FMET-tRNA peptides

If a translation reaction directed by the universal 027IF2Cp(A) mRNA is carried out in the presence of four precharged aminoacyl-tRNAs (fMet-tRNA, Phe-tRNA, Thr-tRNA, and Ile-tRNA) in amounts sufficient to ensure the synthesis of the 027 peptide (which contains only these amino acids) even in the presence of an aminoacyl-tRNA inhibitor, the system will be able to detect an aminoacylation inhibitor in a library of natural or synthetic products through the selective inhibition of IF2C domain synthesis. Thus, if the synthesis of the 027 and IF2C peptides is measured in parallel, a general inhibitor of translation would be expected to inhibit the synthesis of both products, while an aminoacylation inhibitor would inhibit... 

Protocol The test for the identification of aminoacyl-tRNA synthetase inhibitors requires the availability of precharged fMet-tRNA, Phe-tRNA, Thr-tRNA, and Ile-tRNA, which correspond to the amino acids present in the 027 peptide. The preparation of fMet-tRNA is described in the accompanying chapter by Milon et al., (2007), while the preparation of the other aminoacyl-tRNAs has been described previously. 

In the following section, we describe protocols for tests aimed at screening for compounds capable of interfering with some of the main activities of this factor, such as (a) recognition and binding of initiator tRNA (b) codon-dependent ribosomal binding of fMet-tRNA leading to the formation of a 30S or 70S initiation complex (c) ribosome-dependent hydrolysis of GTP and (d) accommodation of fMet-tRNA in the ribosomal P-site and formation of the first peptide bond (initiation dipeptide formation). 

Once the initiating fMet-tRNA of bacteria or the eukaryotic Met-tRNA is in place in the P site of a ribosome and is paired with the initiation codon in the mRNA, peptide chain growth can commence. Amino acid residues are added in turn by insertion at the C-terminal end of the growing peptide chain. Elongation requires three processes repeated over and over until the entire peptide is formed. 

Next, fMet-tRNA will deliver the carboxyl terminus of fMet to the amino terminus of the amino acid linked to the tRNA at the A site (a peptidyl transfer reaction), with the subsequent formation of a peptide bond between the two amino acids. At this point, the tRNA at the A site is covalently linked to a dipeptide (Fig. 23-5). 

Further study of the specificity of aminoacyl-tRNA binding of EF-Tu resulted in the solution of the following problem the chain initiator in E. coli is fMet-tRNA( which is synthesized by formylation of Met-tRNAj. There is a different Met-4RNA species, Met-tRNA which provides methionyl residues for internal positions of the peptide chain. Furthermore as initiator codons in E. coli, GUG, as well as AUG, spedfy fMet-tRNAt, whereas as codons for internal aminoacyl residues GUG stands for Val-tRNA and AUG for Met-tRNA . 

Another phase of protein synthesis in which I became interested at Yale is peptide-chain initiation. In 1966, J. Eisenstadt and I proved that the chain initiator fMet-tRNAf is strictly required for the translation of f2 bacteriophage RNA, a natural messenger RNA in a crude extract from E. coli. This requirement was, however, only manifested when the magnesium ion concentration in the extract was low. For this demonstration, we had to deplete the pool, and block the formation, of fMet-tRNAf in the extract. We found two compounds which accomplished this by making formyltetrahydrofolate, the source of the formyl residue in fMet-tRNA(, unavailable trimethoprim, an inhibitor of dihydrofolate reductase, and hydroxylamine which was shown by Bertino to react with methylenetetra-hydrofolate and thereby deplete the pool of formyltetrahydrofolate, produced the result desired. 

Protein synthesis begins with the N-terminal amino acid and proceeds from this point. In some bacteria, yeast, and higher organisms, this first aminoacyl-tRNA is known to be iV-formylmethionyl-tRNA Formylation of the amino function can be considered as a protecting group to prevent participation of the amino function is peptide bond formation. The fMet-tRNA is then the first aminoacyl-tRNA to bind to the ribosome and mRNA. After the protein is synthesized, the formyl group is removed by enzymatic cleavage (formylase). 

Fig. 6). The lack of a free amino group in fMet-tRNA prevents its insertion into a protein anywhere but at the N-terminal position. Furthermore, the presence of an amide bond targets fMet-tRNA for the P-site of the ribosome. Only the initiator tRNA enters the P-site directly— all other aminoacyl-tRNAs enter the ribosome at the A-site and are moved to the P-site after peptide bond formation. This targeting is achieved both by the presence of the amide bond of fMet-tRNA and by the uniquely rigid anticodon stem of the initiator tRNA, which contains three G C base pairs. Progressive substitution of these three G C pairs has been shown to weaken binding of the initiator tRNA to the P-site. 

This claim is based on the following observations (1) Like that of their prokaryotic ancestors, mitochondrial translation uses fMet-tRNA (rather than the Met-tRNA used by cytoribosomes) as the initiator. (2) It can do so successfully because the transformylase which converts the Met-tRNA into its fMet derivative is mitochondrial in its localization. (3) The formation of the initiation complex can be monitored by the transfer of labeled formate (f ) to f Met to puromycin, resulting in its quantitative conversion to f Met-puro —a reaction that is restricted both in vitro and in vivo to the mitochondrial fraction and can be shown to go on with linear kinetics for extended periods. (4) Retention of f Met on nascent polypeptide chains is restricted to mitochondrial polyribosomes and can be used as a specific means for the identification and characterization of the latter. (5) Mitochondria, at least of the yeast species examined by us, appear to be deficient in both a deformylase capable of removing formate from fMet, whether free or on polypeptides, as well as in peptidases capable of removing either this component itself or small peptides from the N-terminal end. (6) Initiation by fMet is absent in p" mutants. In principle then, presence of formate as N-terminal fMet in a polypeptide provides an unambiguous means for its identification as having been synthesized on mitoribosomes. In practice, although feasible, as will be shown in the next section, this is difficult because it requires the prior... 

Correct answer = D. Because fMet-Phe is made, the ribosomes must be able to complete initia tion, bind Phe-tRNA to the A-site, and use pep tidyltransferase activity to form the first peptide bond. Because the ribosome is not able to pro ceed any further, ribosomal movement (translo cation) is most likely the inhibited step. The ribosome is, therefore, frozen before it reaches the stop codon of this message. 

At the start of the first round of elongation (Fig. 5), the initiation codon (AUG) is positioned in the P site with fMet-tRNAfMet bound to it via codon-anticodon base-pairing. The next codon in the mRNA is positioned in the A site. Elongation of the polypeptide chain occurs in three steps called the elongation cycle, namely aminoacyl-tRNA binding, peptide bond formation and translocation ... 

The process continues until a stop codon ends up in the A site at which point a protein release factor binds to the stop codon, the peptide (H3N+-fMet-Gly-Ser— HN-CH(R,i)COO )-tRNA bond is hydrolysed, the completed polypeptide is released and the ribosomal subunits separate. 

The mechanism responsible for the initiation of peptide chain synthesis has been studied in bacteria [for review, see (L14)]. The first step appears to be the binding of N-formylmethionyl tRNA (fMet-tRNAf) at a unique initiating codon (GUG) in the mRNA. At least two species of methionyl-tRNA are known one, known as Met-tRNAf, can be for-mylated but the second (Met-tRNA, ) recognizes only the internal AUG codon and thus incorporates methionyl residues into the polypeptide chain at internal positions. 

The elongation phase of polypeptide synthesis is depicted in Fig. 9-12. The 508 subunit is a peptidyl transferase that catalyzes the formation of a peptide bond between amino acids. The 238 rRNA is responsible for this catalytic activity. As its name implies, it transfers the fMet (and in later reactions a peptide) from the tRNA that occnpies the P site to the amino acid on the tRNA in the A site. To do this, the amino group of... 

Fig. 2. Formation of a stable initiation complex between a 70 S ribosome and messenger RNA. In the final complex fMet-tRNAf " is in the correct position for the formation of a peptide bond. IF-1, IF-2, and IF-3 are the protein initiation factors and fMet-tRNAf " is the formyl methionyl tRNA which is used for the initiation of protein synthesis in prokaryotes. The process in animal cells is thought to be substantially the same, the initiation factors being termed IF-Ml, IF-M2, and IF-M3 and the initiator tRNA, Met-tRNAt . The methionine attached to this tRNA species is not normally formylated but can be so modified by enzymes from bacterial cells.

The amino acid carried by this tRNA bonds to the peptide chain, and the elongation process is repeated. This occurs over and over until the entire polypeptide chain is synthesized. The elongation process is represented in Figure 21.21 for the synthesis of the tripeptide fMet—Phe— Val. 

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