Transfer RNA aminoacylation

The genetic information in DNA is converted into the linear sequence of amino acids in polypeptides in a two-phase process. During transcription, RNA molecules are synthesized from a DNA strand through complementary base pairing between the bases in DNA and the bases in free ribonucleoside triphosphate molecules. During the second phase, called translation, mRNA molecules bind to ribosomes that are composed of rRNA and ribosomal proteins. Transfer RNA-aminoacyl complexes position their amino acid cargo in the catalytic site within the ribosome in a process that involves complementary base pairing between the mRNA codons and tRNA anticodons. Once the amino acids are correctly positioned within the catalytic site, a peptide bond is formed. After the mRNA molecule moves relative to the ribosome, a new codon enters the ribosome s catalytic site and base pairs with the appropriate anticodon on another aminoacyl-tRNA complex. After a stop codon in the mRNA enters the catalytic site, the newly formed polypeptide is released from the ribosome. 

The genetic code (which includes the codon) serves as a basis for establishing how genes encoded in DNA are decoded into proteins. A critical interaction in protein synthesis is the interaction between the codon in messenger RNA (mRNA) and the anticodon in an aminoacyl-transfer RNA (aminoacyl-tRNA). 

The mechanism whereby RNA is translated into protein is complex, and the cell devotes considerable resources to the translational machinery. The components include 20 different amino acids, transfer RNAs, aminoacyl-tRNA synthetases, ribosomes, and a number of protein factors which cycle on and off the ribosomes and facilitate various steps in initiation of translation, elongation of the nascent polypeptide chain, and termination of synthesis with release of the completed polypeptide from the ribosome. The process depends on a supply of energy provided by ATP and GTP. The rate of protein synthesis is typically in the range of 6 (immature red blood cells of the rabbit) to 20 Escherichia coli growing optimally) peptide bonds per sec. at 37°C. 

Since 2001, 40 non-natural amino acids have been added into protein by creating a unique codon (recoding) and a corresponding transfer-RNA aminoacyl - tRNA-synthetase pair to encode it with diverse physicochemical and biological properties in order to be used as a tool to exploring protein structure and function or to create novel or enhanced proteins. 

Transfer RNA (tRNA) serves as a carrier of amino acid residues for protein synthesis. Transfer RNA molecules also fold into a characteristic secondary structure (marginal figure). The amino acid is attached as an aminoacyl ester to the 3 -terminus of the tRNA. Aminoacyl-tRNAs are the substrates for protein biosynthesis. The tRNAs are the smallest RNAs (size range—23 to 30 kD) and contain 73 to 94 residues, a substantial number of which are methylated or otherwise unusually modified. Transfer RNA derives its name from its role as the carrier of amino acids during the process of protein synthesis (see Chapters 32 and 33). Each of the 20 amino acids of proteins has at least one unique tRNA species dedicated to chauffeuring its delivery to ribosomes for insertion into growing polypeptide chains, and some amino acids are served by several tRNAs. For example, five different tRNAs act in the transfer of leucine into... 

Figure 8.4 outlines the proeess of protein synthesis involving the ribosome, ruRNA, a series of aminoacyl transfer RNA (tRNA) moleeules (at least one for eaeh amino aeid)... 

L. Feng D. Tumbula-Hansen B. Min S. Namgoong J. Salazar O. Orellana D. Soil, Transfer RNA-Dependent Amidotransferases Key Enzyme for Asn-tRNA and GIn-tRNA Synthesis in Nature. In The Aminoacyl-tRNA Synthetases M. Ibba,... 

Many examples of catalytic nucleic acids obtained by in vitro selection demonstrate that reactions catalyzed by ribozymes are not restricted to phosphodiester chemistry. Some of these ribozymes have activities that are highly relevant for theories of the origin of life. Hager et al. have outlined five roles for RNA to be verified experimentally to show that this transition could have occurred during evolution [127]. Four of these RNA functionalities have already been proven Its ability to specifically complex amino acids [128-132], its ability to catalyze RNA aminoacylation [106, 123, 133], acyl-transfer reactions [76, 86], amide-bond formation [76,77], and peptidyl transfer [65,66]. The remaining reaction, amino acid activation has not been demonstrated so far. 

Transfer RNAs are small, single-stranded polynucleotides (70-90 bases long). The tRNA molecule is linked to its specific amino acid in a reaction that is catalysed by the enzyme tRNA-aminoacyl synthetase. It occurs in two stages ... 

Although all tetracyclines have a similar mechanism of action, they have different chemical structures and are produced by different species of Streptomyces. In addition, structural analogues of these compounds have been synthesized to improve pharmacokinetic properties and antimicrobial activity. While several biological processes in the bacterial cells are modified by the tetracyclines, their primary mode of action is inhibition of protein synthesis. Tetracyclines bind to the SOS ribosome and thereby prevent the binding of aminoacyl transfer RNA (tRNA) to the A site (acceptor site) on the 50S ri-bosomal unit. The tetracyclines affect both eukaryotic and prokaryotic cells but are selectively toxic for bacteria, because they readily penetrate microbial membranes and accumulate in the cytoplasm through an energy-dependent tetracycline transport system that is absent from mammalian cells. 

The translation of the mRNA into proteins is the final step in the biological flow of information (see Fig. 6.1). Similar to other macromolecular polymerizations, protein synthesis can be divided into initiation, chain elongation, and termination. Critical players in this process are the aminoacyl transfer RNAs (tRNAs). These molecules form the interface between the mRNA and the growing polypeptide. Activation of tRNA involves the addition of an amino acid to its acceptor stem, a reaction catalyzed by an aminoacyl-tRNA synthetase. Each aminoacyl-tRNA synthetase is highly specific for one amino acid and its corresponding tRNA molecule. The anticodon loop of each aminoacyl-tRNA interacts... 

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. 

The second key advance was made by Mahlon Hoagland and Zamecnik, when they found that amino acids were activated when incubated with ATP and the cytosolic fraction of liver cells. The amino acids became attached to a heat-stable soluble RNA of the type that had been discovered and characterized by Robert Holley and later called transfer RNA (tRNA), to form aminoacyl-tRNAs. The enzymes that catalyze this process are the aminoacyl-tRNA synthetases. 

A third mechanism of synthesis, which was only recently recognized, appears to provide the sole source of asparagine for many bacteria.98b The asparagine-specific transfer RNA tRNAAsn is "mischarged" with aspartic acid to form Asp-tRNAAsn. This compound is then converted to the properly aminoacylated Asn-tRNAAsn by a glutamine-dependent amidotransferase. (The entire ATP-dependent sequence is shown in Eq. 29-6.) The activated asparaginyl group is then transferred from Asn-tRNAAsn into proteins as they are synthesized. 

Overview of reactions in protein synthesis. (aab aa2, aa3 = amino acids l, 2, 3.) Protein synthesis requires transfer RNAs for each amino acid, ribosomes, messenger RNA, and a number of dissociable protein factors in addition to ATP, GTP, and divalent cations. First the transfer RNAs become charged with amino acids, then the initiation complex is formed. Peptide synthesis does not start until the second aminoacyl tRNA becomes bound to the ribosome. Elongation reactions involve peptide bond formation, dissociation of the discharged tRNA, and translocation. The elongation process is repeated many times until the termination codon is reached. Termination is marked by the dissociation of the messenger RNA... 

Identity elements in four tRNAs. Each circle represents one nucleotide. Filled circles indicate nucleotides that serve as recognition elements to the appropriate aminoacyl-tRNA synthase. It is possible that other identity elements occur in these structures that are still to be discovered. (From L. H. Schulman and J. Abelson, Recent excitement in understanding transfer RNA identity, Science 240 1591, June 17, 1988. Copyright 1988 by the AAAS. Reprinted by permission.)... 

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. 

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. 

Brown JR, Gentry D, Becker JA, Ingraham K, Holmes DJ, Stanhope MJ (2003) Horizontal transfer of drug-resistant aminoacyl-transfer-RNA synthetases of anthrax and Gram-positive pathogens. EMBO Rep 4 692-698... 

Hendrickson T, Schimmel P. Transfer RNA-dependent amino acid discrimination by aminoacyl-tRNA synthetases. In Translation Mechanisms. Lapointe J, Brakier-Gingras L, eds. 2003. Kluwer Academic/Plenum Publishers, New York. p. 34-64. 

Robertson SA, Ellman JA, Schultz PG. A general and efficient route for chemical aminoacylation of transfer RNAs. J. Am. Chem. Soc. 1991 113 2722-2729. 

A new translation. A transfer RNA with a UGU anticodon is enzymatically conjugated to i C-labeled cysteine. The cysteine unit is then chemically modified to alanine (with the use of Raney nickel, which removes the sulfur atom of cysteine). The altered aminoacyl-tRNA is added to a protein-synthesizing system containing normal components except for this tRNA. The mRNA added to this mixture contains the following sequence ... 

Transfer RNA molecules (tRNAs), messenger RNA, and many proteins participate in protein synthesis along with ribosomes. The link between amino acids and nucleic acids is first made by enzymes called aminoacyl-tRNA synthetases. By specifically linking a particular amino acid to each tRNA, these enzymes implement the genetic code. This chapter focuses primarily on protein synthesis in prokaryotes because it illustrates many general principles and is relatively well understood. Some distinctive features of protein synthesis in eukaryotes also are presented. 

Figure 29.22. Transfer RNA-Binding Sites. (A) Three tRNA-binding sites are present on the 70S ribosome. They are called the A (for aminoacyl), P (for peptidyl), and E (for exit) sites. Each tRNA molecule contacts both the SOS and the 50S subunit. (B) The tRNA molecules in sites A and P are base paired with mRNA.

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