RNA recognition by designed peptide fusion creates \"artificial\" tRNA synthetase

RNA recognition by designed peptide fusion creates ‘‘artificial’’ tRNA synthetase Magali Frugier*, Richard Giege´*, and Paul Schimmel†‡ *De´partement Me´canismes et Macromole´cules de la Synthe`se Prote´ique et Cristallogene`se, Unite´ Propre de Recherche 9002, Institut de Biologie Mole´culaire et Cellulaire du Centre National de la Recherche Scientifique, 15 Rue Rene´ Descartes, F-67084 Strasbourg Cedex, France; and †The Skaggs Institute for Chemical Biology and Departments of Molecular Biology and Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 Contributed by Paul Schimmel, May 8, 2003

T

he development of the genetic code was essential for the transition from the putative RNA world to the theatre of proteins (1, 2). The rules of the modern code are determined in aminoacylation reactions, where each amino acid is linked to the tRNAs bearing the anticodon triplets specific for that amino acid (3). The tRNA itself is comprised (typically) of 76 nucleotides ending at the 3⬘ end with the universal trinucleotide CCA76, where A76 is the amino acid attachment site (Fig. 1A). In three dimensions, the cloverleaf secondary structure is rearranged into two distinct domains fixed at right angles to form an L shape. One domain contains the 12-bp acceptor-T␺C minihelix with the amino acid attachment site at the 3⬘ end. The other domain, at right angles, contains the anticodon at a distance of ⬇75 Å from the amino acid attachment site (4–6). The two domains of tRNA are thought to have arisen independently, with the minihelix being an outgrowth of the earliest substrates for aminoacylation (7–10). Significantly, over half of the synthetases have been shown to charge RNA oligonucleotide substrates based on the minihelix or smaller pieces such as a 7-bp microhelix hairpin that is designed after the tRNA acceptor stem (see below; e.g., refs. 11–16). RNA determinants for specific aminoacylation of microhelices have been referred to as a second genetic code or an operational RNA code (1, 17). The second genetic code may have been the precursor to the modern genetic code (18, 19). Alanyl-tRNA synthetase (AlaRS) is a particularly striking example of an enzyme that catalyzes robust aminoacylation of RNA oligonucleotide substrates based on the acceptor stem of tRNAAla (Fig. 1 A; refs. 11 and 20). A single G:U wobble base pair at position 3:70 in the acceptor stem is a critical determinant for aminoacylation (21, 22). Transfer of G3:U70 into nonalanine tRNA frameworks confers alanine acceptance on them (11, 21, 22). Minor identity elements in the acceptor stem include the A73 ‘‘discriminator’’ nucleotide and a G2:C71 base pair www.pnas.org兾cgi兾doi兾10.1073兾pnas.1332771100

Fig. 1. Domain organizations of E. coli tRNAAla and AlaRS. (A) Sequence and cloverleaf structure of E. coli tRNAAla (Left) and hairpin structure of alanine microhelix (Right). The G3:U70 base pair is highlighted in bold and the acceptor stem sequence is designated by a shaded box (48). (B) Schematic diagram of the full-length E. coli AlaRS where each functional domain is specified (36, 49). The two functional modules used as negative controls (fragment 368N) and positive controls (fragment 461N) are also delineated.

(23–25). In contrast, the anticodon makes no contribution to the specificity or efficiency of recognition of tRNAAla. Indeed, the enzyme makes no physical contact with the anticodon (26). Thus, the determinants that specify alanine acceptance are completely segregated from the triplets that encode alanine. In terms of RNA recognition, AlaRS may be representative of an early synthetase. The catalytic domains of aminoacyl tRNA synthetases are divided into two classes of 10 enzymes each, based on the two distinct architectures of the active sites (27, 28). These domains are ancient and, for virtually all synthetases, basically fixed throughout evolution. Thus, these catalytic domains were established at the time of the last common ancestor to the tree of life (2, 29). The domains contain the site for amino acid activation (aminoacyl adenylate synthesis) and for interaction with the 3⬘ end of tRNA. Other motifs and domains are fused to or inserted into the active-site domains. These elements are essential for specific RNA recognition and, in general, are idiosyncratic to the synthetase and not conserved among enzymes of the same class (30). From the foregoing perspective, the development of RNAspecific aminoacylations came from idiosyncratic acquisitions of RNA-binding elements by preexisting primordial domains for adenylate synthesis. To test the feasibility of this scenario, we Abbreviation: AlaRS, alanyl-tRNA synthetase. ‡To

whom correspondence should be addressed. E-mail: [email protected].

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The genetic code was established through aminoacylations of RNA substrates that emerged as tRNAs. The 20 aminoacyl-tRNA synthetases (one for each amino acid) are ancient proteins, the active-site domain of which catalyzes formation of an aminoacyl adenylate that subsequently reacts with the 3ⴕ end of bound tRNA. Binding of tRNA depends on idiosyncratic (to the particular synthetase) domains and motifs that are fused to or inserted into the conserved active-site domain. Here we take the domain for synthesis of alanyl adenylate and fuse it to ‘‘artificial’’ peptide sequences (28 aa) that were shown previously to bind to the acceptor arm of tRNAAla. Certain fusions confer aminoacylation activity on tRNAAla and on hairpin microhelices modeled after its acceptor stem. Aminoacylation was sensitive to the presence of a specific G:U base pair known to be a major determinant of tRNAAla identity. Aminoacylation efficiency and specificity also depended on the specific peptide sequence. The results demonstrate that barriers to RNA-specific aminoacylations are low and can be achieved by relatively simple peptide fusions. They also suggest a paradigm for rationally designed specific aminoacylations based on peptide fusions.

Fig. 3. Schematic diagrams and sequences of fusion proteins. (A) Functional domains are indicated with different colors: 368N is shown in gray, linker is shown in green, RNA-binding motifs (RGG boxes) are shown in red, and the 10-aa recognition sequence from MF␤2 is shown in blue. (B) Corresponding primary sequences of fusion peptides of 368N-MF␤21 and 368N-MF␤25.

Fig. 2. Sequence and structure of the selected peptide MF␤2 and assorted variants. (A) The peptide contains two 9-aa RGG motifs (black; ref. 42) that flank the10-aa sequence selected (by phage display methods) to recognize specifically a G:U base pair (red; ref. 32). The standard one-letter abbreviations for amino acids are used. (B) Helical wheel representation of the predicted helical structure corresponding to the parental (‘‘wild-type’’) MF␤2 sequence (Left) and mutated sequences used in this study (Right, mutations are circled in red). In the wild-type sequence on the left, red residues were shown important for specific recognition of the unpaired 2-amino group of G3 that projects into the minor groove.

exploited previous work in which a short peptide was selected by phage display methods (31) to discriminate a G3:U70 base pair from other possibilities (G:C, U:G, and I:U) in microhelixAla (32). The selected peptide, MF␤2, efficiently bound microhelixAla and discriminated G3:U70 with high selectivity (Fig. 2A). Recognition depended on the presence of the unpaired 2-amino group of G3 that projects into the minor groove. This same amino group is critical for recognition by native AlaRS. In this work we fused versions of the MF␤2 peptide to the catalytic domain of the 875-aa Escherichia coli AlaRS (33). A fragment of the N-terminal 368 aa (fragment 368N) encodes the domain for adenylate synthesis and has determinants for contacting the 3⬘ end of tRNAAla (34, 35; Fig. 1B). Fragment 368N lacks polypeptide determinants needed for recognition of the G3:U70 base pair and consequently for binding tRNAAla or microhelixAla (36, 37). (These determinants are contained in a 93-aa segment that extends from T369 to D461.) Peptides based on MF␤2 were joined through a linker to 368N and tested for their capacity for aminoacylation and for specificity of base-pair recognition at the 3:70 position. The results show that base pair-sensitive aminoacylation systems based on peptide fusions are accessible and suggest a paradigm for the rational design of specific aminoacylation systems. Materials and Methods Preparation of Artificial AlaRSs. AlaRS fragments 461N and 368N

and the different fragment-MF␤2 fusions were overexpressed and purified in E. coli. Genes were cloned into expression vectors pQE70 and pQE30 (Qiagen, Courtaboeuf, France) fused to a Cor N-terminal His6 tag, respectively. Induction (1 mM isopropyl ␤-D-thiogalactoside) was performed for 3 h at 37°C under moderate shaking (180 rpm). AlaRS variants were purified on a nickel-nitrilotriacetic acid column according to the Qiagen protocol. Proteins were quantified by UV measurement at 280 7472 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.1332771100

nm by using a calculated extinction coefficient ␧ ⫽ 48,220 M䡠cm⫺1 for fragment 368N and for the various fusion proteins (which have no chromophores that absorb at 280 nm). Proteins were stored at ⫺20°C in 50 mM phosphate buffer (pH 7.2)兾150 mM KCl兾50% glycerol兾10 mM 2-mercaptoethanol. All AlaRS constructs yielded one band on SDS兾PAGE and from an overloading of material the purity was estimated as ⬎99%. Fragment 461N with an N-terminal His6 tag had an activity reduced compared with the C-terminal His6-tagged protein. However, all N-terminal-tagged 368N-MF␤2 fusions were inactive. Thus, C-terminal His6-tagged 461N was used as a positive control to compare with N-terminal-tagged 368N and fusions of 368N with peptides. The DNA sequence coding for 368N was cloned into pQE30, between SpHI and HindIII sites. The linker and the different peptide sequences were subsequently fused by using overlapping oligonucleotides with specific restriction sites. The restriction sites led to minor sequence variations in the fusion proteins: substitution of R368 in 368N with the sequence K368L in the fusion proteins and insertion of the dipeptide sequence Glu-Phe between the linker and the MF␤2-encoding peptides (Fig. 3B). Finally, all proteins contain a C-terminal sequence AAAKLN encoded by the plasmid pQE30. In a test experiment, this hexapeptide extension did not have an effect on the activity. Preparation and Aminoacylation of RNAs. RNA microhelices were

chemically synthesized on a Pharmacia (Piscataway, NJ) gene assembler synthesizer as described and subsequently purified on a 16% polyacrylamide gel. Concentrations of RNAs were determined by absorbance at 260 nm using calculated extinction coefficients based on base compositions. After optimization, aminoacylation of alanine microhelices variants was performed at 37°C in 25 mM Tris䡠HCl (pH 7.5)兾 10 mM MgCl2兾5 mM ATP兾75 mM NaCl兾1 mM dithioerythritol兾0.5 ␮g/␮l BSA兾0.01 units/␮l inorganic pyrophosphatase (Sigma–Aldrich)兾50 ␮M L-[3H]alanine (Amersham Pharmacia, Orsay, France) and appropriate amounts of RNA microhelices. Before aminoacylation, transcripts were renatured in H2O by heating at 85°C for 90 s and fast cooling on ice prior to the addition of MgCl2 (to a final concentration of 50 ␮M). Aminoacylation reactions were initiated by the addition of appropriate amounts of enzyme diluted in 100 mM HepesKOH (pH 7.4)兾10% glycerol兾1 mM dithioerythritol兾5 ␮g/␮l BSA. Reactions were stopped (after 2.5-, 5-, 10-, and 20-min incubations at 37°C) by the addition of 5% trichloroacetic acid Frugier et al.

and treated in the conventional way by filtration of precipitates on Whatman 3 MM paper (38). Kinetic constants (kcat and Km) were derived from Lineweaver–Burk plots. RNA concentrations extended from 20 to 100 ␮M, and protein concentrations ranged from 30 nM for fragment 461N to 5 ␮M for 368N and for the fusion proteins 368N-MF␤21 and 368N-MF␤25. Kinetic plots and plateaus represent an average of at least two independent experiments. Values of kcat兾Km for replicate experiments varied by at most 10%. Comments on Optimization of Aminoacylation Conditions. In the first

Fig. 4. Aminoacylation of microhelixAla by fusion proteins. Sequences of the different fusion constructs are shown in Fig. 3. The activity of one representative of each group is shown: 368N-MF␤21, most efficient; 368N-MF␤25, moderately active; 368N-MF␤27, low activity; and 368N, no activity. (Inset) Activity of all other constructs (368N-MF␤22, 368N-MF␤23, 368N-MF␤24, and 368N-MF␤26) compared with 368N-MF␤21. Aminoacylation reactions were run at pH 7.5 and 37°C with 30 ␮M microhelixAla and 5 ␮M of each fusion protein.

helix substrates were measured by polyacrylamide coelectorphoresis using the protocol described in ref. 39. Samples of radiolabeled microhelix (0.1 pmol of 5⬘ 32P-labeled microhelix were mixed with 20 pmol of E. coli total tRNA in a final volume of 20 ␮l), 10% glycerol, 50 mM Tris䡠HCl (pH 7.5), and 10 mM MgCl2 were electrophoresed for 90 min (70 V at 4°C) through a 10% acrylamide兾bisacrylamide gel (37.5:1; 200 ⫻ 100 ⫻ 1.5 mm3) containing increasing concentrations of fragment 461N or fusion proteins 368N-MF␤21 and 368-MF␤25 (25–50 ␮M) in Tris buffer (100 mM Tris base兾100 mM boric acid). Gels were dried and analyzed on a FUJIX BAS 2000 bioimaging analyzer (Raytest, Courbevoie, France) with WORK STATION 1.1 software.

library) for its ability to recognize the acceptor arm of tRNAAla in a G3:U70-dependent way (32). Peptide MF␤2 consists of a central decapeptide flanked with two RGG-containing motifs (KRGGKRGGK) that enhance general RNA binding (refs. 41 and 42; Fig. 2 A). The peptide forms a specific complex with microhelixAla with a Kd of 300 nM (32). The decapeptide central region was strongly predicted to form an ␣-helix. By mutational analysis 4 of the 10 residues were demonstrated important for specific recognition of the G3:U70 base pair (Fig. 2B). These residues are S2, A4, E6, and N9. Three of these (S2, E6, and N9) are on the same side of the helix. Using a linker (four repeats of a GS dipeptide), we fused 368N to MF␤2-containing peptides. Altogether, seven fusion proteins (368N-MF␤21 to 368N-MF␤27) were constructed. All contained fragment 368N, the linker, and the G:U-specific decapeptide MF␤2. The number and localization of the RGG boxes neighboring the selected decapeptide were varied to generate the seven different constructs (Fig. 3).

Results

Aminoacylation with Fusion Constructs. By using optimized condi-

Measurements of Affinities for MicrohelixAla. Affinities for micro-

Rational Design of Fusion Proteins. The overall architecture of E.

coli AlaRS is based on four modular units responsible for alanyl-adenylate synthesis, tRNA recognition, tRNA-dependent editing, and assembly of the tetrameric structure (Fig. 1B). Although the native enzyme is tetrameric, fragment 368N is a monomer that catalyzes synthesis of alanyl adenylate with the same efficiency as that of the native enzyme (34, 40). Fragment 368N has no activity for aminoacylation or tRNA binding. Aminoacylation of tRNAAla requires the segment extending from T369 to D461 to give fragment 461N (36). All contacts with the acceptor stem are encoded by 461N. As a consequence, 461N aminoacylates microhelixAla with the same catalytic efficiency as does the native enzyme. [The native enzyme has a smaller Km for charging tRNAAla than does 461N because of contacts with tRNAAla that lie outside the acceptor arm that are contributed by portions of AlaRS that lie on the C-terminal side of D461 and therefore are missing from 461N (40).] Thus, in our work we sought to replace the 93-aa segment from T369 to D461, which is needed for acceptor helix contacts. This replacement was a short, ‘‘artificial’’ peptide that was selected (from a phage display Frugier et al.

tions, the seven fusion constructs were tested for aminoacylation of microhelixAla and compared (Fig. 4). The first construct, 368N-MF␤25, had one RNA-binding motif located on the N-terminal side of the G-U-recognizing peptide and had significant activity for charging of microhelixAla. In contrast, under the same conditions no aminoacylation was observed with fragment 368N (as expected). Each of the six other constructs also showed activity, with the different combinations of RGG motifs leading to more or less efficient enzymes. The most efficient fusion constructs were 368N-MF␤21 and 368N-MF␤22, whereas the least efficient were 368N-MF␤26 and 368N-MF␤27. The latter fusion was virtually inactive. The most efficient constructs corresponded to fusions with four (368N-MF␤21) or three (368N-MF␤22) RGG boxes, whereas low activities were associated with 368N-MF␤26 and 368N-MF␤27, which had one or no RGG box, respectively. However, 368N-MF␤25 had only one RGG box and showed a relatively strong activity. This construct charged microhelixAla more effectively than did either 368NMF␤23 ␱r 368N-MF␤24 that each hold two RGG motifs. Thus, the presence of only one RGG motif in the artificial PNAS 兩 June 24, 2003 兩 vol. 100 兩 no. 13 兩 7473

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set of experiments, 368N-MF␤25 was chosen to optimize the aminoacylation conditions. Under starting conditions (25 mM Tris䡠HCl, pH 7.5兾75 mM NaCl兾10 mM MgCl2兾1 mM dithioerythritol) this fusion charged up to 1.2% of the RNA substrate present in the assay. (This level was low compared with the plateaus obtained in the positive control with fragment 461N that reach 80%.) The low activity was explicitly associated with the presence of the peptide MF␤2 in the fusion, because the negative control (fragment 368N) did not show any charging activity (data not shown). Four parameters then were varied to optimize the activity of 368N-MF␤25: the presence of inorganic pyrophosphatase and BSA, concentration of alanine (25–100 ␮M), and temperature. Three of these parameters significantly affected the overall rate. Thus, the presence of 0.01 units兾␮l inorganic pyrophosphatase together with 0.5 ␮g兾␮l BSA increased the rate by 3-fold, whereas raising the temperature from 25 to 37°C increased the rate by 5-fold. Thus, by combining the two sets of variables, a gain of 15-fold in aminoacylation rate and ⬇10-fold in yield was achieved over that realized with the starting conditions.

Table 1. Binding and aminoacylation parameters of microhelixAla with 461N and fusion proteins 368N-MF␤21 and 368N-MF␤25 Construct

Kd, ␮M

Km, ␮M

461N (C-terminal 6-His) Fusion proteins 368N-MF␤21 368N-MF␤25

3.100

100

0.075 1.500

150 150

kcat, min⫺1 100 0.25 0.1

kcat兾Km, 10⫺3 1,000 1.66 0.66

Loss in efficiency 1 1兾600 1兾1,500

Kinetic parameters were determined at pH 7.5, 37°C. Kd values were determined at 4°C.

AlaRS is enough to form an active complex with microhelixAla. To act efficiently as an anchor of the RNA, it seems that an RGG motif has to be localized at the N-terminal side of the critical decapeptide (compare 368N-MF␤25 with 368N-MF␤26). Interestingly, the most active construct, 368N-MF␤21, encoded the original 28-mer selected in the earlier study (32). Binding and Catalytic Parameters. To put these results on a quan-

titative footing, two of the most active fusion constructs were chosen for further investigation. Affinity coelectorphoresis of microhelixAla binding and the microhelixAla concentration dependence of aminoacylation were investigated for 368N-MF␤21, 368N-MF␤25, and the control fragment 461N (Table 1). Apparent kinetic parameters for the two fusion proteins yielded kcat兾Km values that were reduced 1兾600 (368N-MF␤21) and 1兾1,500 (368N-MF␤25) compared with fragment 461N. These values correspond to differences in the apparent free energy of activation of 4–5 kcal䡠mol⫺1 compared with the free energy of activation for 461N. Interestingly, most of the difference from fragment 461N lies in the kcat parameter, unsurprisingly suggesting that positioning of the 3⬘ end of microhelixAla is not as optimal when bound to either of the fusion proteins as when bound to the natural active site. In contrast, Km values for the fusion proteins are comparable to that for 461N. In addition, microhelixAla binds (Kd values) more tightly to the two fusion proteins than to 461N. The lower Kd values for the artificial enzymes are possibly related to the nonspecific RNA-binding components (RGG motifs) that are built into the MF␤2 peptides that were fused to 368N.

(S2L兾N9A), and the triple substitution (S2L/E6A兾N9A). Aminoacylation activity progressively dropped as more substitutions were included (Fig. 5B). For example, charging efficiency dropped almost 2-fold when either the S2L or N9A substitution was introduced and 5-fold when both substitutions were combined (S2L兾N9A). With the triple substitution, aminoacylation efficiency was reduced 10-fold. Both single mutants lost specificity, showing an increase in efficiency of charging of G3:C70-, U3:G70-, or I3:U70-containing microhelices relative to charging of native microhelixAla. This loss of specificity was seen also with the double and triple substitutions, each of which had a further decline in aminoacylation activity. Thus, the charging activity and specificity of the fusion 368N-MF␤25 construct strongly depends on the sequence of the previously selected central decapeptide sequence.

Specificity of Microhelix Aminoacylation. Three constructs (368NMF␤21, 368N-MF␤22, and 368N-MF␤25) were tested for their capacity to discriminate between a G3:U70 base pair versus G:C, U:G, and I:U pairs (Fig. 5A). Thus, RNA microhelices differing only at the 3:70 position were used as substrates. Of the three constructs, 368N-MF␤25 was the most efficient for discriminating between G:U and the other three base pairs. Indeed, all three alternatives, G:C, U:G, and I:U, had the same reduction in activity with 368N-MF␤25. ⌻he other two constructs, 368NMF␤21 and 368N-MF␤22, were more efficient at charging the G3:U70-containing substrate but also had higher activities with substrates harboring any of the other three base pairs. Still, these fusion constructs were most efficient with native microhelixAla. Mutational Analysis of Fusion Peptide. To test whether the efficiency of aminoacylation depended on the central decapeptide sequence that contained the putative determinants for binding microhelixAla, substitutions were introduced at three of the four positions previously shown to be important for recognition of G3:U70. These residues are S2, E6, and N9 (Fig. 2B). S2 was replaced by leucine. (Introduction of an alanine at this position led to the production of a nonsoluble fusion protein.) E6 and N9 were replaced with alanines. Different combinations were tested in the context of the sequence of 368N-MF␤25: single substitutions at position 2 (S2L) or 9 (N9A), double substitutions at positions 2 and 9 7474 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.1332771100

Fig. 5. Specificity of aminoacylation of microhelixAla with various substitutions at the critical 3:70 position, which is a G:U pair in most tRNAAlas throughout evolution (50). Aminoacylation efficiencies were calculated as pmol䡠min⫺1 of aminoacylated microhelix synthesized during a 2.5-min incubation. (B) Discrimination at the 3:70 position by mutant forms of 368N-MF␤25, with mutations placed at specific positions in the 10-aa recognition motif of the free MF␤2 peptide. Mutations correspond to those shown previously to diminish specificity of binding of MF␤2 to microhelixAla (32).

Frugier et al.

Discussion ‘‘Fusing’’ to 368N a 93-aa natural AlaRS peptide segment from T369 to D461 confers RNA-binding activity sufficient for specific aminoacylation of microhelixAla or tRNAAla (36, 37). In previous work, large nonspecific RNA-binding domains of 175 and 228 aa (C-terminal domain of Saccharomyces cerevisiae Arc1p and N-terminal appended domain of S. cerevisiae glutaminyl-tRNA synthetase, respectively) were fused to fragment 368N (43). Significantly, the resulting chimeric proteins catalyzed aminoacylation of microhelixAla. Unsurprisingly, no preference for the critical G3:U70 pair was seen (indeed, A3:U70 was somewhat preferred over G3:U70). These large protein fusions gave early support to the idea that a simple fusion to a domain for adenylate synthesis could result in the acquisition of aminoacylation function. Not clear was whether much smaller motifs (⬍75–100 aa) could also confer aminoacylation and, if so, whether even a modest specificity could be achieved. Here peptide motifs of just 28 or 15 aa, fused via a linker of 8 aa to 368N, generated an enzyme that catalyzed aminoacylation with a clear preference for a G3:U70 pair in a microhelix substrate (368N-MF␤21 and 368N-MF␤25, respectively). Importantly, the two components, adenylate synthesis and specific RNA binding, were generated independently. In particular, the MF␤2 peptide framework used in this work was generated through reiterative selections imposed on a phage display library (31). Selection was specifically to obtain a sequence that bound with strong preference to G3:U70 in the context of microhelixAla (32). Thus, the results are consistent with the idea that early tRNA synthetases arose from small, idiosyncratic RNA-binding elements being fused to domains for adenylate synthesis. These RNA-binding elements might have developed originally to bind and protect ribozymes (to give early ribonucleopeptides or ribonucleoproteins; refs. 44–47). The fusions of RNA-binding peptides to domains for adenylate

synthesis may have been the first step in developing proteinbased synthetases that overcame the ribozyme-based system of aminoacylation. The 28-aa MF␤2 peptide obtained in the earlier selections bound to microhelixAla in a 1:1 complex with a Kd of 300 nM at pH 7.5, 4°C, by using the same affinity coelectorphoresis procedure adopted for the present work. For the 368N-MF␤21 peptide fusion, the resulting Kd was of a similar order (75 nM; see Table 1). Thus, most of the RNA-binding energy for the fusion construct almost certainly came from the 28-aa peptide element. This result suggests that the cost of a fusion, in terms of lost RNA-binding activity compared with the free peptide, is inconsequential. More significant, however, is the reduction in specificity. In a competition assay, U3:G70-substituted microhelixAla was the best competitor for binding of the MF␤2 free peptide to microhelixAla. In particular, the peptide bound microhelixAla by 20- to 25-fold over U3:G70-containing microhelixAla. The specificity for G:U versus U:G at the 3:70 position of microhelixAla was reduced ⬇5-fold for 368N-MF␤25 relative to the free MF␤2 peptide. This reduction probably reflects the serendipitous weak nonspecific RNA-binding determinants in fragment 368N that affect the context for RNA binding by the fusion peptide (35). Further selections could be placed on the fusion proteins to ameliorate these effects. For example, the disposition of the fusion peptide relative to the active site within 368N might be most affected by variations in the length and character of the linker sequence.

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PNAS 兩 June 24, 2003 兩 vol. 100 兩 no. 13 兩 7475

BIOCHEMISTRY

We thank Professor Ya-Ming Hou for helpful comments on the manuscript. This work was supported by National Institute of Health Grant GM23562, a fellowship from the National Foundation for Cancer Research, and grants from Centre National de la Recherche Scientifique, Ministe`re de l’Education Nationale de la Recherche et de la Technologie (program Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires), and Universite´ Louis Pasteur.