Aminoacyl attachment site

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 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. 

The a-amino group of the new aminoacyl-tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P site (peptidyl or polypeptide site). At initiation, this site is occupied by aminoacyl-tRNA mef. This reaction is catalyzed by a peptidyltransferase, a component of the 285 RNA of the 605 ribosomal subunit. This is another example of ribozyme activity and indicates an important—and previously unsuspected—direct role for RNA in protein synthesis (Table 38-3). Because the amino acid on the aminoacyl-tRNA is already activated, no further energy source is required for this reaction. The reaction results in attachment of the growing peptide chain to the tRNA in the A site. 

Several key concepts are worth remembering. GTP is used as an energy source for translation, but ATP is used to form the aminoacyl-tRNA. The ribosome effectively has two kinds of tRNA binding sites. Only tRNAMet can bind to the P (for peptide) site, and this only occurs during the initial formation of the functional ribosome (initiation). All other aminoacyl-tRNAs enter at the A (for amino acid) binding site. After formation of the peptide bond (this doesn t require GTP hydrolysis), the tRNA with the growing peptide attached is moved (translocated) to the other site (this does require GTP hydrolysis). 

During translation, the amino adds are attached to the 3 ends of their respective tRNAs. The aminoacyl-tRNAs are situated in the P and A sites of the ribosome as shown in Figure 1-4-8, Sotice that the peptide bond forms between the carboxyl group of the amino add (or growing peptide) in the P site and the amino group of the ne rt amino add in the A site. Proteins are syn-desized from the amino to the carboxyl terminus. 

Fig. 11. Comparison of the peptidyl transfer reaction in the ribosome and in the selected peptidyltransferase ribozyme. The ribosome contains a binding site for the peptidyl-tRNA (P-site) and for the aminoacyl-tRNA (A-site). In the selected ribozyme the binding site for the AMP-Met-Bio substrate would be analogous to the P-site. The attacking a-amino group which is bound in the A-site in the ribosome is covalently attached to the 5 -end in the ribozyme. Catalytically active RNAs preferentially attach the biotin tag onto themselves and can thus be separated from the inactive ones...

As the name implies, this class of compounds has four linearly attached six membered rings. Tetracyclines are bacteriostatic and reversibly bind to the 30S ribosomal subunit. They interfere with the binding of aminoacyl tRNA at the A-site of the ribosome. ... 

Chloramphenicol (Chloromycetin) is a nitrobenzene derivative that affects protein synthesis by binding to the 50S ribosomal subunit and preventing peptide bond formation. It prevents the attachment of the amino acid end of aminoacyl-tRNA to the A site, hence the association of peptidyltransferase with the amino acid substrate. Resistance due to changes in the ribosomebinding site results in a decreased affinity for the drug, decreased permeability, and plasmids that code for enzymes that degrade the antibiotic. 

The answer is b. (Hardman, p 1131.) Chloramphenicol inhibits protein synthesis in bacteria and, to a lesser extent, in eukaryotic cells. The drug binds reversibly to the SOS ribosomal subunit and prevents attachment of aminoacyl-transfer RNA (tRNA) to its binding site. The amino acid substrate is unavailable for peptidyl transferase and peptide bond formation. 

While peptide antibiotics are synthesized according to enzyme-controlled polymerization patterns, both proteins and nucleic acids are made by template mechanisms. Tire sequence of their monomer emits is determined by genetically encoded information. A key reaction in the formation of proteins is the transfer of activated aminoacyl groups to molecules of tRNA (Eq. 17-36). Tire tRNAs act as carriers or adapters as explained in detail in Chapter 29. Each aminoacyl-tRNA synthetase must recognize the correct tRNA and attach the correct amino acid to it. The tRNA then carries the activated amino acid to a ribosome, where it is placed, at the correct moment, in the active site. Peptidyltransferase, using a transacylation reaction, in an insertion mechanism transfers the C terminus of the growing peptide chain onto the amino group of... 

The anticodon of the tRNA is base-paired with mRNA in the "decoding site" on the 30S subunit. The 3-CCA end with attached aminoacyl group lies in the peptidyltransferase site in the 50S subunit. (F) and (G) are courtesy of Cate et al.18... 

The ribosomal translocation process is quite complex. As the tRNAs move from A to P to E sites on the 16S RNA platform, the mRNA must also move in discrete single-codon steps. Tire acceptor stems of the tRNAs in the A and P sites must react at the appropriate times in the peptidyltransferase center. Study of protection from chemical probes suggests that tRNAs sometimes lie with the anticodon loop in the A site of the small ribosomal subunit, while the acceptor stem is in the P site of the large subunit (an A/P site as illustrated in Fig. 29-12B). Each aminoacyl-tRNA enters as a complex with EF-Tu and may initially bind with its anticodon in the A site and the acceptor stem with attached EF-Tu in a transient T site, the composite state being A/T. After loss of EF-Tu the acceptor stem can move into the A site to give an A/A state. The peptidyltransferase reaction itself necessarily involves movement at the acceptor stems by 0.1 nm or more. However, additional movement of 1 nm is needed to move the two tRNAs into states A/P and P/E, respectively. Movement of the mRNA then moves the... 

Synthases differ with respect to their site of attachment to tRNA. Some synthases form the 2 ester, some form the 3 ester, and still others produce a mixture of the two. The specificity of the synthases was determined by analyzing their ability to act on tRNA derivatives lacking one or the other terminal hydroxyl group. Once esterified to the terminal ribose, the aminoacyl group can migrate between the vicinal 2 and 3 hydroxyl groups. Thus, in cells, amino-acyl-tRNAs are mixtures of 2 and 3 esters. Only the 3 derivative is a substrate for the subsequent transpeptidation reaction catalyzed by the ribosome. 

Formation of the first peptide linkage. The formylmethionine group is transferred from its tRNA at the P site to the amino group of the second aminoacyl-tRNA at the A site of the ribosome. This involves nucleophilic attack by the amino group of the second amino acid on the carboxyl carbon of the methionine. The resulting bond formation attaches both amino acids to the tRNA at the A site. 

Most transfer RNAs have common parts and uncommon parts. The common parts facilitate binding of the aminoacyl-tRNAs to common sites on the ribosome. The uncommon sites permit specific reactions with charging enzymes that covalently attach the correct amino acids to the correct tRNA. Another uncommon site on the tRNAs is the anticodon, which leads to specific complex formation with the complementary codon site on the messenger. 

The synthetase consists of the three modules E1, E2, and E3 (for a complete description, see Sec. II. A). Each module is composed of an activation site forming the acyl or aminoacyl adenylate, a carrier domain which is posttranslationally modified with 4 -phosphopantetheine (Sp), and a condensation domain (Cl, C2) or, alternatively, a structurally similar epimerization domain (Ep). Activation of aminoadipate (Aad) leads to an acylated enzyme intermediate, in which Aad is attached to the terminal cysteamine of the cofactor (El-Spl-Aad) [reactions (1) and (2)]. Likewise, activation of cysteine (Cys) leads to cysteinylated module 2 [reactions (3) and (4)]. For the condensation reaction to occur between aminoadipate as donor and cysteine as acceptor, both intermediates are thought to react at the condensation site of module 1 (Cl). Each condensation site is composed, in analogy to ribosomal peptide formation, of an aminoacyl and a peptidyl site. In this case of initiation, the thioester of Aad enters the P-site, while the thioester of Cys enters the A-site. Condensation occurs and leaves the dipeptidyl intermediate Aad-Cys at the carrier protein of the second module [reaction (5)]. The third amino acid valine is activated on module 3, and Val is attached to the carrier protein 3 [reactions (6) and (7)]. Formation of the tripeptide occurs at the second condensation site C2, with the dipeptidyl intermediate entering the P-site and the valiny 1-intermediate the A-site [reaction (8)]. 

Deoxyribonucleic acid is the genetic material such that the information to make all the functional macromolecules of the cell is preserved in DNA (Sinden, 1994). Ribonucleic acids occur in three functionally different classes messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA) (Simons and Grun-berg-Manago, 1997). Messenger RNA serves to carry the information encoded from DNA to the sites of protein synthesis in the cell where this information is translated into a polypeptide sequence. Ribosomal RNA is the component of ribosome which serves as the site of protein synthesis. Transfer RNA (tRNA) serves as a carrier of amino acid residues for protein synthesis. Amino acids are attached as aminoacyl esters to the 3 -termini of the tRNA to form aminoacyl-tRNA, which is the substrate for protein biosynthesis. 

In the first step of the peptidyl transferase reaction, a peptidyl tRNA molecule is bound in the P-site with its nascent peptide extending down the peptide exit tunnel (Fig. 4.1). An elongation factor binds to a factor binding site (FBS) and positions an aminoacyl-tRNA in the A-site. The a amino group of the aminoacyl-tRNA nucleophilically attacks the ester bond which connects the peptide to the tRNA bound in the P-site (Fig. 4.2). The ester bond is broken as an amide bond forms, and the peptide becomes one amino acid longer, and is now attached to the tRNA that in the A-site. Translocation of the products follows peptide bond formation, as the newly formed deacylated- tRNA of the P-site moves into the E-site, and as the newly elongated peptidyl-tRNA moves from the A-site into the P-site. 

Tetracyclines inhibit bacterial protein synthesis by binding to the 30S snbnnit of the ribosome, thns blocking the attachment of aminoacyl-tRNA to the receptor site on the messenger RNA-ribosome complex. 

Figure 5.31. Symbolic Diagram of an Aminoacyl-tRNA. The amino acid is attached at the 3 end of the RNA. The anticodon is the template-recognition site.

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