Tertiary structure of tRNA

The tertiary structure of all tRNAs are likewise similar. All known tRNAs are roughly L-shaped, with the anticodon on one end of the L and the acceptor stem on the other. Each stem of the L is made up of two of the stems of the cloverleaf, arranged so that the base pairs of each stem are stacked on top of each other. The parts of the molecule that are not base-paired are involved in other types of interactions, termed tertiary interactions. The tertiary structures of tRNAs thus reflect the dual functions of the molecule The anticodons are well-separated from the acceptor stems. This feature allows two tRNA molecules to interact with two codons that are adjacent on an mRNA molecule. See Figure 10-4. 

Just as main-chain NH 0=C hydrogen bonds are important for the stabilization of the a-helix and / -pleated sheet secondary structures of the proteins, the Watson-Crick hydrogen bonds between the bases, which are the side-chains of the nucleic acids, are fundamental to the stabilization of the double helix secondary structure. In the tertiary structure of tRNA and of the much larger ribosomal RNA s, both Watson-Crick and non-Watson-Crick base pairs and base triplets play a role. These are also found in the two-, three-, and four-stranded helices of synthetic polynucleotides (Sect. 20.5, see Part II, Chap. 16). 

Vlassov, V.V., Giege, R. and Ebel, J.P. (1981). Tertiary structure of tRNAs in solutions monitored by phosphodiester modification with ethylni-trosourea. Eur. J. Biochem. 119, 51-59. 

Figure 26.8 shows the conventional two-dimensional "cloverleaf configuration of a typical tRNA (see also the tertiary structure of tRNA in Chapter 2). Based on the characterization of a large number of tRNAs, it is clear that both the anticodon sequence (with base modifications), and the nucleotides present in the backbone of the tRNA molecule (also with modifications), are sufficiently different to distinguish the tRNAs from each other without grossly affecting the overall structure. Note that the 30 end contains the sequence CCA and that the amino acid is linked to the adenine residue. As described in Chapter 25, this terminal CCA sequence can be added... 

Figure 4.27 How self-complementarity dictates the tertiary structure of tRNA. 

The task of assigning a plausible pattern of base pairing is greatly simplified if sequences are available for different species of RNA known to possess similar structures and functions. For example, the cloverleaf structure and the L-shaped fold have served as good approximations for modeling the secondary and tertiary structures of tRNA respectively. DNA and RNA sequences can be submitted to respective DNA mfold (http //bioinfo.math.rpi.edu/ nnfold/dna) and RNA mfold (http //bioinfo.math.rpi.edu/ mfold/ma) for fold predictions. 

As soon as It became apparent that all tRNA sequences could be assembled in a cloverleaf (12,13) (Fig. 8.5), a number of studies were performed to define the tertiary structure of tRNA (14,15). A proliferation of models for tRNA appeared, more or less well incorporating the chemical data known at the time. It is this evidence that will now be evaluated. 

The interaction between tRNA and its corresponding synthetase is, in this respect, much simpler, in particular due to the probably precise tertiary structure of tRNA and the relatively comparable, much smaller size of the two reactants. In this case the kinetic barrier is not prohibitive and a diffusion-controlled process could account for this reaction. 

It was earlier considered that all the amino acid-activating synthetases were derived from a single primeval synthetase , so that all synthetases would have similar structures. Surprisingly, however, this is not the case. When the primary sequences, and in part the secondary and tertiary structures, of all the synthetases had been determined, clear differences in their construction became obvious. The aminoacyl-tRNA synthetases consist either of one single polypeptide chain (a) or of two or four identical polypeptides (ot2 or 04). In addition, there are heterogeneously constructed species with two sets of two identical polypeptide chains (OC2P2). This nomenclature indicates that, for each synthetase, a or P refers to a primary structure. The number of amino acids can vary from 334 to more than 1,000. 

To say that RNA molecules are single-stranded molecules is not the same as saying that they have no higher-order structures, hi fact they have several. The formation of Watson-Crick complementary base pairs is a driving force for formation of higher-order structures. These include the stem-loop and hairpin secondary structures, as well as more complex tertiary structures. Of particular note, are the complex structures for transfer RNAs, tRNAs. Examples are provided in figure 12.5 (note that there are several nnnsnal bases in these structnres this is typical of tRNAs but not of RNA molecnles in general). These strnctures are intimately related to the function of these molecnles as adaptors in the process of protein synthesis, as developed in the next chapter. 

The base sequence and the tertiary structure of the yeast tRNA specific for phenylalanine (tRNA " ) is typical of all tRNAs. The molecule (see also p.86) contains a high proportion of unusual and modified components (shaded in dark green in Fig. 1). These include pseudouridine (T), dihydrouridine (D), thymidine (T), which otherwise only occurs in DNA, and many methylated nucleotides such as 7-methylguanidine (m G) and—in the anticodon—2 -0-methylguanidine (m G). Numerous base pairs, sometimes deviating from the usual pattern, stabilize the molecule s conformation (2). 

The start of protein synthesis is signalled by specific codon-anticodon interactions. Termination is also signalled by a codon in the mRNA, although the stop signal is not recognized by tRNA, but by proteins that then trigger the hydrolysis of the completed polypeptide chain from the tRNA. Just how the secondary and tertiary structures of the proteins are achieved is not yet clear, but certainly the mechanism of protein synthesis, which we have outlined here, requires little modification to account for preferential formation of particular conformations. 

The tertiary structure of yeast phenylalanine tRNA. (a) The full tertiary structure. Purines are shown as rectangular slabs, pyrimidines as square slabs, and hydrogen bonds as lines between slabs. (Source From G. J. Quigley and A. Rich, Structural domains of transfer RNA molecules, Science, 194 796, 1976.) (b) Nucleotide sequence. Residues... 

Removal of internal sequences in eukaryotes is not restricted to mRNA processing. It also occurs in the processing of rRNA and some tRNAs. In tRNAs the mechanism appears to be different in that the signal for splicing originates not from the primary sequence but from the secondary or tertiary structure of the pre-tRNA. 

Base-stacking interactions are an important stabilizing force in nucleic acid structures. Describe how base stacking contributes to the tertiary structure of the tRNA molecule. 

You can see the common structural features of tRNAs at both the secondary and tertiary levels. Only a few sequences or bases are common to all tRNAs. The common secondary structure of tRNAs is the cloverleaf pattern, where the 5 and 3 sequences are base-paired, and then the other three stem-loops of the cloverleaf are formed by... 

Figure 5 Protein-RNA interactions of aaRSs. The cloverleaf secondary structure of tRNA " folds into an L-shaped tertiary molecule. The tRNA can bind in an aminoacylation complex, where the 3 end is located in the canonical Class I or Class II core as shown in the upper right for the P. horikoshii LeuRS-tRNA - aminoacylation complex. In aaRSs that edit, a second complex can be formed, where the 3 end interacts with a separate domain such as the connective polypeptide insertion (CPI) that contains a hydrolytic active site as shown in the lower right for the T. thermophilus LeuRS-tRNA - editing complex. (Table (1) PDB files 1WZ2 and 2BYT).

In some cases, more is known about tertiary structure in RNA than in DNA. The full three-dimensional structure is known for some tRNA molecules from X-ray crystallography data. The structure involves several stem-loop regions of secondary structure which interact with one another to give a specific tertiary structure. The tRNA structure shown in Figure 2.8 includes stem-loop structures, unusual bases, pyrimidine-pyrimidine base pairs and... 

The enzyme A -isopentenyl pyrophosphate tRNA A -isopentenyl transferase has been further characterized. It has a molecular weight of about 55 000, needs dimethylallyl pyrophosphate (3), and is highly stereospecific in its action. The tertiary structure of the tRNA is necessary before a reaction will occur, and the enzyme then modifies the adenosine unit adjacent to the 3 -end of the anticodon. 

Helical stacking Helical stacking is one strategy for packing RNA helices into a tertiary structure. The secondary structure of tRNA consists of four short helices that radiate from the center in a cloverleaf-like shape. In its three-dimensional structure, two pairs of helices coaxially stack and perpendicularly align to yield the L-shaped tertiary structure. Coaxial stacking of helices is observed in ribozymes leading to extensive tertiary interactions between helical subdomains. 

We have tried to resolve these ambiguities g the low ten erature assignments by using tRNAs other than yeast tRNA having the same modified nucleoside in the same position of the primary sequence [ IC), but a simpler spectrum. This approach relies on the assumption that the tertiary structure of different tRNA species is similar [11]. Therefore a particular modified nucleotide located in the defined position of the sequence should have a similar chemical shift regardless of the tRNA species. 

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