Peptidyl transferase reaction

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... 

Iordanov, M. S. et al. Ribotoxic stress response Activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA. Mol. Cell. Biol. 17, 3373, 1997. 

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 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. 

In the first step of the peptidyl transferase reaction, a peptidyl tRNA molecule is bound in the P-site with its nascent peptide extending down the peptide exit tunnel (Fig. 4.1). An elongation factor binds to a factor binding site (FBS) and positions an aminoacyl-tRNA in the A-site. The a amino group of the aminoacyl-tRNA nucleophilically attacks the ester bond which connects the peptide to the tRNA bound in the P-site (Fig. 4.2). The ester bond is broken as an amide bond forms, and the peptide becomes one amino acid longer, and is now attached to the tRNA that in the A-site. Translocation of the products follows peptide bond formation, as the newly formed deacylated- tRNA of the P-site moves into the E-site, and as the newly elongated peptidyl-tRNA moves from the A-site into the P-site. 

F. 4.2 Peptidyl transferase reaction. Left The a amino group of an A-site substrate, attacks (arrow) the ester bond that links a P-site substrate tRNA to its nascent peptide chain. The first 73 nucleotides of tRNA are represented by ribbons, C74 and C75 are represented by the letter C, and A76 and the peptide are represented by chemical drawings. Center During the nucleophilic attack. 

Most antibiotics that inhibit the function of the SOS subunit bind near its peptidyl transferase center (Fig. 4.4) and block peptide bond formation. Crystal structures are available for several such antibiotics bound to the ribosome (Fig. 4.5). They appear to inhibit the peptidyl transferase reaction either by competing directly with its substrates for binding, or indirectly by blocking the exit tunnel. 

The bound macrolides almost completely occlude the peptide exit tunnel, which explains the two distinctive characteristics of macrolide inhibition. Firstly, the reason that macrolides do not inhibit ribosomes that are already actively making protein is that the nascent peptide in the exit tunnel blocks access to the macrolide binding site. Secondly, the reason macrolides do not directly inhibit the peptidyl transferase reaction is that they bind near to, but not directly at, the active site. Only after a few peptide bonds are formed will the peptide contact the bound macrolide and be blocked from further elongation. As a result, short di-, tri- and tetra-peptides will accumulate. 

Seila AC, Okuda K, Nunez S, Seila AF, Strobel SA. Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction. Biochemistry 2005 44 4018-4027. 

Chloramphenicol binds to the 50S subunit of ribosomes and appears to act by inhibiting the movement of ribosomes along mRNA, probably by inhibiting the peptidyl transferase reaction by which the peptide chain is extended. 

The second step in the elongation of protein synthesis is the peptidyl-transferase reaction in which a peptide bond is formed between the amino acid in the P site and the amino acid coupled to aminoacyl-tRNA in the A site. This reaction is facilitated by the intrinsic activity of the ribosome. As a result of this reaction, the growing polypeptide chain becomes elongated without moving forward. The movement or translocation of the dipeptide-tRNA from the A site to the P site is achieved by the action of EF-2, while another molecule of GTP is hydrolyzed. This process results in the relative movement of the mRNA by three nucleotides, so that a new codon becomes readable in the A site. The deacylated tRNA is pushed out of the ribosome after a transient halt at the so-called exit (E) site. At this point, all the components... 

In the second step of elongation, the a-amino group of the amino acid in the A site (AA2) acts as a nucleophile and attacks the carbonyl group of AA1 (in this case fMet). This reaction leads to the formation of a dipeptidyl-tRNA in the A site and a deacylated-tRNAfMet in the P site (Fig. 26.12). As shown by Harry Noller in 1992, this peptidyl transferase reaction is catalyzed by ribozyme activity present in the 23 S rRNA of the 50S ribosome subunit (and the 28S rRNA in the 60S ribosome subunit in eukaryotes), rather than by ribosomal proteins, as originally thought. 

Peptidyl transferase reaction in the elongation phase of protein synthesis. 

A good example of translational inhibition is the mechanism by which puromycin mimics the structure of the aminoacyl group of aminoacyl-tRNA, as shown in Figure 26.15. Puromycin is an aminoacyl-tRNA analogue that is able to bind to the A site of both prokaryotic and eukaryotic ribosomes, even without a corresponding tRNA, and thus act as an acceptor for the peptidyl transferase reaction. 

Protein synthesis requires three discrete phases initiation, elongation, and termination. Initiation involves the assembly of an initiation complex consisting of initiation factors, ribosomal subunits, mRNA, the initiator methionine tRNA, and a GTP hydrolysis step. Elongation is a continuous cycling of aminoacylated tRNAs into the A site, GTP hydrolysis, and a peptidyl transferase reaction catalyzed by the ribozyme activity encoded within the large rRNA. Termination occurs when one of the three termination codons is present in the A site and no tRNA is available to function as the acceptor in the peptidyl transferase reaction. 

Kubota, K., Okuyama, A., and Tanaka, N. (1972). Differential effects of antibiotics on peptidyl transferase reactions. Biochem. Biophys. Res. Commun. 47, 1196-1202. 

In the fragment reaction, the ability of puromycin to mimic the aminoacyl-tRNA in the peptidyl transferase reaction was exploited to measure catalytic activity. Puromycin was subsequently used to design a transition-state analog for the peptidyl transferase reaction, known as the Yarns inhibitor, in which it is linked to the oligonucleotide CCdA by a phosphoramide group [73]. In a complex with the 50S ribosomal subunit, the Yarns inhibitor was used to define the catalytic site in a high-resolution crystal structure. No protein was found within 18 A of this site [74]. This result demonstrated conclusively that the catalytic activity indeed resides in the ribosomal RNA. 

The ribosome is actually a ribozyme. There are no amino acids at the active site where the peptidyl transferase reaction occurs. Specific bases on the rRNA are believed to catalyze the reaction. 

Peptidyl transferase center (PTC), a ribo-somal complex catalyzing peptide bond formation. The ribosomal PTC resides in the large ribosomal subunit and catalyzes both peptide bond formation and peptide release. The peptidyl transferase reaction involves aminolysis by the depro-tonated a-amino function of the A-site aminoacyl-tRNA of the ester bond linking the nascent peptide to the 3 hydroxyl of the 3 terminal ribose of the P-site tRNA. The formed short-lived tetrahedral reaction intermediate decomposes by donation a proton back to the leaving oxygen, yielding... 

Puromycin, though not a direct inhibitor of peptid-yl-transferase, inhibits normal peptide chain elongation by participating in the peptidyl transferase reaction. 

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