Biological Chemistry - Berezov, T. T., & Korovkin, B. F. 1998

Protein Biosynthesis
Translation and general requirements for protein synthesis in a cell-free system
Transfer RNAs

M. Hoagland's laboratory demonstrated that when a ¹⁴C-amino acid was incubated with the soluble cytoplasmic fraction in the presence of ATP, followed by The addition of trichloroacetic acid, no radioactive label was detected in the resulting protein precipitate. These findings led to the Conclusion that the labeled amino acid is not incorporated into the protein molecule. Instead, the label was found to be covalently bound to RNA contained within the protein-free filtrate. Further research revealed that the RNA to which the labeled amino acid attaches has a low molecular weight and is localized in the soluble fraction; consequently, it was initially termed soluble RNA, and later adaptor or Transfer RNA (tRNA). tRNA accounts for about 10–15% of total cellular RNA. To date, over 60 different tRNAs have been discovered. There is at least one specific tRNA in The Cell for each amino acid (for several Amino Acids, more than one has been found: specifically, 6 different tRNAs for Serine, leucine, and Arginine, and 4 different tRNAs each for Alanine, Threonine, and Glycine, although in these cases as well, each tRNA is associated with a specific aminoacyl-tRNA synthetase). The Molecular Weight of most tRNAs ranges from 24,000 to 29,000. They contain 75 to 85 NUCLEOTIDES, of which 8 or more are modified bases. Amino acids ultimately attach to the free 3'-OH group (see above regarding Class 1 aminoacyl-tRNA synthetase Enzymes) of the terminal mononucleotide, which in all tRNAs is AMP (adenylic acid), via an ester bond. Interestingly, almost all tRNAs share not only remarkably similar Functions, but also a very similar three-dimensional Structure (Fig. 14.3).

The Introduction/19.html">Primary Structure of nearly all 60 discovered tRNAs has been established (Fig. 14.4). Knowledge of The nucleotide sequence, and consequently the composition of tRNAs, has provided researchers with a wealth of valuable information regarding the Biological Role of individual tRNA components. tRNAs also share a common native three-dimensional structure determined by X-ray crystallography, originally termed the cloverleaf conformation; in reality, this conformation adopts an inverted L-shape (see Fig. 14.3). Elucidation of tRNA Structure by this method revealed several distinct features. Helical regions, unusual Hydrogen Bonds, and hydrophobic interactions within non-helical regions were discovered in the tRNA molecule. It has been shown that tRNA contains a pseudouridine loop, formed by nucleotides containing pseudouridine (TψC), and a dihydrouridine loop. Both loops participate in forming the corner of the L-shape. The 3'-OH terminus features a CCA-OH triplet sequence identical in all tRNAs, to which the specific amino acid is attached via an ester bond. Binding occurs primarily through the 3'-OH group of the terminal adenylyl nucleotide, although, as noted, evidence exists for the possibility of preliminary amino acid attachment via its 2'-OH group as well.

The specific roles of individual tRNA regions are not yet fully understood. In particular, the pseudouridine loop appears to mediate the binding of aminoacyl-tRNA to the ribosome, while the dihydrouridine loop is most likely required as a recognition site for the specific enzyme, aminoacyl-tRNA synthetase. Additionally, there is an extra loop whose composition varies among Different types of tRNA molecules; its function remains unknown. An essential region with a fully elucidated function is the anticodon loop, which bears the triplet known as the anticodon and is located on the opposite side of the molecule from The amino acid-attachment end. The anticodon loop consists of 7 nucleotides: three occupy the central position and form the highly specific anticodon itself, two nucleotides are located on either side of it—including a modified purine and a variable base on one side, and two pyrimidine bases on the other. The anticodon is specific and complementary to the corresponding mRNA codon, with both showing antiparallel complementarity.

Careful analysis of The nucleotide sequences of various tRNAs has shown that they all contain an identical 5'-terminal nucleotide—GMP—with a free 5'-phosphate group. The adaptor function of tRNA molecules consists in binding each tRNA molecule to its specific amino acid. However, since there is no structural correspondence or affinity between the nucleic acid and the specific Functional groups of amino acids, this recognition function—acting essentially as a molecular intermediary between tRNA and the amino acid—must be performed by the protein molecule of the enzyme. The interaction between aminoacyl-tRNA synthetase and tRNA is commonly referred to as the "second Genetic Code," emphasizing its pivotal role in ensuring The fidelity of Protein Synthesis. Moreover, the coding rules are likely more complex than those of the "first" genetic code (see below).