Acceptor arm, of tRNA

FIGURE 4 Domain organization of synthetases and tRNAs. The class-defining domain of the aminoacyl-tRNA synthetase contains conserved structural features and contacts the acceptor arm of tRNA. These contacts are mediated by additions to the classdefining catalytic domain. Appended nonconserved protein domains interact with other portions of the tRNA, including in many cases the anticodon (as indicated by the dotted line). [From Schim-mel, R, and Ribas de Pouplana, L. (1995). Cell81, 983-986.]... 

All tRNA molecules contain four main arms. The acceptor arm terminates in the nucleotides CpCpAoH. These three nucleotides are added posttranscription-ally. The tRNA-appropriate amino acid is attached to the 3 -OH group of the A moiety of the acceptor arm. 

The regions of the tRNA molecule teferred to in Chapter 35 (and illustrated in Figure 35-11) now become important. The thymidine-pseudouridine-cyti-dine (T PC) arm is involved in binding of the amino-acyl-tRNA to the ribosomal surface at the site of protein synthesis. The D arm is one of the sites important for the proper recognition of a given tRNA species by its proper aminoacyl-tRNA synthetase. The acceptor arm, located at the 3 -hydroxyl adenosyl terminal, is the site of attachment of the specific amino acid. 

All tRNAs in solution fold into a three-dimensional L-shaped structure like that of tRNAphe (fig. 29.5b). This structure is composed of two helical arms joined at right angles. The ribose moiety to which the amino acid is joined is at the end of one arm, identifying it as the acceptor arm. The anticodon is at the end of the other arm, identifying it as... 

The bases within the base-paired region of each arm of the tRNA cloverleaf stack in a manner similar to the base stacking described in Chapter 25 for DNA. In addition to the base stacking within base-paired regions, there is also stacking of one helix on top of another in the tRNA molecule. In particular, the acceptor stem stacks with the T PC stem and loop to form one nearly continuous stacked double helix. 

Regarding the stmctural features that enable EF-Tu and SelB to distinguish their tRNAs, an extensive mutational analysis was performed and synthetic RNA minihelices were analyzed for binding to EF-Tu and SelB. It was found that both the length of the aminoacyl-tRNA acceptor stem and the basal helical part of the acceptor arm were crucial. ... 

All tRNAs have a similar three-dimensional structure that includes an acceptor arm for attachment of a specific amino acid and a stem-loop with a three-base anticodon sequence at its ends (see Figure 4-22). The anticodon can base-pair with its corresponding codon in mRNA. 

One of the four arms of the tRNA secondary structure is the acceptor arm. It consists of seven base pairs and a single-stranded 3 -terminus. At the 3 -terminal nucleotide (invariably A76), the amino acid is specifically attached. The aminoacylation of the tRNA occurs by esterification of the amino acid to the 2 or 3 -OH group of the ribose of adenosine A76 [15]. This reaction is catalyzed by aminoacyl-tRNA synthetases. 

Earlier biochemical studies have demonstrated that in many cases the main identification features or recognition elements necessary for the correct recognition of tRNAs by their cognate aminoacyl-tRNA synthetases are localized in the acceptor stem [16-19]. Frequently, these recognition sites are referred to as identity elements since they make this particular tRNA unique and distinguishable from all other tRNAs for the corresponding aminoacyl-tRNA synthetase. For the tRNA from E. coli it could be shown by the groups of Schim-mel [20, 21] and McClain [19, 22-24] that a wobble base pair G3-U70 at position 3 of the acceptor arm represents the major identity element. 

Subsequently, it was demonstrated that even a truncated tRNA, consisting only of the acceptor arm with the single-stranded 3 -end, is recognized as a substrate by the alanyl-tRNA synthetase (ARS) and correctly aminoacylated with alanine [21, 25], provided that the G3-U70 base pair is present at the correct location. 

Figure 19.4 Imino region of the H NMR spectra of the acceptor arm duplex derived from yeast tRNA ° before (lower trace) and after (upper trace) addition of 7.5 pM MnCl2 (RNA concentration 1.65 mM, 7.5 mM MgCy at 277 K.

Though one-dimensional H NMR studies of the tRNA acceptor arm gave hints to the special role of the G-U base pair, detailed information on the possible modifications of the local helical geometry in the vicinity of the G-U pair caimot be obtained by these experiments. 

Figure 19.5 Schematic representation of the observed intemucleotide NOESY contacts in the E. coli tRNA derived acceptor arm duplex (18mer/GU) for mixing time of 300 ms at 303 K.

Figure 19.7 Model of the tRNA acceptor arm structure as determined on the basis of the 300 ms NOESY spectrum using the IRMA procedure [45] and restrained molecular dynamics. Three structures resulting from different calculations are superimposed. The continuation of the helix geometry into the single-stranded terminus is clearly visible. The black sphere marks the most probable location of the bound manganese ion in the vicinity of G3-U70 base pair.

The results presented above demonstrate that there is a deviation from the regular A-hehcal geometry in the wild-type tRNA acceptor arm that is mainly characterized by a displacement of the U70 base. Thereby the base plane overlap between C71 and U70 is dis-... 

The other arms of the tRNA cloverleaf also have distinctive conserved features. The modified base dihydrouridine (D) is typically present in the loop that closes off a short 3- or 4-bp stem following the acceptor stem. This stem and loop are therefore called the D-arm. The anticodon arm consists of a 5-bp helix closed by a loop that contains the trinucleotide anticodon. Following the anticodon arm is the variable loop, which can contain 3-21 nucleotides, with a stem as long as 7 bp, depending on the particular tRNA. The modified bases pseudouridine (4>) and ribo-thymidine (T) are usually present in the loop of the T FC arm, so named because of the presence of this highly conserved sequence. 

The two portions of the L can be considered distinct domains with separate contributions to protein synthesis. The minihelix containing the acceptor arm includes the site of amino acid attachment (the 3 -OH). It is considered by many investigators to be related to the historical or early form of tRNA. The anticodon trinucleotide is located at the other end of the L, approximately 75 A away. This... 

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. 

The first orthogonal E. coli tRNA-synthetase pair generated from archaeal bacteria was derived from the tyrosyl pair taken from Methanococcus janmschii In vitro experiments showed that the major recognition elements of M. jannaschii tRNA" include the discriminator base A73 and the first base pair, C1-G72, in the acceptor stem (Figure 2(a)). The anticodon triplet participates only weakly in identity determination. By contrast, E. coli uses A73, G1-C72, a long variable arm, and the anticodon as identity elements. The M. 

Each tRNA has a cloverleaf secondary structure containing an anticodon arm, a D (or DHU) arm, a T or TTC arm, and an amino acid acceptor stem to which the relevant amino acid becomes covalently bound, at the 3 OH group. Some tRNAs also have a variable (or optional) arm. The three-dimensional structure is more complex because of additional interactions between the nucleotides. 

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