tRNA structure and evolution and standardization to the three nucleotide genetic code

Abstract

Cloverleaf tRNA with a 75 nucleotide (nt) core is posited to have evolved from ligation of three 31 nt minihelices followed by symmetric internal deletions of 9 nt within ligated acceptor stems. Statistical tests strongly support the model. Although the tRNA anticodon loop and T loop are homologs, their U-turns have been treated as distinct motifs. An appropriate comparison, however, shows that intercalation of D loop G19 between T loop bases 4 and 5 causes elevation of T loop base 5 and flipping of T loop bases 6 and 7 out of the 7 nt loop. In the anticodon loop, by contrast, loop bases 3-7 stack tightly to form a stiff connection to mRNA. Furthermore, we identify ancient repeat sequences of 3 (GCG), 5 (UAGCC) and 17 nt (∼CCGGGUUCAAAACCCGG) that comprise 75 out of 75 nts of the tRNA cloverleaf core. To present a sufficiently stiff 3-nt anticodon, a 7-nt anticodon loop was necessary with a U-turn between loop positions 2 and 3. Cloverleaf tRNA, therefore, was a radical evolutionary innovation essential for the 3-nt code. Conservation of GCG and UAGCC repeat sequences indicates that cloverleaf tRNA is at the interface between a strange RNA repeat world and the first evolution of molecules that fold to assume biologic functions. We posit that cloverleaf tRNA was the molecular archetype around which translation systems evolved.

Keywords: D loop; T loop; T-loops; U-turn; acceptor stems; anticodon loop; evolution of tRNA; genesis of the 3 nucleotide code; last universal common cellular ancestor; repeat sequences; tRNA.

Figure 1.

The tRNA structure. The 7-nt paired As are green (5′-As: 1–7; 3′-As: 69–75). The 5-nt As* are cyan (D loop 25–29 and V loop 47–51). The homologous Ac and T loop 17-nt microhelices are red (Ac loop: 30–46; T loop: 52–68). The D loop 17-nt microhelix is magenta (8–24). 3′-CCA is white. Blue dots indicate the anticodon (37–39). Blue and orange dots indicate stacked bases (37–41) in the Ac loop. Yellow dots indicate elevated base 61 (loop nt 5) and flipped out bases 62 and 63 (loop nts 6 and 7) in the T loop. G19 and G20 (magenta) and C59 (red) that help form the elbow are indicated. Homologous Ac and T loops are numbered for loop positions (1–7). Numbering is based on a 75-nt cloverleaf tRNA core (Fig. 2).

Figure 2.

“Typical” cloverleaf diagrams. (A) Pyrococcus (archaeal) tRNAs (three species). (B) Archaeal tRNAs. (C) Bacterial tRNAs. Only 5 nts of the V loop (47–51) are considered (red line) in the model because additional nts result from inserts. The model is numbered for a 75-nt core, neglecting the 3′-CCA, where the amino acid is covalently attached. A previous 72-nt numbering system was based on tRNAs with 3 nt deletions in the D loop. In (B) U-turns are indicated. D loop G19 and G20 interactions with the T loop are shown. Blue dots indicate the anticodon (37–39). Orange dots indicate stacked bases in the Ac loop (40–41). Yellow dots indicate elevated base 61 and flipped out bases 62 and 63 in the T loop (loop positions 5–7) (colored dot labels as in Fig. 1).

Figure 3.

A model for cloverleaf tRNA evolution. Three 31-nt minihelices are ligated to form a 93-nt precursor. Two symmetric 9 nt deletions within ligated acceptor stems generate the 75-nt tRNA core. Colors as in Fig. 1. Parentheses indicate base pairing. Asterisks indicate unpaired bases. / indicates a U-turn. / indicates no U-turn.

Figure 4.

Comparisons of 17-nt microhelix structures and their interactions. (A) Comparison of an Ac loop and a T loop. Overlay of Saccharomyces cerevisiae tRNA^PHE Ac and T loops (PDB 4TRA). Loop positions are numbered. The Ac loop was colored for chemistry. The T loop is transparent white. Two views. (B) Interactions of the D loop with the T loop. The paired stem is green. The 7-nt T loop is white. The D loop is colored for chemistry. (C) Comparison of an Ac loop from a ribosome- and mRNA-bound tRNA (5DOY_W) (A site; colored for chemistry) and an Ac loop from an unbound tRNA (4TRA) (transparent white). Two views. (D) Overlay of an Ac loop from a P site (colored for chemistry) and an Ac loop from an A site (transparent white) tRNA. Two views. Blue dots indicate anticodon positions. Orange dots indicate stacked bases 6 and 7 of the Ac loop. Yellow dots indicate elevated base 5 and flipped out bases 6 and 7 in the T loop (A and B only). RMSD is for backbone atoms.

Figure 5.

Homology of acceptor stems and acceptor stem remnants. The dotted line indicates the observed alignment evolutionary distance score. The gray histogram indicates 1000 randomly permuted sequences of the same base composition. In the sequence logo, blue arrows indicate homologous positions. Parentheses indicate base pairing. Asterisks indicate loops. mH indicates minihelices or microhelices. CL indicates cloverleaf fold.

Figure 6.

The T loop and the Ac loop 17-nt microhelices are homologs, but the D loop microhelix is distinct in sequence. Symbols for sequence logos are the same as in Fig. 5. The U-turn is indicated by a red arrow.

Figure 7.

Complementary sequences in the tRNA cloverleaf support the model for tRNA evolution. J is a negative control test for I. Because these sequences are complementary but not homologous, the posited D loop minihelix stems test as homologs vs. their stem complement (Fig. 8I) but as non-homologs (stem vs. stem) in a direct sequence comparison (Fig. 8J).

Figure 8.

Breaking the 17-nt D loop microhelix into TAGCC₄ repeats. In the sequence logo, different modes of folding the D loop microhelix are indicated (TAGCCTAGCCTAGCCTA repeat folding; folding in a 17-nt microhelix or a 31-nt minihelix (mH); folding in the cloverleaf tRNA (CL)). Parentheses indicate base pairing. Asterisks indicate unpaired bases. Tests A–I are predicted to test as similar for a perfect repeat. Degeneracy in the third repeat (invariant A14 vs. invariant G19) causes the test in Fig. 9H to test as negative for similarity.

Figure 9.

Acceptor stems may be generated from a (GCG)₃ repeat. Tests B–D are consistent with the (GCG)₃ repeat model. Test A is a trivial test of the complementarity of the 5′ and 3′ acceptor stems. Tests E–H are negative control tests for the tests directly above.