FMET-tRNA

The test aimed at determining whether the target of a translational inhibitor is tRNA aminoacylation requires four precharged aa-tRNAs (f Met-tRNA, Thr-tRNA, Ile-tRNA, and [14C] Phe-tRNA). Here, we present the protocol for the aminoacylation of the elongator tRNAs, while fMet-tRNA preparation is described in detail in the accompanying chapter by Milon et al, (2007). 

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. 

An example of the results that can be obtained using the microtiter format is shown in Fig. 12.5A, which illustrates the inhibition of fMet-tRNA binding to 022 mRNA-programmed 30S ribosomal subunits caused by increasing concentrations of GE81112, the inhibitor of 30SIC formation characterized in Brandi et al. (2006b). 

Kinetics of fMet-tRNA binding to 30S ribosomal subunit Inhibition of ribosomal binding of fMet-tRNA by an antibiotic may reduce the level of initiation complex formed at equilibrium. However, if the effect of the inhibitor consists mainly of slowing down the binding reaction, its effect may appear less dramatic after a relatively long incubation time. For this... 

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). 

This essential property of IF2 can be tested in at least three different ways, all of which require the availability of f[3H]Met-tRNA and IF2, which are prepared according to the protocol detailed in Milon et al. (2007). However, all the tests described in this section can make use of the sturdier and smaller C domain of Bacillus stearothermophilus IF2, since this domain contains all molecular determinants for the IF2-fMet-tRNA interaction (Guenneugues et al, 2000 Spurio et al, 2000). The method for the preparation and purification of B. stearothermophilus IF2C is essentially that described by Spurio et al. (1993). The concentration of the protein... 

The activity of IF2 in binding fMet-tRNA was measured quantifying the protection conferred by these proteins on the initiator tRNA with respect to spontaneous hydrolysis occurring at alkaline pH (Gualerzi et al., 1991 Petersen et al., 1979). Reaction mixtures (50 pi) in Buffer F contained 22 pM f 35S]Met-tRNA, an appropriate amount of protein that is capable of protecting approximately 80% of the initiator tRNA after 60 min incubation as well as increasing concentrations of the antibiotic to be tested. Samples (20 pi), withdrawn after 0 and 60 min of incubation at 37°, are spotted on Whatman 3MM paper discs for determination of the acid-insoluble radioactivity by the cold TCA procedure, described previously. 

Caskey CT, Beaudet AL, Scolnick EM, Rosman M (1971) Hydrolysis of fMet-tRNA by peptidyl transferase. Proc Natl Acad Sci USA 68 3163-3167... 

Fig. 1. 55. The function of eIF-2 in eucaryotic translation. eIF-2, the initiator protein for the translation is a regulatory GTPase that occurs in an active GTP-form and in an inactive GDP form (see ch. 5). The active eIF-2 GTP forms a complex with the initiator-tRNA, fMet-tRNA "" and the 40S subunit of the ribosome. This complex binds to the cap structure of mRNA to initiate the scanning process. eIF-2 undergoes an activation cycle typical for regulatory GTPases the inactive eIF-2 GDP fom is activated with the assistance of the eIF-2B protein into the active elF-2 GTP form. eIF-2B acts as a G-nucleotide exchange factor in the cycle (see ch. 5).

Prevents premature binding of tRNAs to A site Facilitates binding of fMet-tRNA 61 to 30S ribosomal subunit Binds to 30S subunit prevents premature association of 50S subunit enhances specificity of P site for fMet-tRNA 61... 

IF1, which is essential to the viability of bacteria, binds and partially occludes the A site of the ribosome, preventing the initiator fMet-tRNA from incorrectly occupying the 30S A site.70,296a,3°2 Binding of IF1 also causes the functionally important bases A1492 and A1493 of 16S RNA (Fig. 29-2) to be flipped out of helix 44 and to bind to pockets in IF1. This induces further... 

Some information about spatial arrangements of the ribosomal proteins involved in initiation was provided by the fact that antibodies against proteins SI 9 and S21 block the formation of a complex with fMet-tRNA, while antibodies against S2, S18, and S20 block the binding of IF3. Crosslinking experiments showed that IF2 and S19 are close together and that IF3 is close to S12 (Fig. 29-1A). 

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. 

E. coli has three initiation factors (fig. 29.13) bound to a small pool of 30S ribosomal subunits. One of these factors, IF-3, serves to hold the 30S and 50S subunits apart after termination of a previous round of protein synthesis. The other two factors IF-1 and IF-2, promote the binding of both fMet-tRNA,Mel and mRNA to the 30S subunit. As we noted before, the binding of mRNA occurs so that its Shine-Dalgarno sequence pairs with 16S RNA and the initiating AUG sequence with the anticodon of the initiator tRNA. The 30S subunit and its associated factors can bind fMet-tRNAjMet and mRNA in either order. Once these ligands are found, IF-3 dissociates from the 30S, permitting the 50S to join the complex. This releases the remaining initiation factors and hydrolyzes the GTP that is bound to IF-2. The initiation step in prokaryotes requires the hydrolysis of one equivalent of GTP to GDP and Pj. 

FIGURE 3.7 A cryo-EM map of the Escherichia coli ribosome (complexed with fMet-tRNAf Met and mRNA) where fMet = formylmethionine obtained from 73,000 particles at a resolution of 11.5 A. (a-d) Four views of the map, with the ribosome 30S subunit painted in yellow, the ribosome 50S subunit in blue, helix 44 of 16S RNA in red, and fMet-tRNA at the P site in green. Inset on top juxtaposes the experimental tRNA mass (green, on left) with the appearance of the X-ray structure of tRNA at 11 A resolution (on right). Arrows mark points at which tRNA contacts the surrounding ribosome mass. Landmarks h = head and sp = spur of the 30S subunit. CP = central protuberance LI = LI stalk and St = L7/L12 stalk base of the 50S subunit. 

Several protein factors are involved in the initiation process. These factors aren t usually part of the ribosome instead, they help form an active initiation complex. Initiation factor 3 (IF3) helps keep the 30S subunit dissociated from the 50S subunit and available for protein synthesis. IF1 binds to the isolated 30S subunit and helps form the complex between the RBS and 16S rRNA. IF2 forms a complex with frnet-tRNAme i and GTP, releasing IF3. After the complex contains mRNA and initiator fmet-tRNA, the following things occur GTP is hydrolyzed to GDP, the initiation factors are released from the ribosome, and the 50S subunit associates with the complex to form an elongating ribosome, as shown in Figure 11-5. 

The binding of GTP to IF-2, which allows mRNA and fMet-tRNA to join the complex with the release of IF-3. 

In addition to mRNA, fMet-tRNA tc , and the ribosome subunits, three initiation factors (proteins) and GTP are involved in the initiation of polypeptide synthesis. The process is described in Example 17.7. 

The first step of RNA translation begins with the initiation of polypeptide synthesis (Fig. 17-10). GTP is bound into the 30S initiation complex and is subsequently hydrolyzed and released upon binding to the 50S subunit. The fMet-tRNA eI occupies what is known as the peptidyl (P) site of the ribosome (Fig. 17-9) another site (A), capable of accommodating an aminoacyl-tRNA, is empty at this stage. It is aligned with the next codon (shown as xxx in Fig. 17-10) in the mRNA. 

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). 

To remove the now uncharged fMet-tRNA from the P site, the ribosomal complex will shift toward the 3 end of the mRNA by three bases, positioning a new codon in the A site and ejecting the spent fMet-tRNA. At this point, the dipeptide bound by the tRNA that formerly occupied the A site now occupies the P site. This final step in translation elongation is fueled by the hydrolysis of another GTP molecule by a translocase enzyme called elongation factor G (EF-G, Fig. 23-5). 

This process frees the A site on the ribosome to accept the next aminoacyl tRNA. Again, the next aminoacyl tRNA is aided by EF-Tu, which hydrolyzes one molecule of GTP to GDP + Pi. The unchanged fMet tRNA is ejected from the ribosome. 

The first amino acid for reaction is jV-formylmethionine (fMet) which has a specific tRNA (tRNA 1 1) (as opposed to the Met-specific tRNA tRNAmMl 1). Using GTP hydrolysis as an energy source, the 30S subunit complexes with initiation factors IF1, IF2 and IF3. This complex binds the mRNA with the anticodon (3 -UAC-5 ) of the jV-formylmethionyl-tRNA M t (fMet-tRNAf 11 1) hydrogen bonding to the start codon (5 -AUG — 3 ) of the mRNA, the fMet-tRNA -Ml 1 binding at the so-called P site with release of IF3. 

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