Aminoacyl-RNA synthetases

The first step, the amino-acid activation reaction (1), is catalysed by a specific activating enzyme or aminoacyl-RNA synthetase. With the reaction between the carboxyl group of the aa.i and the pyrophosphoryl group of ATP. a mixed... 

As reviewed by Stulberg and Novelli in 1962, several methods are available to measure the activity of the aminoacyl-RNA synthetases. The kinetic parameters are generally derived using the ATP exchange reaction, and the isolation of the aminoacyl-transfer RNAs. 

When a wrong amino acid is placed on the transfer RNA, for example when Cys-tRNA is chemically reduced to Ala-tRNA > t. the synthetase (Cys) becomes unable to remove the wrong amino acid (Ala), finally resulting in the synthesis of a wrong protein. A correct insertion is thus entirely dependent upon the conformable sites involved in the mutual recognition of the transfer RNA, and the aminoacyl-RNA synthetase. 

The information contained in the DNA (i.e., the order of the nucleotides) is first transcribed into RNA. The messenger RNA thus formed interacts with the amino-acid-charged tRNA molecules at specific cell organelles, the ribosomes. The loading of the tRNA with the necessary amino acids is carried out with the help of aminoacyl-tRNA synthetases (see Sect. 5.3.2). Each separate amino acid has its own tRNA species, i.e., there must be at least 20 different tRNA molecules in the cells. The tRNAs contain a nucleotide triplet (the anticodon), which interacts with the codon of the mRNA in a Watson-Crick manner. It is clear from the genetic code that the different amino acids have different numbers of codons thus, serine, leucine and arginine each have 6 codewords, while methionine and tryptophan are defined by only one single nucleotide triplet. 

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,... 

Building on earlier work of Osawa and co-workers [55], Oliver and Kowal [52] tested the feasibility of introducing a noncoded amino acid at an unassigned codon in M. luteus. DNA templates were prepared which coded for 19-mer polypeptides containing either the unassigned codon AGA(Arg) or the termination codon TAG at position 13 under the control of a T7 RNA polymerase promoter. The corresponding tRNAs, produced as described in Sect. 2, were based on tRNA and acylated with phenylalanine. The tRNA was modified to prevent recognition by the alanine aminoacyl-tRNA synthetase and to increase translational efficiency. 

A ribozyme activity that led to RNA-modifications that are analogous to the 5 -5 pyrophosphate caps of eukaryotic RNA transcripts was selected by Huang and Yarns [84]. Actually the author s intention was to isolate ribozymes which catalyze the formation of a mixed anhydride between an amino acid carboxylate and a 5 -terminal phosphate of an RNA, an activity that is chemically analogous to the activation of amino acids by ATP catalyzed by aminoacyl tRNA synthetases. However, while the selected ribozymes did... 

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 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. 

Among the many proteins that bind to RNA molecules437 39 are the aminoacyl-tRNA synthetases, a variety of other well known enzymes,440 the ribosomal proteins discussed in Chapter 29, and various proteins with dual functions of catalysis and regulation of... 

Not all aminoacyl-tRNA synthetases have editing sites. The cysteinyl- and tyrosyl-tRNA synthetases bind the correct substrates so much more tightly than their competitors that they do not need to edit.13,14 Similarly, since the accuracy of transcription of DNA by RNA polymerase is better than the overall observed error rate in protein synthesis at about 1 part in 104, RNA polymerases do not need to edit.15 The same should be true for codon-anticodon interactions on the ribosome. However, it is possible that accuracy has been sacrificed to achieve higher rates in this case, which is analogous to a change from Michaelis-Menten to Briggs-Haldane kinetics, and so an editing step is required.16... 

Several systems based on the potentialities of amino acid-phosphoric acid mixed anhydrides have been devised to check the idea that the genetic code developed from an early pathway of RNA-dependent peptide synthesis in an RNA world [168]. RNA sequences have thus been selected that are capable of self-aminoacylation using amino acid adenylates, catalyzing a reaction chemically similar to the aminoacylation of fRNA by the protein aminoacyl fRNA synthetases [169]. 

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... 

Jhe synthesis of proteins, as characterized by the in vitro incorporation of amino acids into the protein component of cytoplasmic ribonu-cleoprotein, is known to require the nonparticulate portion of the cytoplasm, ATP (adenosine triphosphate) and GTP (guanosine triphosphate) (15, 23). The initial reactions involve the carboxyl activation of amino acids in the presence of amino acid-activating enzymes (aminoacyl sRNA synthetases) and ATP, to form enzyme-bound aminoacyl adenylates and the enzymatic transfer of the aminoacyl moiety from aminoacyl adenylates to soluble ribonucleic acid (sRNA) which results in the formation of specific RNA-amino acid complexes—see, for example, reviews by Hoagland (12) and Berg (1). The subsequent steps in pro-... 

Knorre, D. G., Lavrik, O. I. Affinity labeling of aminoacyl-t-RNA synthetases, in Theory and Practice in Affinity Techniques, (ed. Sundaram, P. V., Eckstdn, F.) Academic Press, Lonson 1978, p. 169... 

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. 

Salazar JC, Ambrogelly A, Crain PF, Me Qoskey JA, Soil D. A truncated aminoacyl-tRNA synthetase modifies RNA. Proc. Natl. Acad. Sci. U.S.A. 2004 101 7536-7541. 

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. 

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