Ribosomes aminoacyl-tRNA binding

J. Tetracyclines bind to the A-site of the prokaryotic ribosome and prevent aminoacyl-tRNAs from binding. Thus protein synthesis is halted becanse new amino acids cannot be added to the growing protein. 

Prevents the aminoacyl-tRNA from binding to the ribosome Prevents the incorporation of new amino acids into the protein Inhibits the initiation of protein synthesis Prevents the new peptide bond from being formed... 

Figure 1.84 Schematic of translation. The mRNA codons are read and converted from nucleoside sequences to protein primary structure by means of cognate aminoacyl-tRNAs. All mRNA codons are translated at a ribosome (prepared from rRNA) that has two cognate aminoacyl-tRNA binding sites P (peptidyl) and A (aminoacyl). All tRNAs are "adaptors" that can bind a particular mRNA codon through their anticodon loop, using Watson-Crick base pairing, and also associate covalently with the appropriate amino acid residue coded for by the corresponding mRNA codon When two cognate aminoacyl-tRNA molecules bind mRNA in P and A sites (a), then both are close enough for peptide link formation to take place with the emergence of a peptide chain (b). As amino acyl tRNA molecules continue to dock sequentially onto mRNA codons (in the direction 5 (c), and amino acid residues continue to be added (W —> C ) (d),...

C-labeled histidine-tRNA. Only the aminoacyl-tRNA whose binding is directed by the trinucleotide codon will become bound to the ribosomes and retained on the nitrocellulose filter. The amount of radioactivity retained by the filter is a measure of trinucleotide-directed binding of a particular labeled aminoacyl-tRNA by ribosomes. Use of this binding assay to test the 64 possible codon trinucleotides against the 20 different amino acids quickly enabled researchers to assign triplet code words to the individual amino acids. The genetic code was broken. 

Protein synthesis begins with the N-terminal amino acid and proceeds from this point. In some bacteria, yeast, and higher organisms, this first aminoacyl-tRNA is known to be iV-formylmethionyl-tRNA Formylation of the amino function can be considered as a protecting group to prevent participation of the amino function is peptide bond formation. The fMet-tRNA is then the first aminoacyl-tRNA to bind to the ribosome and mRNA. After the protein is synthesized, the formyl group is removed by enzymatic cleavage (formylase). 

Fig. 2.1. Peptide bond formation in the biological system. The entire process takes place on the ribosome and involves two binding sites, the P (peptidyl) and A (aminoacyl) sites. The reaction is catalyzed by the enzyme peptidyl transferase. It is the message carried by mRNA (which in turn is dictated by the genetic material DNA) which determines by specific interactions as to which aminoacyl-tRNA will bind at the P and...

Fig. 2.2. Structure of the antibiotic puromycin. Puromycin interrupts the synthesis of the protein chain by mimicing an aminoacyl-tRNA complex, binding to the ribosome, attacking a peptidyl-tRNA (at the P site) with its amino function, but then not undergoing attack by an aminoacyl-tRNA, hence terminating the synthesis. In the upper right corner is shown the adenylic acid terminus of tRNA.

This conformation is both able to bind aminoacylated tRNA and bind to the ribosome. Unfortunately the fragmented protein binds neither GTP nor tRNA. However, it is known that the aminoacyl end of the tRNA interacts with domain I of EF-Tu (Jonak et al., 1979 Duffy et al., 1981). Thus His-66 can be crosslinked to N -bromoacetyl-lysyl-tRNA and chemical blocking of Cys-81 prevents tRNA from binding. tRNA and short fragments of its aminoacy 1-acceptor end prevent this cysteine from reacting. This interaction site for tRNA is neither far from the assumed site for the of GTP nor far from the site of proteolytic cleavage (Arg-58) near the connection to domain III. 

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). 

Tetracycline and its derivative doxycycline are antibiotics widely used in the treatment of bacterial infections. They also exert an antimalarial activity. Tetracyclines inhibit the binding of aminoacyl-tRNA to the ribosome during protein synthesis. 

The regions of the tRNA molecule teferred to in Chapter 35 (and illustrated in Figure 35-11) now become important. The thymidine-pseudouridine-cyti-dine (T PC) arm is involved in binding of the amino-acyl-tRNA to the ribosomal surface at the site of protein synthesis. The D arm is one of the sites important for the proper recognition of a given tRNA species by its proper aminoacyl-tRNA synthetase. The acceptor arm, located at the 3 -hydroxyl adenosyl terminal, is the site of attachment of the specific amino acid. 

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. 

Of the fonr possible optical isomers of chloramphenicol, only the o-threo form is active. This antibiotic selectively inhibits protein synthesis in bacterial ribosomes by binding to the 50S subunit in the region of the A site involving the 23 S rRNA. The normal binding of the aminoacyl-tRNA in the A site is affected by chloramphenicol in such a... 

Several key concepts are worth remembering. GTP is used as an energy source for translation, but ATP is used to form the aminoacyl-tRNA. The ribosome effectively has two kinds of tRNA binding sites. Only tRNAMet can bind to the P (for peptide) site, and this only occurs during the initial formation of the functional ribosome (initiation). All other aminoacyl-tRNAs enter at the A (for amino acid) binding site. After formation of the peptide bond (this doesn t require GTP hydrolysis), the tRNA with the growing peptide attached is moved (translocated) to the other site (this does require GTP hydrolysis). 

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