Activated tRNA

Activation tRNAs, ATP, Mg2+ aminoacyl-tRNA synthetases tRNAs, ATP, Mg2+ aminoacyl-tRNA synthetases... 

It has recendy been shown that EF-Tu is involved in another level of translation fidelity. When the correct amino acid is bound to the correct tRNA, EF-Tu is efficient at delivering the activated tRNA to the ribosome. If the tRNA and amino acid are mismatched, then either the EF-Tu does not bind the activated tRNA very well, in which case it does not deliver it well to the ribosome, or it binds the activated tRNA too well, in which case it does not release it from the ribosome. See the articles by LaRiviere et al. and Ibba cited in the bibliography at the end of this chapter for more on this topic. 

Trimming is necessary to obtain RNA transcripts of the proper size. Frequently, several tRNAs are transcribed in one long RNA molecule and must be trimmed to obtain active tRNAs. 

All tRNAs must have similar overall dimensions because, during translation, all activated tRNAs interact singly and very precisely with the same sites on the ribosome. The anticodon is positioned at one end of the tRNA to enable interaction of tRNA with the bound mRNA, and the amino acid at the other end is precisely located on the surface of the ribosome with respect to the location of the bound peptidyl transferase. 

Figure 11.15 An activated tRNA general structure and a schematic representation. 

No activated tRNA matches the stop codon so ribosome breaks apart and polypeptide chain is freed... 

In addition to their critical role in protein synthesis, it has become clear that AARSs are involved in several other cellular pathways. Some AARSs regulate their own transcription and translation, while others contribute to splicing activities in mitochondria. Nuclear aminoacylation of tRNAs by imported AARSs is thought to be a quality control mechanism to ensure that only mature, fully active tRNAs are released efficiently to the cytoplasm for protein synthesis. Programmed cell death (apoptosis) also appears to have an AARS component—human tyrosyl-tRNA synthetase can be proteolytically cleaved into two polypeptides with distinct cytokine activities, despite the lack of such activity in the full-length TyrRS. It is likely that in time many more nontranslational functions of AARS will be identified. 

FIGURE 17.13 An activated tRNA with anticodon AGU bonds to serine at the acceptor stem. 

Cellular protein biosynthesis involves the following steps. One strand of double-stranded DNA serves as a template strand for the synthesis of a complementary single-stranded messenger ribonucleic acid (mRNA) in a process called transcription. This mRNA in turn serves as a template to direct the synthesis of the protein in a process called translation. The codons of the mRNA are read sequentially by transfer RNA (tRNA) molecules, which bind specifically to the mRNA via triplets of nucleotides that are complementary to the particular codon, called an anticodon. Protein synthesis occurs on a ribosome, a complex consisting of more than 50 different proteins and several stmctural RNA molecules, which moves along the mRNA and mediates the binding of the tRNA molecules and the formation of the nascent peptide chain. The tRNA molecule carries an activated form of the specific amino acid to the ribosome where it is added to the end of the growing peptide chain. There is at least one tRNA for each amino acid. 

Selenocysteine was identified in 1976 (57) in a protein produced by Clostridium stricklandii, and it is thought to be the form in which selenium is incorporated, stoichiometricaHy, into proteins. Studies with rats show that over 80% of the dietary selenium given them is incorporated into proteins, thus selenocysteine takes on metaboHc importance. Selenoproteins having known enzymatic activities contain selenocysteine at the active sites. Two other forms of metabohc selenium are recognized methylated selenium compounds are synthesized for excretion, and selenium is incorporated into some transfer ribonucleic acids (tRNAs) in cultured cells (58). Some of the more important seleno-compounds are Hsted in Table 4. Examples of simple ring compounds are shown in Eigure 4. 

Amino-3 -deoxyadenosine. 3 -Amino-3 -deoxyadenosine (17) is elaborated by Cordyceps militarise Aspergillus nidulanSe and Helminthosporium (3,4). The biosynthesis proceeds direcdy from adenosine. Compound (17) inhibits RNA polymerase, but not DNA polymerase, and replaces the adenosyl residue at the 3 -terminus of tRNA. Phenylalanyl-(3 -amino-3 -deoxyadenosyl)-tRNA has acceptor but not donor activity (31,32). Compound (17) also inhibits retroviral RNA-dependent DNA polymerase (33). 

Puromycin. Puromycin (19), elaborated by S. alboniger (1—4), inhibits protein synthesis by replacing aminoacyl-tRNA at the A-site of peptidyltransferase (48,49). Photosensitive analogues of (19) have been used to label the A-site proteins of peptidyltransferase and tRNA (30). Compound (19), and its carbocycHc analogue have been used to study the accumulation of glycoprotein-derived free sialooligosaccharides, accumulation of mRNA, methylase activity, enzyme transport, rat embryo development, the acceptor site of human placental 80S ribosomes, and gene expression in mammalian cells (51—60). 

Aminohexose Nucleosides. The 4-aminohexose nucleosides (128—140) are Hsted in Table 7 (1—4,240—242). A biosynthetic relationship between the 4-aminohexose peptidyl nucleoside antibiotics and the pentopyranines has been proposed (1). The 4-aminohexose pyrimidine nucleoside antibiotics block peptidyl transferase activity and inhibit transfer of amino acids from aminoacyl-tRNA to polypeptides. Hikizimycin, gougerotin, amicetin, and blasticidin S bind to the peptidyl transferase center at overlapping sites (243). 

In recent year s, clinical studies on the role of uiinai y luodified nucleosides as the biochemical mai kers of various types of cancer have been actively undertaken. Most of the urinai y modified nucleosides ai e piimai ily originated by methylation of either the base part, the sugar hydroxyl par t, or in some cases, both par ts of the course of biodegradation of tRNA molecules. Hence, their isolation and identification plays a major role in biochemical analysis. 

Figure 4.15 Schematic diagram of the enzyme tyrosyl-tRNA synthetase, which couples tyrosine to its cognate transfer RNA. The central region of the catalytic domain (red and green) is an open twisted a/p stmcture with five parallel p strands. The active site is formed by the loops from the carboxy ends of P strands 2 and S. These two adjacent strands are connected to a helices on opposite sides of the P sheet.

Figure 4.16 A schematic view of the active site of tyrosyl-tRNA synthetase. Tyrosyl adenylate, the product of the first reaction catalyzed by the enzyme, is bound to two loop regions residues 38-47, which form the loop after p strand 2, and residues 190-193, which form the loop after P strand 5. The tyrosine and adenylate moieties are bound on opposite sides of the P sheet outside the catboxy ends of P strands 2 and 5.

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

Tetracycline and its derivative doxycycline are antibiotics widely used in the treatment of bacterial infections. They also exert an antimalarial activity. Tetracyclines inhibit the binding of aminoacyl-tRNA to the ribosome during protein synthesis. 

Scheme 18 Possible carboxylate group participation in the activation of tyrosine in tyrosyl-tRNA synthetase...

Selenocysteine, an essential active site residue in several mammahan enzymes, arises by co-translational insertion of a previously modified tRNA. 

A ribosome is a cytoplasmic nucleoprotein stmcture that acts as the machinery for the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and tRNA molecules interact to translate into a specific protein molecule information transcribed from the gene. In active protein synthesis, many ribosomes are associated with an mRNA molecule in an assembly called the polysome. 

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