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

Abstract

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 3' 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. 1.

Domain organizations of E. coli tRNA^Ala and AlaRS. (A) Sequence and cloverleaf structure of E. coli tRNA^Ala (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.

Fig. 2.

Sequence and structure of the selected peptide MFβ2 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'')MFβ2 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.

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 MFβ2 is shown in blue. (B) Corresponding primary sequences of fusion peptides of 368N-MFβ21 and 368N-MFβ25.

Fig. 4.

Aminoacylation of microhelix^Ala 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-MFβ21, most efficient; 368N-MFβ25, moderately active; 368N-MFβ27, low activity; and 368N, no activity. (Inset) Activity of all other constructs (368N-MFβ22, 368N-MFβ23, 368N-MFβ24, and 368N-MFβ26) compared with 368N-MFβ21. Aminoacylation reactions were run at pH 7.5 and 37°C with 30 μM microhelix^Ala and 5 μM of each fusion protein.

Fig. 5.

Specificity of aminoacylation of microhelix^Ala with various substitutions at the critical 3:70 position, which is a G:U pair in most tRNA^Alas throughout evolution (50). Aminoacylation efficiencies were calculated as pmol·min^–1 of aminoacylated microhelix synthesized during a 2.5-min incubation. (B) Discrimination at the 3:70 position by mutant forms of 368N-MFβ25, with mutations placed at specific positions in the 10-aa recognition motif of the free MFβ2 peptide. Mutations correspond to those shown previously to diminish specificity of binding of MFβ2 to microhelix^Ala (32).