Yeast tRNA-Asp
Created by Thomas McConnel
While only a small molecule, the aspartic acid transfer RNA (tRNA-Asp) [PBD ID: 1VTQ] plays a vital role in the growth and metabolism of one of the most well studied species of yeast, Saccharomyces cerevisiae.
At the most basic level, tRNAs function in between the genetic code (specifically mRNA) and amino acids in the process of protein synthesis. This happens through the catalyzed attachment of an amino acid to a tRNA followed by anticodon-codon pairing of the tRNA to mRNA within the ribosome. As a result, the amino acids become incorporated into forming polypeptide chains in the order dictated by the genetic code,1 and the tRNA dissociates for additional use.
As a single chain composed of 75 nucleotides and weighing in at 24181.50 Da2, yeast Asp-tRNA is a fairly typical tRNA; what sets it apart from most others functionally is the amino acid that it associates with, aspartic acid.
Although this molecule is fairly well understood, recent discoveries such as surprisingly stable Asp-tRNA complexes3 (via interactions of the anticodons) and large numbers of tRNA isodecoders in the human genome1 have made it clear that the molecule is not an exhausted scientific topic.
Yeast Asp-tRNA shares considerable homology with most tRNAs. Phe-tRNA is one of the most historically significant transfer RNAs, and serves as a good model for comparison. Considered here is Phe-tRNA of Saccharomyces cerevisiae [PDB ID: 1EHZ]
In terms of primary structure Phe-tRNA similar to Asp-tRNA but far from identical: yeast Asp-tRNA has just over 50% of the same nucleotide bases in rough alignment with yeast Phe-tRNA. Some notable similarities in primary structure included the presence of the anticodon triplet (positions 34-36), the CCA tail shared by all tRNAs to which an amino acid becomes linked during translation, and the G10 base noted for playing a role in aminoacyl-tRNA synthetase binding. However the corresponding base at position 25 was U (as expected) in Asp-tRNA, but was found to be C25 in Phe-tRNA, possibly indicating a role in the specificity of the tRNA-aminoacyl-tRNA synthetase interaction. Phe-tRNA shares a similar secondary structure with Asp-tRNA and an essentially identical tertiary one.4
The comparison with Phe-tRNA, and the discussion so far has mainly focused on the the primary structure of the molecule, and while it seems basic, it is the most important level of structure since it determines the interactions that will take place within the molecule leading to the development of the higher levels of organization. The base content (and positioning) also is the basis for interactions with other molecules, including aminoacyl-tRNA synthetases and strands of mRNA to be translated.
Specifically, yeast tRNA-Asp has five features that have been noted to play important roles in intermolecular interactions [some of which were noted in the comparison with tRNA-Phe]; they are the three residues of the anticodon triplet, the nucleotide base G73, and the base pair G10-U25.5The three residues of the anticodon (G34, U35, C36) determine the sequence(s) of mRNA that tRNA-Asp can bind to during translation, and also are capable of anticodon-anticodon binding when in contact with another tRNA-Asp molecule.6 Base G73 is known as the discriminator base, because the base in this position is always invariant for tRNAs corresponding to a certain amino acid; it is recognized by the aminoacyl-tRNA synthetase. The G10-U25 pair [it can be noted that this pairing via hydrogen bonds constitutes the beginnings of secondary structure] plays a slightly different role in the fact that the tRNA-synthetase actually does not interact with it but the pairs adjacent to it, but it must remain intact so that these interactions can occur: when this base pair is mutated or disrupted, significant decreases in the binding rate is observed.5 Additionally,the A residue of the acceptor stem (of which G73 is a part), is where the carboxyl group of an amino acid becomes linked when aspartyl-tRNA synthetase “loads” the tRNA-Asp.7 When these features of primary structure on tRNA-Asp are mutated, a severe loss of aspartylation activity is observed, suggesting that the determined structure is the wild-type.8
As previously mentioned, the primary structure paves the road for the formation of higher structural organization, most immediately, secondary structure. In tRNA-Asp, the primary structure is such that many intramolecular hydrogen bonds form (21 intramolecular base pairs), resulting in the formation of secondary structure motifs such as hairpin turns, double helices, and stem-loops.9 The overall shape created by the base pairing in tRNA is thought of as a cloverleaf, consisting of an acceptor stem (previously discussed), the D loop (named for its dihydrouridine bases), the T loop (similarly named), and the anticodon loop. The “loops” that form to allow complementary stretches of the single stranded RNA to align (the stems), while the helical regions result from an intrinsic twisting of the hydrogen bonded region.7 It is important to remember that all of this occurs spontaneously because of the tendency for molecules to reside in their most stable form (net increase in entropy). Most of these secondary structures function to enable the formation of tertiary structure in a way that keeps the “active sites” available for intermolecular interactions and increase the stability of the molecule.
It is almost impossible to consider the secondary structures of an entire molecule without bringing up its tertiary structure, its overall 3-dimensional structure. The tertiary structure results from interactions (typically base-pairing) between elements of the secondary structure. The most important of these interactions are generally understood to occur between the D and T-Loops causing the cloverleaf to fold over on itself creating an L-shaped (also sometimes referred to as trefoil shaped10) 3D structure. The region where these two loops meet at corner of the molecule is known to have several unique characteristics including some unusual base pairs: the T54-A58 reverse-Hoogsteen base pair, G18-C55 and G19-C56 inter-loop base pairs; as well as 4- high stack of purine bases. These interactions are also found in other closely related tRNAs indicating their importance to the proper function of the molecule11. The list goes on; in this region there are also a number of conserved base pairs such as the G15-C48 reverse Watson-Crick base pair that indicate [since functional importance necessitates widespread conservation] the importance of the tertiary structure in the function of the molecule. The exact role of most of these interactions is not entirely clear, but they are generally understood to provide structural support and stability. On the other hand, the locations of the “active sites” on tRNA-Asp have a clearly visible functional significance in the fact that they allow the sites to exist on opposite ends of the L so that they can interact/be attached to both an amino acid and mRNA at the same time in the process of translation: the two [free, single stranded] ends of the RNA reside at the more pointed end of the L where an amino acid can be added while the rounded end of the L shape is home to the anticodon10. The structural analysis of tRNA-Asp, has indeed shed a lot of light on the function of these molecules, and continued to provide new insights about the mechanisms employed. For example, it was previously discovered that the anticodon-anticodon interactions between two tRNA-Asp molecules were over 100 times stronger than expected from the base sequences alone, but when the structural basis for this strength was examined it was discovered that the modified G residue at position 37 actually stacks on top of the G-C base pair of the anticodons, accounting for this stabilization.12 Furthermore, it has been discovered that in the before mentioned anticodon-anticodon base pairing, the bases form a minihelix. It is believed that these structural findings can be extrapolated to the understanding of t-RNA anticodon-mRNA codon interactions as well.6 Truly, structure is at the heart of understanding biological function.