TRNA, anticodon loop function

On each of the tRNA molecules, one of the single-stranded loops contains a trinucleotide sequence that is complementary to the triplet codon sequence used in the genetic code to specify a particular amino acid. This loop on the tRNA is known as the anticodon loop, and it is used to match the tRNA with a complementary codon on the mRNA. In this way the amino acids carried by the tRNA molecules can be aligned in the proper sequence for polymerization into a functional protein. 

Modified nucleosides incorporated into small RNA model systems can also be used to investigate the global versus individual effects of modified nucleotides on natural RNAs, such as rRNA or tRNA. For example, in some early studies, Yarian et al. (44) demonstrated that pseudouridine (Table 1) leads to increased thermal stability of the tRNA anticodon stem-loop region. Later, Meroueh et al. (45) demonstrated that pseudouridines have opposing effects on rRNA helix 69 stability, which depends on their specific locations and sequence contexts. These effects on stability may be important for conformational switching mechanisms in functional RNAs (46, 47). 

The structure of tRNA allows it to perform two critical functions involving the most important structural components the 3 -terminus and the anticodon loop. 

Modification of the 2 -hydroxyl moiety on the ribose can also occur in the anticodon loop of tRNA. Although methylation of nucleosides is widespread, 2 -0-ribose methylation is found occasionally in the first position of the anticodon but not the second or third. Because the modification of nucleosides in and adjacent to the anticodon loop of tRNA is commonplace, Satoh et examined the decoding efficiency of tRNA in a cell-free expression system when either the first, second, or third nucleosides in the anticodon (either C-G-A or C-U-C) were methylated at the 2 -hydroxyl. While 2 -0-methylcytosine (Cm) in the first position increased the translational efficiency, methylation of both the second and third nucleoside (both the double modification as well as individual methylations at either position) resulted in dramatic reductions in activity. This study serves as a reminder that although methylation may seem simple and diverse throughout the RNA sequence, we still have much to learn about the functionality and biological importance of these modified nucleosides. 

Post Synthetic Modification.— Various methods have been used to modify tRNA in order to relate its structure to its function in the reactions of protein synthesis. Excision of the Y nucleotide from yeast tRNA using the method reported last year, followed by resealing of the anticodon loop with T4 RNA ligase gives modified tRNA , with six nucleotides in the anticodon loop instead of seven, which is virtually non-chargeable with phenylalanine, although the half-molecules used to prepare the modified tRNA were chargeable. A... 

They prevent intramolecular hydrogen bonding (which occurs in tRNA via the usual A-U and C-G associations), thus permitting loops that are critical for function, the most important being the anticodon loop. 

Reaction of the APA probe with if has been described above (Section III,A,2). It should be possible to carry out this reaction on intact tRNA, and by suitable choice of a tRNA containing only the in loop IV, introduce a specific affinity probe into that loop. Reaction with can be obviated by choice of a tRNA lacking S, by conversion of to U, " or by chemical or photochemical blocking, i.e., by reaction with iodoaceta-mide -" or by S -C,3 cross-linking.- Other possible residues which should react with APA and similar compounds are the 2-thiouridine derivatives found in the anticodons of a number of tRNAs, 2-thiocytidine found in the anticodon loop, which can be introduced at the acceptor end of tRNA, - and 2-thiothymidine located in loop IV of some thermophilic bacteria. It is essential that attachment of the APA or APAA groups at these sites does not impair the function of tRNA. This wdll have to be tested for each individual case. 

The aminoacyl domain of tRNA composed of the CCA end, seven base pairs of the aminoacyl stem and five base pairs of the T stem, is sufficient to promote efficient binding to the EF-Tu GTP [68]. About 10 base pairs of the aminoacyl domain are bound to domain III of EF-Tu [79]. This binding is probably governed by ionic interactions of the RNA-phos-phate backbone. The sequence of nucleotides, however, also plays some role in this process. EF-Tu GTP serves for aminoacyl-tRNA not only as a vehicle transporting it to the ribosome but in addition as a matrix setting the correct conformation of the L shaped molecule [80]. The precise distance between the anticodon loop and aminoacyl-residue is evidently required for aminoacyl-tRNA functioning during translation [50]. 

As discussed earlier, AARSs have core catalytic domains that perform the functions of aminoacyl adenylate formation and transfer of the amino acid to the cognate tRNA. The sequences and structures of these domains also differentiate the enzymes as belonging to Class I or II. In addition to this class-defining active site domain, most AARSs also have one or more appended domains that are unique. These idiosyncratic domains often make specific contacts with recognition elements outside the tRNA acceptor stem, for example, at the anticodon or variable loop of the tRNA molecule (Fig. 4). In addition to the two-domain (or more) organization of the AARS enzymes, tRNAs can also be viewed as modular structures. As mentioned earlier, the acceptor stem and T4 C arm coaxially stack to form one portion of the L-shaped tRNA structure, while the D and anticodon arms stack to make the other tRNA arm (Fig. 2). The acceptor arm makes contacts with the catalytic core of the enzyme and contains the amino acid attachment site, while the anticodon, located on the second arm of the tRNA, is recognized by an appended domain. 

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