Translation peptidyl tRNA

In the translocation step, the ribosome moves exactly three nudeotides (one codon) along the message. This moves the growing peptidyl-tRNA into the P site and aligns the next codon to be translated with the empty A site. 

In the E. coli system, it is important to stop the in vitro translation reaction by rapid cooling on ice. The reaction is usually diluted severalfold in prechilled buffer containing the components for stabilization of the ribosomal complexes. In the E. coli system, the ribosomal complexes can be very efficiently stabilized by low temperature and by high Mg2+ concentrations (50 mM), and then used for affinity selection. It is believed that high Mg2+ condenses the ribosome by binding totherRNA, making it difficult for the peptidyl-tRNA to dissociate or be hydrolyzed. The low temperature probably slows down the hydrolysis of the peptidyl-tRNA ester bond, and perhaps also the thermal motions, which would facilitate dissociation of the peptidyl-tRNA. Such complexes are stable for up to several days. 

Fig. 4. Role of the stop codon and lOSa-RNA in E. coli translation. (A) When a stop codon is encountered, a complex of two release factors, RF-1 and RF-3 or RF-2 and RF-3, binds instead of the tRNA. The release factor RF-1 recognizes the stop codons UAA and UAG, while RF-2 recognizes UAA and UGA. The binding of the release factor complex results in hydrolysis of the peptidyl-tRNA and release of the peptide. (B) The role of lOSa-RNA. If truncated mRNA without a stop codon is translated in E. coli, the ribosome stops at the end of the mRNA. lOSa-RNA can then bind to the ribosomal A site and lOSa-RNA can act as tRNA by transferring an alanine to the truncated protein. Subsequently, lOSa-RNA acts as mRNA and a peptide tag with the indicated sequence is added to the truncated protein. lOSa-RNA encodes a stop codon and therefore the protein is released and then degraded by proteases specifically recognizing this C-terminal tag.

An extreme example of translational bypassing occurs in the translation of the bacteriophage T4 gene 60 (13). The ribosome reads the first 46 codons of the mRNA, pauses at a UAG stop codon, hops over 47 nucleotides (a 50-nucleotide coding gap), and resumes translation. This bypass requires matched codons. The peptidyl-tRNA pairs first with one codon, slips, and then pairs with the matching codon, which results in only one... 

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... 

After the correct aminoacyl-lRNA has been placed in the A site, the transfer of the polypeptide chain from the tRNA in the P site is a thermodynamically spontaneous process, driven by the formation of the stronger peptide bond in place of the ester linkage. However, protein synthesis cannot con-linue without the translocation of the mRNA and the tRNAs within the ribosome. The mRNA must move by a distance of three nucleotides so that the next codon is positioned in the A site for interaction with the incoming aminoacyl-tRNA. At the same time, the deacylated tRNA moves out of the P site into the E site on the 30S subunit and the peptidyl-tRNA moves out of the A site into the P site on the 30S subunit. The movement of the peptidyl-tRNA into the P site shifts the mRNA by one codon, exposing the next codon to be translated in the A site. 

The second stage of translation is chain elongation. This occurs in three steps that are repeated until protein synthesis is complete. We enter the action after a tetrapeptide has already been assembled, and a peptidyl tRNA occupies the P-site (Figure 24.19b). 

The first event is binding of an aminoacyl-tRNA molecule to the empty A-site. Next, peptide bond formation occurs. This is catalyzed by an enzyme on the ribosome called peptidyl transferase. Now the peptide chain is shifted to the tRNA that occupies the A-site. Finally, the tRNA in the P-site falls away, and the ribosome changes positions so that the next codon on the mRNA occupies the A-site. This movement of the ribosome is called translocation. The process shifts the new peptidyl tRNA from the A-site to the P-site. The chain elongation stage of translation requires the hydrolysis of GTP to GDP and Pj. Several elongation factors are also involved in this process. 

The last stage of translation is termination. There are three termination codons— UAA, UAG, and UGA—for which there are no corresponding tRNA molecules. When one of these "stop" codons is encountered, translation is terminated. A release factor binds the empty A-site. The peptidyl transferase that had previously catalyzed peptide bond formation hydrolyzes the ester bond between the peptidyl tRNA and the last amino acid of the newly s)mthesized protein (Figure 24.19c). At this point the tRNA, the newly synthesized peptide, and the two ribosomal subunits are released. 

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