Ribosomes protein formation

The peptidyl transferase centre of the ribosome is located in the 50S subunit, in a protein-free environment (there is no protein within 15 A of the active site), supporting biochemical evidence that the ribosomal RNA, rather than the ribosomal proteins, plays a key role in the catalysis of peptide bond formation. This confirms that the ribosome is the largest known RNA catalyst (ribozyme) and, to date, the only one with synthetic activity. Adjacent to the peptidyl transferase centre is the entrance to the protein exit tunnel, through which the growing polypeptide chain moves out of the ribosome. 

FIGURE 10.9 Normal protein formation sequence. First, mRNA is formed from the cell s DNA (A). Second, the mRNA enters into the cell s cytoplasm (B). Third, ribosome forms around the mRNA (C) resulting in the formation of the desired protein (D). 

Inside the cell, aminoglycosides bind to specific 30S-subunit ribosomal proteins (S12 in the case of streptomycin). Protein synthesis is inhibited by aminoglycosides in at least three ways (Figure 45-3) (1) interference with the initiation complex of peptide formation (2) misreading of mRNA, which causes incorporation of incorrect amino acids into the peptide and results in a nonfunctional or toxic protein and (3) breakup of polysomes into nonfunctional monosomes. These activities occur more or less simultaneously, and the overall effect is irreversible and lethal for the cell. 

Phosphorylated IRS-1 activates a second signaling pathway by interacting with an 85-kDa SH2-containing protein that is a subunit of phophatidylinositol 3-kinase.384 386 This activates the 110-kDa catalytic subunit of the 3-kinase, which catalyzes formation of phosphatidylinositol 3-phosphate as well as Ptdlns (3,4)P2 and Ptdlns (3,4,5)P3.387/387a These compounds, which remain within membranes, activate other branches of the signaling cascade, some of which may converge with those of the MAP kinase cascade. However, there appears to be specific activation of a ribosomal Ser/Thr kinase that, among other activities, phosphorylates ribosomal protein S6, a component of the small ribosomal subunit.388 It also phosphorylates some isoforms of protein kinase C and other enzymes. Ptdlns 3-kinase may also activate 6-phosphofructo-2-kinase (Fig. 11-2, step ti).384/388... 

Initiation (Figs. 29-10 and 29-11), elongation (Fig. 29-12), and termination are three distinct steps in the synthesis of a protein. A variety of specialized proteins are required for each stage of synthesis. Their sequential interaction with ribosomes can be viewed as a means of ensuring an orderly sequence of steps in the synthesis cycle. The rate of protein formation will depend upon the concentrations of amino acids, tRNAs, protein factors, numbers of ribosomes, and kinetic constants. The formation of specific proteins can also be inhibited by translational repressors, proteins that compete with ribosomes for binding to target mRNAs.287... 

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). 

The synthetase consists of the three modules E1, E2, and E3 (for a complete description, see Sec. II. A). Each module is composed of an activation site forming the acyl or aminoacyl adenylate, a carrier domain which is posttranslationally modified with 4 -phosphopantetheine (Sp), and a condensation domain (Cl, C2) or, alternatively, a structurally similar epimerization domain (Ep). Activation of aminoadipate (Aad) leads to an acylated enzyme intermediate, in which Aad is attached to the terminal cysteamine of the cofactor (El-Spl-Aad) [reactions (1) and (2)]. Likewise, activation of cysteine (Cys) leads to cysteinylated module 2 [reactions (3) and (4)]. For the condensation reaction to occur between aminoadipate as donor and cysteine as acceptor, both intermediates are thought to react at the condensation site of module 1 (Cl). Each condensation site is composed, in analogy to ribosomal peptide formation, of an aminoacyl and a peptidyl site. In this case of initiation, the thioester of Aad enters the P-site, while the thioester of Cys enters the A-site. Condensation occurs and leaves the dipeptidyl intermediate Aad-Cys at the carrier protein of the second module [reaction (5)]. The third amino acid valine is activated on module 3, and Val is attached to the carrier protein 3 [reactions (6) and (7)]. Formation of the tripeptide occurs at the second condensation site C2, with the dipeptidyl intermediate entering the P-site and the valiny 1-intermediate the A-site [reaction (8)]. 

They contain many unusual bases, typically between 7 and 15 per molecule. Some are methylated or dimethylated derivatives of A, U, C, and G formed by enzymatic modification of a precursor tRNA (Section 28.1.8). Methylation prevents the formation of certain base pairs, thereby rendering some of the bases accessible for other interactions. In addition, methylation imparts a hydrophobic character to some regions of tRNAs, which may be important for their interaction with synthetases and ribosomal proteins. Other modifications alter codon recognition, as will be discussed shortly. 

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