants examined here, both functional and non-functional, appeared folded when they were assayed for activity (not shown). Their folding was evaluated by electrophoresis on polyacrylamide gels under native conditions that have been used to examine folding of RNA ribozymes.32,33 Recent studies have shown that pathways of RNA folding initiates with a collection of non-speci®c compact structures that arise from local favorable interactions, followed by sequential assembly of stable sub-structures to arrive at the native state.34 ± 36 The non-functional mutations may have mis-folded intermediates that are unable to rearrange to the native structure during the transition state of aminoacylation. To test this idea and to gain better insights into the core in aminoacylation, further kinetic studies of RNA folding of several tRNA mutants will be necessary.

Materials and Methods Preparation of tRNA transcripts and aminoacylation with cysteine The tRNA genes used in this study were constructed in plasmid pTFMa (a derivative of pUC18).12,37 Mutations in tRNA genes were created by site-directed mutagenesis with the Kunkel procedure.38 Transcription of tRNA genes by T7 RNA polymerase and puri®cation of T7 transcripts was as described.12 The concentration of each transcript was determined by aminoacylation to a plateau level and calculated based on 1600 pmol/A260 unit. We used puri®ed T7 RNA polymerase for T7 transcription and puri®ed E. coli cysteine-tRNA synthetase for aminoacylation according to published procedures.39,40 The native E. coli tRNACys was obtained from Subriden. Steady-state conditions were used to derive Km, kcat, and kcat/Km values, according to the Michaelis-Menten equation. Each Km or kcat value was the average of at least three independent determinations. Modification of tRNA transcripts by dimethyl sulfate (DMS) Procedures for DMS modi®cation of N7 of G15 in tRNA transcripts were as described.12,26 DMS (Fluka) was used at 0.4 % with labeled tRNA transcripts ([32P]pCp at the 30 end) and 4 mg of E. coli total tRNA. Each tRNA was tested by three conditions. One was a control (C), where tRNA was not treated with DMS to evaluate non-speci®c cleavage by aniline during the modi®cation procedure. The second was the native condition (N), where tRNA folded in the presence of 10 mM Mg2‡ was probed by DMS. The third was the semidenatured condition (S), where tRNA partially denatured in 10 mM EDTA was probed by DMS. Results of all three conditions were analyzed by 8 % polyacrylamide/7 M urea and band intensities were quanti®ed by phosphorimager (Molecular Dynamics). Structural modeling Modeling of tRNAs using coordinates of E. coli tRNACys and tRNAGln 5,7 obtained from the Protein Data Bank41 was performed with Insight II (Molecular Simulations Inc.). Base substitution was achieved by Biopoly-

An Alternative tRNA Core for Aminoacylation mers and calculation of dynamics was achieved by Discover using the CFF91 force ®eld to satisfy constraints of torsion angle and bond length. Energy minimization (excluding solvent) was achieved by 500 cycles of the steepest algorithm, 0.01 derivative, and no charges, cross charges, or morse. The difference in the potential energy between the starting structure and the ®nal was no more than 2 kcal/mol.

Acknowledgments We thank Dr John J. Perona for critically reading the manuscript. This work was supported in part by a grant from the NIH (GM56662 to YMH) and a grant from the Jefferson Medical College (to Y.M.H.). R.S.A.L. is the postdoctoral fellowship recipient of the American Heart Association-Pennsylvania-Delaware Division.

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An Alternative tRNA Core for Aminoacylation

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Edited by D. Draper (Received 24 July 2000; received in revised form 15 September 2000; accepted 18 September 2000)