Initiator aminoacyl-tRNA

Stage 2 Initiation The mRNA bearing the code for the polypeptide to be made binds to the smaller of two ri-bosomal subunits and to the initiating aminoacyl-tRNA. The large ribosomal subunit then binds to form an initiation complex. The initiating aminoacyl-tRNA base-pairs with the mRNA codon AUG that signals the beginning of the polypeptide. This process, which requires GTP, is promoted by cytosolic proteins called initiation factors. 

Amino acids are activated by specific aminoacyl-tRNA synthetases in the cytosol. These enzymes catalyze the formation of aminoacyl-tRNAs, with simultaneous cleavage of ATP to AMP and PPj. The fidelity of protein synthesis depends on the accuracy of this reaction, and some of these enzymes carry out proofreading steps at separate active sites. In bacteria, the initiating aminoacyl-tRNA in all proteins is A-formylmethionyl-tRNAfMet. 

Initiation in prokaryotes involves binding of mRNA by small ribosomal subunit (30S), followed by association of the fMet-tRNAmet (initiator formyl-methionyl-tRNAmel) that recognizes the initiation codon. Large ribosomal subunit (50S) then joins to form the 70S initiation complex. In eukaryotes, the initiator aminoacyl-tRNA is not formylated. Instead Met-tRNAf161 forms 40S preinitiation complex with small ribosomal subunit (40S) in the absence of mRNA. The association of mRNA results in a 40S preinitiation complex, which forms an 80S initiation complex after large ribosomal subunit (60S) joins. 

Initiation involves binding of mRNA to the small ribosomal snbnnit, followed by association of initiator aminoacyl-tRNA that recognizes the first codon. 

In eukaryotes, the initiator aminoacyl-tRNA is not formylated but instead is a unique tRNA functioning only in initiation as Met-tRNAi . Eukaryotic initiation requires a set of eukaryotic initiation factors (elFs), as listed in Table 13.9, and proceeds in three steps ... 

The a-amino group of the new aminoacyl-tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P site (peptidyl or polypeptide site). At initiation, this site is occupied by aminoacyl-tRNA mef. This reaction is catalyzed by a peptidyltransferase, a component of the 285 RNA of the 605 ribosomal subunit. This is another example of ribozyme activity and indicates an important—and previously unsuspected—direct role for RNA in protein synthesis (Table 38-3). Because the amino acid on the aminoacyl-tRNA is already activated, no further energy source is required for this reaction. The reaction results in attachment of the growing peptide chain to the tRNA in the A site. 

After formation of the initiation dipeptide, the first EF-G-dependent translocation allows binding of the third aminoacyl-tRNA in the A-site so that a tripeptide is formed. The apparent rate of this event may depend upon the nature of the initiation complex initially formed, being slower, for instance, with those containing mRNAs with an extended SD sequence than with those having either very short or no SD complementarity (C. O. G. and M. Rodnina, unpublished results). Furthermore, very powerful translocation inhibitors may block tripeptide formation to such an extent that they mimic translation initiation inhibitors. 

Several key concepts are worth remembering. GTP is used as an energy source for translation, but ATP is used to form the aminoacyl-tRNA. The ribosome effectively has two kinds of tRNA binding sites. Only tRNAMet can bind to the P (for peptide) site, and this only occurs during the initial formation of the functional ribosome (initiation). All other aminoacyl-tRNAs enter at the A (for amino acid) binding site. After formation of the peptide bond (this doesn t require GTP hydrolysis), the tRNA with the growing peptide attached is moved (translocated) to the other site (this does require GTP hydrolysis). 

Figure 5 Kinetic mechanism of aminoacyl-tRNA selection by the ribosome. The aminoacyl-tRNAs are delivered to the ribosome in the form of a ternary complex with EF-Tu-GTP. Incorrect aminoacyl-tRNAs can either dissociate as a ternary complex in the initial selection phase or later as free aminoacyl-tRNA in the proofreading phase. The two selection phases are separated through the irreversible GTP hydrolysis by EF-Tu. Discrimination against incorrect tRNAs is achieved by increased dissociation rate constants (/r 2 and kj) as well as decreased forward rate constants (ks and ks) compared to cognate tRNAs.

In the initial step of the so-called indirect (transamidation) pathway, an ND-GluRS aminoacylates tRNA with glutamate or an ND-AspRS aminoacylates tRNA " with aspartate in the second step, the incorrectly... 

The first stage in protein synthesis is initiation, in which an aminoacyl-tRNA is positioned on one of the subunits of the ribosome. The attached amino acid will be the N-terminus of the completed protein. 

Translation involves three stages initiation, elongation and termination. A brief summary of these processes is provided below. However, the first step in polypeptide synthesis, from intracellular amino acids, is the formation of aminoacyl-tRNA. This reaction is particularly important so that the biochemistry is discussed in some detail. In addition, it is also important in the regulation of the rate of translation (see below). 

Figure 20.25 Regulation of the activities of the aminoacyl-tRNA synthetases by the concentrations of free tRNAs (i.e. uncharged tRNA). Changes in the concentrations of free tRNAs provide the mechanism for communication between control via the initiation factor (Figure 20.20) and ribosomal protein kinase (steps 6 and 7) and the flux-generating step.

Initially, the amino acid is activated by an ATP-dependent process, producing an aminoacyl-AMP. A hydroxyl group in ribose, part of a terminal adenosine group of tRNA, then reacts with this mixed anhydride. In this way, the amino acid is bound to tRNA via an ester linkage as an aminoacyl-tRNA. The tRNA involved will be specific for the particular amino acid. A detailed mechanism for this process has been considered in 8ection 13.5.1. 

The mRNA is bound to the smaller 30S subunit of the bacterial ribosome. The mRNA is a transcription of one of the genes of DNA, and carries the information as a series of three-base codons. The message is read (translated) in the 5 to 3 direction along the mRNA molecule. The aminoacyl-tRNA anticodon (UAC) allows binding via hydrogen bonding to the appropriate codon (AUG) on mRNA. In prokaryotes, the first amino acid encoded in the sequence is A-formylmethionine (fMet). Although the codon for initiation (A-formylmethionine) is the same as... 

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... 

At one point or another during protein synthesis, several other proteins will be associated with the ribosome. These include factors that help in initiating the synthetic process, others that help in elongating the peptide chain, and yet others that play a role in terminating the synthesis of a peptide chain. Beyond this, there is also the mRNA to consider, as well as the aminoacylated tRNA molecules. Finally, since protein biosynthesis consumes energy, there is the hydrolysis of ATP and GTP to AMP and GDP, respectively, by the ribosome. 

Functional 70S ribosome (initiation complex) Aminoacyl-tRNAs specified by codons Elongation factors (EF-Tu, EF-Ts, EF-G)... 

MECHANISM FIGURE 27-14 Aminoacylation of tRNA by aminoacyl-tRNA synthetases. Step is formation of an aminoacyl adenylate, which remains bound to the active site. In the second step the aminoacyl group is transferred to the tRNA. The mechanism of this step is somewhat different for the two classes of aminoacyl-tRNA synthetases (see Table 27-7). For class I enzymes, (2a) the aminoacyl group is transferred initially to the 2 -hydroxyl group of the 3 -terminal A residue, then (3a) to the 3 -hydroxyl group by a transesterification reaction. For class II enzymes, ( the... 

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