Aminoacyl-tRNA binding to ribosomes

PS (inhibits aminoacyl tRNA binding to ribosome) [antibacterial]... 

The ribosome includes three sites for tRNA binding called the A (aminoacyl) site, the P (peptidyl) site, and the E (exit) site. With a tRNA attached to the growing peptide chain in the P site, an aminoacyl-tRNA binds to the A site. A peptide bond is formed when the amino group of the aminoacyl-tRNA nucleophically attacks the ester carbonyl group of the peptidyl-tRNA. On peptide-bond formation, the tRNAs and mRNA must be translocated for the next cycle to begin. The deacylated tRNA moves to the E site and then leaves the ribosome, and the peptidyl-tRNA moves from the A site into the P site. 

Chlortetracyclin inhibits protein synthesis by binding to the 30S subunit of ribosomes and prevents the aminoacyl-tRNA binding to the A site on the ribosome. This prevents the codon-anticodon interaction from taking place. Protein release is also inhibited. 

Fig. 15.11. Recycling of EFl in eukaryotes. Note that EFl is a heterotrimeric G protein. Its a-subunit binds GTP and activates the process whereby an aminoacyl-tRNA binds to the A site of the ribosome. GTP is hydrolyzed, and EFla binds to the EFipy subunits, releasing GDP. GTP binds to the a subunit, the (Jy subunits are released, and EFla GTP is ready for another round. In prokaryotes, EFla is EF-Tu and the protein corresponding to (Jy is EF-Ts.

One ATP is used for activation of the tRNA, and then one GTP is hydrolyzed on binding of the aminoacyl-tRNA binding to the A site on the ribosome, and another GTP is hydrolyzed on translocation. Thus the equivalent of three ATPs are used for each amino acid incorporated, ignoring initiation. Note that in amino acid activation, the products are AMP and PPi, the latter being hydrolyzed to Pi to drive the reaction to... 

Elongation. The next aminoacyl-tRNA (in Fig. 70.1 it is glycinyl-tRNA ) binds to the A site attracted by the next codon (CAG) of the mRNA. NB Initiator methionyl-tRNAi (the special one ) binds to the ribosomal P site whereas all other aminoacyl tRNAs bind to the... 

Stage 2 Initiation The mRNA bearing the code for the polypeptide to be made binds to the smaller of two ri-bosomal subunits and to the initiating aminoacyl-tRNA. The large ribosomal subunit then binds to form an initiation complex. The initiating aminoacyl-tRNA base-pairs with the mRNA codon AUG that signals the beginning of the polypeptide. This process, which requires GTP, is promoted by cytosolic proteins called initiation factors. 

The final step in elongation is known as translocation (fig. 29.17). This reaction, like aminoacyl-tRNA binding, is catalyzed by a factor (the translocation factor, known as EF-G in prokaryotic systems and EF-2 in eukaryotic systems) that cycles on and off the ribosome and hydrolyzes GTP in the process. The overall purpose of translocation is to move the ribosome physically along the mRNA to expose the next codon for 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. 

Several sites on the ribosome interact with tRNA (Fig. 4.1) and also are targeted by antibiotics. The peptidyl-tRNA binding site (P-site) of the large subunit binds to the 3 end of peptidyl-tRNA. The aminoacyl-tRNA binding site (A-site) of the... 

Chloramphenicol inhibits protein synthesis by binding to the 50S subunit of the bacterial ribosome and blocking aminoacyl-tRNA binding. 

The cycle of peptide-chain elongation continues until one of the three stop codons (UAA, UAG, UGA) is reached. There is no aminoacyl-tRNA complementary to these codons, and instead a termination factor or a release factor (RF) with bound GTP binds to the ribosome and induces hydrolysis of both the aminoacyl-linkage and GTP, thereby releasing the completed polypeptide chain from the ribosome. The 475 amino acid-long sequence of rabbit liver RF has been deduced from its cDNA sequence, and it shows 90% homology with mammalian trypto-phanyl-tRNA synthetase (Lee et al., 1990). It has also been reported that for efficient and accurate termination, an additional fourth nucleotide (most commonly an A or a G) after the stop codon is required (Tate and Brown, 1992). The exact role of the fourth nucleotide in the termination of protein synthesis is not fully understood at present. 

The genetic information in DNA is converted into the linear sequence of amino acids in polypeptides in a two-phase process. During transcription, RNA molecules are synthesized from a DNA strand through complementary base pairing between the bases in DNA and the bases in free ribonucleoside triphosphate molecules. During the second phase, called translation, mRNA molecules bind to ribosomes that are composed of rRNA and ribosomal proteins. Transfer RNA-aminoacyl complexes position their amino acid cargo in the catalytic site within the ribosome in a process that involves complementary base pairing between the mRNA codons and tRNA anticodons. Once the amino acids are correctly positioned within the catalytic site, a peptide bond is formed. After the mRNA molecule moves relative to the ribosome, a new codon enters the ribosome s catalytic site and base pairs with the appropriate anticodon on another aminoacyl-tRNA complex. After a stop codon in the mRNA enters the catalytic site, the newly formed polypeptide is released from the ribosome. 

The mode of action of kasugamycin is similar to that of blasticidin S. It inhibits protein synthesis in fungi and bacteria. It inhibits binding of the aminoacyl-tRNA complex to the ribosome (Tanaka et al., 1966), but does not disturb the synthesis of nucleic acids. The inhibitory effect is considerably lower in the liver tissue of rats. The selectivity of toxicity may be due to the particular sensitivity to kasugamycin of the ribosomes of each type of organism. This may also explain the relatively rapid development of resistance to kasugamycin. 

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