Ribosomes dissociation

Example polysome profiles from sucrose gradient fractionation of HeLa cell lysates, either untreated or treated with EDTA, are shown in Fig. 6.4A and B. The polysome profile of untreated HeLa lysates shows three defined peaks in less dense fractions (6 to 9), which correspond to the 80S, 60S, and 40S peaks (Fig. 6.4A). Treatment of lysates with 30 /iM EDTA results in ribosome dissociation leaving predominantly free 60S and 40S subunits... 

Chang, C. W. Effect of ozone on sulfhydryl groups of ribosomes in pinto bean leaves. Relationship with ribosome dissociation. Biochem. Biophys. Res. Commun. 44 1429-1435, 1971. 

FIGURE 27-26 Termination of protein synthesis in bacteria. Termination occurs in response to a termination codon in the A site. First, a release factor, RF (RF-1 or RF-2, depending on which termination codon is present), binds to the A site. This leads to hydrolysis of the ester linkage between the nascent polypeptide and the tRNA in the P site and release of the completed polypeptide. Finally, the mRNA, de-acylated tRNA, and release factor leave the ribosome, and the ribosome dissociates into its 30S and 50S subunits. 

The appearance of a UAA or UAG termination (stop) codon in the A site causes release factor RF1 to bind whereas RF2 recognizes UGA. RF3 assists RF1 and RF2. The release factors trigger peptidyl transferase to transfer the polypeptide to a water molecule instead of to aminoacyl-tRNA. The polypeptide, mRNA, and free tRNA leave the ribosome and the ribosome dissociates into its subunits ready to begin a new round of translation. 

Eventually, one of three termination codons (also called Stop codons) becomes positioned in the A site (Fig. 7). These are UAG, UAA and UGA. Unlike other codons, prokaryotic cells do not contain aminoacyl-tRNAs complementary to Stop codons. Instead, one of two release factors (RF1 and RF2) binds instead. RF1 recognizes UAA and UAG whereas RF2 recognizes UGA. A third release factor, RF3, is also needed to assist RF1 or RF2. Thus either RF1 + RF3 or RF2 + RF3 bind depending on the exact termination codon in the A site. RF1 (or RF2) binds at or near the A site whereas RF3/GTP binds elsewhere on the ribosome. The release factors cause the peptidyl transferase to transfer the polypeptide to a water molecule instead of to aminoacyl-tRNA, effectively cleaving the bond between the polypeptide and tRNA in the P site. The polypeptide, now leaves the ribosome, followed by the mRNA and free tRNA, and the ribosome dissociates into 30S and 50S subunits ready to start translation afresh. 

Four major steps are required to initiate translation ribosome dissociation, formation of a preinitiation complex, formation of the 40S initiation complex and formation of the SOS initiation complex. 

When a stop codon appears at the A site translation is terminated. There are no tRNA s that recognize stop codons. Instead releasing factors, eRF, recogiiize the stop codon. The releasing factors along with peptidyl transferases and GTP catalyze the hydrolysis of the bond between the polypeptide chain and the tRNA. The protein and tRNA disassociate from the P site and the ribosome dissociates into the 40S and 60S subunits releasing the mRNA. 

Dissociation of the 708 ribosome. The 708 ribosome dissociates into 308 and 508 subunits, which may start synthesis of another polypeptide chain. 

The answer is b. (Murray, pp 452-467. Scriver, pp 3-45. Sack, pp 1-40. Wilson, pp 101-120.) During the course of protein synthesis on a ribosome, peptidyl transferase catalyzes the formation of peptide bonds. However, when a stop codon such as UAA, UGA, or UAG is reached, aminoacyl-tRNA does not bind to the A site of a ribosome. One of the proteins, known as a release factor, binds to the specific trinucleotide sequence present. This binding of the release factor activates peptidyl transferase to hydrolyze the bond between the polypeptide and the tRNA occupying the P site. Thus, instead of forming a peptide bond, peptidyl transferase catalyzes the hydrolytic step that leads to the release of newly synthesized proteins. Following release of the polypeptide, the ribosome dissociates into its major subunits. 

The 70S ribosome is now unstable in the presence of a protein called ribosome recycling factors, as well as the initiation factors IF3 and IFl. Consequently, the 70S ribosome dissociates to SOS and 30S subunits and is ready for another round of translation. 

Ribosomes and ribosomal RNA have been studied extensively via sedimentation coefficients. Most research on prokaryotic systems has been done with the bacterium Escherichia coli, which we shall use as an example here. An E. coli ribosome typically has a sedimentation coefficient of 70S. When an intact 70S bacterial ribosome dissociates, it produces a light SOS subunit and a heavy 50S subunit. Note that the values of sedimentation coefficients are not additive, showing the dependence of the S value on the shape of the particle. The 30S subunit contains a 16S rRNA and 21 different proteins. The 50S suhunit contains a 5S rRNA, a 23S rRNA, and 34 different proteins (Figure 9.26). For comparison, eukaryotic ribosomes have a sedimentation coefficient of SOS, and the small and large subunits are 40S and 60S, respectively. The small suhunit of eukaryotes contains an 18S rRNA, and the large suhunit contains three types of rRNA molecules 5S, 5.8S, and 28S. 

FIGURE 12.17 Simultaneous protein synthesis on polysomes. A single mRNA molecule is translated by several ribosomes simultaneously. Each ribosome produces one copy of the polypeptide chain specified by the mRNA. When the protein has been completed, the ribosome dissociates into subunits that are used in further rounds of protein synthesis. 

Four bands light, heavy, a hybrid of light 305 and heavy 505, and a hybrid of heavy 305 and light 505. Recall that ribosomes dissociate into 305 and 505 subunits after the polypeptide product is released. As protein synthesis continues, 70S ribosomes are reformed from the various heavy and light subunits. 

Dissociation of SOS ribosome subunit IF-3 binds to the 30S subunit to promote the dissociation of the inactive 70S ribosomes, while IF-1 assists this binding and therefore facilitates the rate of ribosome dissociation. 

The chain elongation process continues and polypeptide synthesis continues until the ribosome complex reaches a stop codon (UAA, UAG, or UGA) on the mRNA. At that point, a specific protein known as a termination factor binds to the stop codon and catalyzes the hydrolysis of the completed polypeptide chain from the final tRNA. The empty ribosome dissociates and can then bind to another strand of mRNA to once again begin the process of protein synthesis. 

Russell, D. W., and Spremulli, L. L., 1979, Purification and characterization of ribosome dissociation factor (eIF-6) from wheat germ, J. Biol. Chem. 254 8796. 

Chain termination, on the other hand, is signaled by the codons UAG, UAA, or UGA, to which normally no tRNA corresponds only in suppressor mutants, do certain tRNA s (like su -tyr-tRNA) permit the reading of termination signals. The 70 S ribosome dissociates into 50 S and 30 S subunits upon chain termination. 

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