Reaction Ribosomal subunit

Perhaps the most significant case of catalysis by RNA occurs in protein synthesis. Harry F. NoIIer and his colleagues have found that the peptidyl transferase reaction, which is the reaction of peptide bond formation during protein synthesis (Figure 14.24), can be catalyzed by 50S ribosomal subunits (see Chapter 12) from which virtually ail of the protein has been removed. These... 

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 ribosome is a ribozyme this is how Cech (2000) commented on the report by Nissen et al. (2000) in Science on the successful proof of ribozyme action in the formation of the peptide bond at the ribosome. It has been known for more than 30 years that in the living cell, the peptidyl transferase activity of the ribosome is responsible for the formation of the peptide bond. This process, which takes place at the large ribosome subunit, is the most important reaction of protein biosynthesis. The determination of the molecular mechanism required more than 20 years of intensive work in several research laboratories. The key components in the ribosomes of all life forms on Earth are almost the same. It thus seems justified to assume that protein synthesis in a (still unknown) common ancestor of all living systems was catalysed by a similarly structured unit. For example, in the case of the bacterium E. coli, the two subunits which form the ribosome consist of 3 rRNA strands and 57 polypeptides. Until the beginning of the 1980s it was considered certain that the formation of the peptide bond at the ribozyme could only be carried out by ri-bosomal proteins. However, doubts were expressed soon after the discovery of the ribozymes, and the possibility of the participation of ribozymes in peptide formation was discussed. 

As an alternative, it is possible to use a mixture of purified 30S and 50S ribosomal subunits or 70S monomers (0.25 fiMfinal concentration) and 4 to 8 1/reaction tube of SI 00 post-ribosomal supernatant as well as initiation factors IF1, IF2, and IF3 in a 1.5-to-l ratio with the ribosomes. After 30 min incubation at 20°, the activity of the synthesized luciferase is determined as described later. 

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... 

The origin of the idea that a ribosome might be a ribozyme is derived from the experiment in which peptidyl transferase activity was observed even after digestion of protein components of the ribosome [15]. This was surprising because the most important biological function involved in the synthesis of proteins is catalyzed by RNA. Recently, a large ribosomal subunit from Haloarcula marismortui was determined at a resolution of 2.4 A [16, 155]. Importantly, because of the absence of proteins at the active site, it was concluded that the key peptidyl transferase reaction is accomplished by the ribosomal RNA (rRNA) itself, not by proteins. How does it work ... 

Chloramphenicol is able to inhibit the peptidyl transferase reaction and so bacterial protein synthesis by binding reversibly to the 50s ribosomal subunit. Resistance can occur due to the plasmid-mediated enzyme chloramphenicol acetyltransferase which inactivates the drug by acetylation. Such resistance is often a part of plasmid-mediated multidrug resistance. Resistance can also occur by an altered bacterial permeability. However in most instances resistance to chloramphenicol only develops slowly and remains partial. 

The enzymatic activity that catalyzes peptide bond formation has historically been referred to as peptidyl transferase and was widely assumed to be intrinsic to one or more of the proteins in the large ribosomal subunit. We now know that this reaction is catalyzed by the 23S rRNA (Fig. 27-9), adding to the known catalytic repertoire of ribozymes. This discovery has interesting implications for the evolution of life (Box 27-3). 

The last initiation step (step/, Fig. 29-11) is the reaction of the 60S ribosomal subunit with the 48S initiation complex to form the 80S initiation complex. Initiation factors 3,4C, the eIF2 GDP complex, and inorganic phosphate are all released in this process, which is promoted by IF5. This monomeric 60-kDa protein355 356 also stimulates conversion of the GTP bound to IF2 into GDP and P . IF5 is unique as the only known protein containing hypusine, Ne -(4-amino-2-hydroxybutyl)lysine, a posttranslationally modified lysine. It occurs only at position 50 in the 17-kDa protein.356-358 Hypusine is not present in eubacte-ria but is essential for viability of both eukaryotes and archaeobacteria358 and is present within an invariant 12-residue sequence. 

The ribosomal translocation process is quite complex. As the tRNAs move from A to P to E sites on the 16S RNA platform, the mRNA must also move in discrete single-codon steps. Tire acceptor stems of the tRNAs in the A and P sites must react at the appropriate times in the peptidyltransferase center. Study of protection from chemical probes suggests that tRNAs sometimes lie with the anticodon loop in the A site of the small ribosomal subunit, while the acceptor stem is in the P site of the large subunit (an A/P site as illustrated in Fig. 29-12B). Each aminoacyl-tRNA enters as a complex with EF-Tu and may initially bind with its anticodon in the A site and the acceptor stem with attached EF-Tu in a transient T site, the composite state being A/T. After loss of EF-Tu the acceptor stem can move into the A site to give an A/A state. The peptidyltransferase reaction itself necessarily involves movement at the acceptor stems by 0.1 nm or more. However, additional movement of 1 nm is needed to move the two tRNAs into states A/P and P/E, respectively. Movement of the mRNA then moves the... 

Peptide bond formation the second step, peptide bond formation, is catalyzed by peptidyl transferase, part of the large ribosomal subunit. In this reaction the carboxyl end of the amino acid bound to the tRNA in the P site is uncoupled from the tRNA and becomes joined by a peptide bond to the amino group of the amino acid linked to the tRNA in the A site (Fig. 5). A protein with peptidyl transferase activity has never been isolated. The reason is now clear in E. coli at least, the peptidyl transferase activity is associated with part of the 23S rRNA in the large ribosomal subunit. In other words, peptidyl transferase is a ribozyme, a catalytic activity that resides in an RNA molecule (see also Topic G9). 

The steps of initiation occur on the isolated small subunit (30S) of the prokaryotic ribosome. Ribosomes contain two subunits, a 30S and 50S subunit, which associate to form a 70S particle. (The S values refer to the rate at which each component sediments in the ultracentrifuge they don t always add up.) In general, the 30S subunit is mostly involved in the decoding and tRNA-mRNA interaction process, while the 50S subunit is involved in actual peptide bond synthesis. Ribosomal subunits are dissociated prior to the initiation reaction. 

The drug binds to the bacterial 50S ribosomal subunit and inhibits protein synthesis at the peptidyl transferase reaction. Because of the similarity of mammalian mitochondrial ribosomes to those of bacteria, protein synthesis in these organelles may be inhibited at high circulating chloramphenicol levels, producing bone marrow toxicity. 

By contrast, eucaryal mRNAs are translated after extensive modifications of the primary transcripts that yield mature (generally capped and polyadenylated) monocistronic mRNAs. Recognition of translation start sites does not rely upon Shine-Dalgarno recognition instead, the small ribosomal subunit (generally) binds to the capped 5 end of mRNA and scans its nucleotide sequence until the initiator AUG codon is encountered. The polypeptide chains are initiated by a non-formylated methionine and the initiation reactions are aided by as many as 8-10 protein factors, some of which possess ATPase activity and perform functions not encountered in bacteria, such as cap recognition and mRNA unwinding (for a detailed review see ref [4]). 

Fig. 3. Protein composition of Sulfolobus 50S ribosomal subunits. The primary RNA-binding proteins are evidenced as black spots. The solid arrows indicate proteins found in the low-temperature reconstitution intermediate and in the 42S hybrid particles obtained from the reaction between Sulfolobus TP 50 and either E. coli or H. mediterranei 23 S RNA. The open arrows indicate proteins found in the 42S hybrid particles but not in the low-temperature reconstitution intermediate.

The peptidyltransferase activity of ribosomes can be segregated from other translation reactions by the so-called puromycin reaction [129] which monitors the formation of [acetyl-aminoacyl]-puromycin or [peptidyl]-puromycin from puromycin and either [acetyl-aminoacyl]-tRNA or [peptidyl]-tRNA. In its simplest form (termed uncoupled or 30S-subunit-independent peptidyltransferase) the reaction requires large ribosomal subunits and an organic solvent (ethanol or methanol) which is presumably needed to promote the binding of tRNA and puromycin to the 50S subunit. In the absence of organic solvents, however, the reaction (then termed coupled or 30S-subunit-dependent... 

A crucial feature of the peptidyltransferase reaction is a particular adenine that is conserved in rRNAs extracted from the large ribosomal subunits of thousands of different organisms from all three kingdoms. 8equence analysis shows that this adenine base (2451 A) always is present in the active site of the ribosome and acts as a general acid-base catalyst by deprotonating the nucleophilic amine as shown below. 

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