Aminoacyl-tRNA binding site

It has been known for some time that tetracyclines are accumulated by bacteria and prevent bacterial protein synthesis (Fig. 4). Furthermore, inhibition of protein synthesis is responsible for the bacteriostatic effect (85). Inhibition of protein synthesis results primarily from dismption of codon-anticodon interaction between tRNA and mRNA so that binding of aminoacyl-tRNA to the ribosomal acceptor (A) site is prevented (85). The precise mechanism is not understood. However, inhibition is likely to result from interaction of the tetracyclines with the 30S ribosomal subunit because these antibiotics are known to bind strongly to a single site on the 30S subunit (85). 

Elongation is a cycUc process on the ribosome in which one amino acid at a time is added to the nascent peptide chain. The peptide sequence is determined by the order of the codons in the mRNA. Elongation involves several steps catalyzed by proteins called elongation factors (EFs). These steps are (1) binding of aminoacyl-tRNA to the A site, (2) peptide bond formation, and (3) translocation. 

As the name implies, this class of compounds has four linearly attached six membered rings. Tetracyclines are bacteriostatic and reversibly bind to the 30S ribosomal subunit. They interfere with the binding of aminoacyl tRNA at the A-site of the ribosome. ... 

Tetracyclines enter microorganisms in part by passive diffusion and in part by an energy-dependent process of active transport. Susceptible cells concentrate the drug intracellularly. Once inside the cell, tetracyclines bind reversibly to the 30S subunit of the bacterial ribosome, blocking the binding of aminoacyl-tRNA to the acceptor site on the mRNA-ribosome complex (Figure 44-1). This prevents addition of amino acids to the growing peptide. 

Tetracyclines inhibit protein synthesis in bacteria by blocking the A site on the ribosome, preventing the binding of aminoacyl-tRNAs. Chloramphenicol inhibits protein synthesis by bacterial (and mitochondrial... 

Ribosomes are large complexes of protein and rRNA. They consist of two subunits. Each ribosome has three binding sites for tRNA molecules, the A, P, and E sites that cover three neighboring codons. The A site codon binds an incoming aminoacyl-tRNA, the P site codon is occupied by peptidyl-tRNA. and the E site is occupied by the empty tRNA as it is about to exit the ribosome. 

The tetracyclines (Fig. 21-10) inhibit the binding of aminoacyl-tRNA at the A site in the 30S ribosomal subunitkk However, this doesn t appear to be a direct effect. Tetracyclines bind to the 16S RNA at two sites. A major site is on helix 34 near the spectinomycin site in the platform region. A second site is on helix 27, the switch helix, which plays a direct role in translocation (see Eq. 29-9).kk Although the basis of the inhibition is not clear,... 

Krauss, G., Pingoud, A., Boehme, D., Riesner, D., Peters, F., Maass, G. (1975) Equivalent and Non-Equivalent Binding Sites for tRNA on Aminoacyl-tRNA Synthetases, Ear. J. Biochem. 55,... 

T. Pape, W. Wintermeyer, and M.V. Rodnina. 1998. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome EMBO J. 11 7490-7497. (PubMed)... 

E. Tetracycline prevents the binding of aminoacyl-tRNAs to the A site on ribosomes. 

EF-Tu in the GDP form must be reset to the GTP form to bind another aminoacyl-tRNA. Elongation factor Ts, a second elongation factor, joins the EF- Fu complex and induces the dissociation of GDP. Finally, GTP binds to EF-lu, and EF-Ts is concomitantly released. It is noteworthy that EF-Tu does not interact loith fMet-tRNAp Hence, this initiator tRNA is not delivered to the A site. In contrast, Met-tRNAn, like all other aminoacyl-... 

The answer is c. (Murray, pp 452-467. Scriver, pp 3-45. Sack, pp 1—40. Wilson, pp 101—120.) Two molecules of GTP are used in the formation of each peptide bond on the ribosome. In the elongation cycle, binding of aminoacyl-tRNA delivered by EF-Tu to the A site requires hydrolysis of one GTE Peptide bond formation then occurs. Translocation of the nascent peptide chain on tRNA to the P site requires hydrolysis of a second GTE The activation of amino acids with aminoacyl-tRNA synthetase requires hydrolysis of ATP to AMP plus PP,. 

Mansouri, S., NouroUahzadeh, E. and Hudak, K.A. (2006) Pokeweed antiviral protein depurinates the sarcin/ricin loop of the rRNA prior to binding of aminoacyl-tRNA to the ribosomal A-site. RNA, 12, 1683-1692. 

Binding of aminoacyl-tRNA in the A-site of the ribosomal complex. 

Termination. During termination the polypeptide chain is released from the ribosome. Translation terminates because a stop codon cannot bind an aminoacyl-tRNA. Instead, a protein releasing factor binds to the A site. Subsequently, pep-tidyl transferase (acting as an esterase) hydrolyzes the bond connecting the now-completed polypeptide chain and the tRNA in the P site. Translation ends as the ribosome releases the mRNA and dissociates into the large and small subunits. 

Before EF-Tu can bind an aminoacyl-tRNA, its GDP moiety must be replaced by GTP. The binding of EF-Ts to EF-Tu (GDP) displaces GDP. EF-Ts is then itself displaced by an incoming GTP. EF-Tu (GTP) then associates with an aminoacyl-tRNA to form an EF-Tu (GTP) aminoacyl-tRNA complex, which proceeds to deliver the aminoacyl-tRNA to the A site of the ribosome. 

Kinetic proofreading is a mechanism that ensures that the correct codon-anticodon pairing occurs in the A site of ribosomes. In eukaryotes eEF-la mediates the binding of aminoacyl-tRNAs to the A site. When the correct pairing occurs eEF-1 a hydrolyses its bound GTP and subsequently exits the ribosome. 

When errors in ammo acid-tRNA binding do occur, they are usually the result of similarities in amino acid structure. Several aminoacyl-tRNA synthetases possess a separate proofreading site that binds incorrect aminoacyl-tRNA products and hydrolyzes them. 

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