MET-tRNA

The binding of the 60S ribosomal subunit to the 48S initiation complex involves hydtolysis of the GTP bound to elF-2 by elF-5. This teaction tesults in telease of the initiation factots bound to the 48S initiation complex (these factots then ate tecycled) and the tapid association of the 40S and 60S subunits to fotm the 80S ribosome. At this point, the met-tRNA is on the P site of the ribosome, ready for the elongation cycle to commence. 

As mentioned above, Met(0) must be converted to Met before it can be incorporated into proteins. There are a wide variety of organisms that have been shown to be capable of enzymatically reducing Met(O) residues. The enzymatic reduction of free Met(O) to Met has been observed in yeast , E. cofi - , Pseudomonas , plants and animal tissues . The enzyme from E. coli has been purified about 1100-fold using a newly developed very sensitive assay . The assay involves first the conversion of [ S]Met(0) to [ S]Met by the Met(O) reductase followed by the measurement of [ S]Met-tRNA after enzymatic acylation of tRNA. Since Met(O) is not a substrate for the acylation reaction , the amount of [ S]Met-tRNA formed is proportional to the amount of [ S]Met(0) converted to [ S]Met. The assay is sensitive to Met levels of less than 1 pmol. 

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). 

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. 

Figure 12-2. Formation of the initiation complex for protein synthesis. Several eukaryotic initiation factors (elFs) ensure proper assembly at each step. The initiator Met-tRNA is bound in the peptidyl site of the SOS complex with its anticodon (black stripes) base paired to the AUG start codon (gray box) of the mRNA.

The distinction between an initiating (5 )AUG and an internal one is straightforward. In bacteria, the two types of tRNA specific for methionine are designated tRNAMet and tRNAfMet. The amino acid incorporated in response to the (5 )AUG initiation codon is A7-formyl-methionine (fMet). It arrives at the ribosome as A7-formylmethionyl-tRNAfMet (fMet-tRNAfMet), which is formed in two successive reactions. First, methionine is attached to tRNAfMet by the Met-tRNA synthetase (which in E. coli aminoacylates both tRNAfMet and tRNAMet) ... 

The transformylase is more selective than the Met-tRNA synthetase it is specific for Met residues attached to tRNAfMet, presumably recognizing some unique structural feature of that tRNA. By contrast, Met-tRNAMet inserts methionine in interior positions in polypeptides. 

Translational regulation has been particularly well studied in reticulocytes. One such mechanism in these cells involves eIF2, the initiation factor that binds to the initiator tRNA and conveys it to the ribosome when Met-tRNA has bound to the P site, the factor eIF2B... 

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. 

McIntosh, B., Ramachandiran, V., Kramer, G., and Hardesty, B. (2000). Initiation of protein synthesis with fluorophore-Met-tRNA(f) and the involvement of IF-2. Biochimie 82, 167-174. 

Eukaryotic ribosomes are larger (80S) and more complex than prokaryotic ribosomes (70S). Initiation is basically similar in prokaryotes and eukaryotes except that in eukaryotes at least nine initiation factors are involved (cf. three factors in prokaryotes), the initiating amino acid is methionine (cf. N-formylmethionine in prokaryotes), eukaryotic mRNAs do not contain Shine-Dalgarno sequences (so the AUG initiation codon is detected by the ribosome scanning instead), and eukaryotic mRNA is monocistronic (cf. some polycistronic mRNAs in prokaryotes). Initiation in eukaryotes involves the formation of a 48S preinitiation complex between the 40S ribosomal subunit, mRNA, initiation factors and Met-tRNA 61. The ribosome then scans the mRNA to locate the AUG initiation codon. The 60S ribosomal subunit now binds to form the 80S initation complex. 

Initiation mRNAs with AUG initiation code fMet-tRNAi IF-1, IF-2, and IF-3 initiation factors, GTP, Mg2+ 30 and 50 S mRNAs with AUG initiation code Met-tRNA eIF3, eIF2, eIF4, eIF5 initiation factors GTP, Mg2+, eIF-2 protein kinase 40 and 60 S ribosomal subunits... 

Polyproanthocyanidin (condensed tannin) Alhagi kirgisorum (Fabaceae) PS - eIF-2 [blocks elF-Met-tRNA-GTP ternary complex formation]... 

The mechanism of N-acetylation of a-crystallin is quite interesting. The N-terminal residue has been identified as N-acetyl methionine. This methionine residue is derived from Met-tRNA et which is responsible for the initiation of the polypeptide chain and not Met-tRNAmet which incorporates the methionine residue in the growing polypeptide chain. It is clear that the N-acetylation is a true posttranslational process since acetyl Met-tRNA cannot replace formyl Met-tRNA et. Moreover, N-acetylation occurs when the polypeptide chain has reached a length consisting of approximately 25 amino acid residues. Other proteins, such as ovalbumin, are also acetylated during the early stages of polymerization on the polysome, and the protein acetyltransferase activity must therefore be associated with the protein-synthesizing apparatus. 

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