Protein synthesis chain initiation

Once protein synthesis is initiated, amino acids are added to the peptide chain corresponding to each triplet in the mRNA until the ribosome encounters a termination or stop codon, whereupon the polypeptide chain is released from the ribosome, and assumes its final configuration. A ribosome covers about 50 bases of an mRNA, which is usually hundreds of bases long. Thus, several ribosomes translate an mRNA consecutively and simultaneously at any instant as shown in Fig. 2.4. A group of ribosomes translating a message is called a polyribosome . 

The methionine residue found at the amino-terminal end of E. coli proteins is usually modified. In fact, protein synthesis in bacteria starts with H-formylmethionine (fMet). A special tRNA brings formylmethionine to the ribosome to initiate protein synthesis. This initiator tRNA (abbreviated as tRNAf) differs from the one that inserts methionine in internal positions (abbreviated as tRNA ). The subscript "f indicates that methionine attached to the initiator tRNA can be formylated, whereas it cannot be formyl-ated when attached to tRNA. In approximately one-half of E. coli proteins, N-formylmethionine is removed when the nascent chain is 10 amino acids long. 

When protein synthesis terminates, the initiator amino acid, Methionine, will have a free amino group. This end of the protein is the N terminus and the last amino acid of the chain has a free carboxy or C terminus. Protein synthesis thus initiates with the amino terminus and proceeds towards the C terminus. Proteins synthesized on the rough ER are transported across a membrane aiad into the cisternal spaces between the sheets of the ER where they are packaged for export. 

Fig. 12.19. The regions of eukaryotic mRNA. The wavy line indicates the polynucleotide chain of the mRNA and the As constituting the poly(A) tail. The 5 -cap consists of a guanosine residue linked at its 5 hydroxyl group to three phosphates, which are linked to the 5 -hydroxyl group of the next nucleotide in the RNA chain. The start and stop codons represent where protein synthesis is initiated and terminated from this mRNA.

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. 

The oxazolidinones have a novel mechanism of action that involves the inhibition of bacterial protein synthesis at the very early stage, prior to chain initiation [55-58]. They are effective against a broad range of Gram-... 

It has long been known that peptides of bacterial origin, such as N-formylat-ed oligopeptides, are potent activators of neutrophils. Bacterial protein biosynthesis is initiated by the codon AUG, which codes for polypeptide chains at the NH2 terminus to start with N-formylmethionine. However, very few mature bacterial proteins actually have this amino acid at the NH2 terminus because Af-formylmethionine is cleaved off by proteolytic processing. Sometimes just this amino acid is cleaved, but often several adjacent residues are also removed with it. These observations formed the basis for the chemical synthesis of a variety of N-formylated oligopeptides and an assessment of their ability to activate neutrophils in vitro. The most potent of these formylated peptides is TV-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). 

Energy requirements in protein synthesis are high. Four energy-rich phosphoric acid anhydride bonds are hydrolyzed for each amino acid residue. Amino acid activation uses up two energy-rich bonds per amino acid (ATP AMP + PP see p. 248), and two GTPs are consumed per elongation cycle. In addition, initiation and termination each require one GTP per chain. 

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... 

Initially, the Boc group was used for reversible a-amine protection and most side-chain functional groups were protected as benzyl derivatives, which are stable to Boc removal in HC1 or TFA. This Boc/Bzl strategy is still frequently used and is the method of choice in several laboratories. It has, however, been replaced in many laboratories by the base-labile Fmoc group, which allows weak acid deprotection of tert-butyl groups from the side chains. For short- and moderate-sized peptides both systems are effective. For protein synthesis, the relative merits have not yet been fully established. 

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. 

The pathway of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the twenty-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5 -end to its 3 -end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNAs often have several coding regions, that is, they are polycistronic (see p. 420). Each coding region has its own initiation codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA codes for only one polypeptide chain, that is, it is monocistronic. The process of translation is divided into three separate steps initiation, elongation, and termination. The polypeptide chains produced may be modified by posttranslational modification. Eukaryotic protein synthesis resembles that of prokaryotes in most details. [Note Individual differences are mentioned in the text.]... 

To form a globular protein, a polypeptide chain must repeatedly fold back on itself. The turns or bends by which this is accomplished can be regarded as a third major secondary structural element in proteins. Turns often have precise structures, a few of which are illustrated in Fig. 2-24. As components of the loops of polypeptide chains in active sites, turns have a special importance for the functioning of enzymes and other proteins. In addition, tight turns are often sites for modification of proteins after their initial synthesis (Section F). 

Hypusine (Ne-(4-amino-2-hydroxybutyl)lysine)242 occurs in mammalian initiation factor 4D, which is utilized in protein synthesis (Chapter 29) and is formed by transfer of the 4-carbon butylamine group from spermidine to a lysine side chain followed by hydroxylation 280 2823 The lupine alkaloid lupinine283 is formed from two C5 units of cadaverine which arises by decarboxylation of lysine. Silaffins (pp. 178, 1381) also contain modified lysines. 

High concentrations of hemin inhibit the transport of ALA synthase into the mitochondria, where one of the substrates, succinyl-CoA, is formed. Thus, heme synthesis is inhibited until enough globin is made to react with any heme already formed. Low concentrations, or the absence, of hemin is the signal that globin is not needed this protein (and, therefore, globin) synthesis is inhibited. In the absence of hemin, a protein kinase is activated this phosphorylates an initiation factor of (eukaryotic) protein synthesis, eIF-2, which then inhibits polypeptide chain initiation (Chap. 17) and hence inhibits globin synthesis. 

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