Protein synthesis is an essential process for the multiplication and survival of all bacterial cells. They key targets for protein synthesis is ribosome. A large number of clinically useful antibiotics targeting this complex translational ribonucleoprotein machinery have been identified. Creative Biolabs has developed an excellent drug discovery platform to determine the mode of action of antibacterial drugs related with protein synthesis.
The prokaryotic ribosome includes a small subunit (30S) containing 16S rRNA chain and a large subunit (50S) containing 23S and 5S rRNA chains. Also, it contains distinct and well-defined binding sites for small molecules. The function of ribosome is translating mRNA into a polypeptide chain in three major stages: initiation, elongation, and termination (Figure 1). Generally, antibiotics that target the ribosome almost exclusively bind to one of the three key sites: the decoding (or A-site) on the 30S, the peptidyl transferase center (PTC) on the 50S, and the peptide exit tunnel on the 50S. Creative Biolabs can help you determine the binding sites on ribosome for novel antibiotics.
Figure 1. The process of protein synthesis
Ribosomal Decoding Site
Ribosomal decoding site (also A-site) is located on the 30S subunit. This site is critical for translation as it recognizes and discriminates codon-anticodon. Within the decoding site, two universally conserved nucleotides, A1492 and A1493, are essential for the monitoring process, particularly at the first two bases of the codon. When there is a cognate fit between the mRNA codon and the tRNA anticodon, the ribose-phosphate backbone adopts a geometry that favors hydrogen bonding with the bases of nucleotides A1492 and A1493. Binding of tRNA to the A site is also accompanied by conformational changes in the 16S rRNA. The changes result in the movement of the head of the small subunit towards the large subunit. The amino acid at the 3' end of the A-site tRNA is then accommodated into the peptidyl-transferase center and becomes optimally aligned for addition to the peptide chain. The bacterial rRNA decoding site is the natural target of some antibiotics, for instance, aminoglycosides. Their binding to A site stabilizes a normal mismatch in codon-anticodon pairing, leading to mistranslations.
Figure 2. Binding sites of antibiotics on the bacterial ribosome. (Poehlsgaard et al. 2005)
Peptidyl Transferase Center (PTC)
The PTC connects all functional cores in the ribosome, including the tRNA entrance and exit regions. In essence, it is the ribosomal active site, catalyzing peptide bond formation by meticulously positioning the reactive partners. Several antibiotics target this key site, including chloramphenicol, clindamycin, tiamulin, sparsomycin, and the streptogramins. These antibiotics either hinder tRNA substrate binding or disrupt peptide bond formation by binding to PTC.
Peptide Exit Tunnel
At the end of protein synthesis, the newly synthesized peptide chain travels through a dynamic tunnel below the PTC, lined with mostly rRNA, and emerges approximately 100 Å out of the 50S subunit. While the exit tunnel is primarily straight, there is a bend 20-35 Å away from the PTC. In this section, two ribosomal proteins constrain the tunnel into its narrowest part. At this region, the peptide sequence can influence gating, or pause elongation and termination. Antibiotics, such as macrolides and ketolides, which bind to the peptide exit tunnel and impede the progression of the nascent peptides.
Other Targets in the Ribosome
Although most antibiotics inhibit translation by binding to the A-site, PTC, and peptide exit tunnel, there are still other targets in the ribosome for antibacterial drugs. Certain classes of antibiotics bind to the 30S ribosomal subunit near the A-site: binding to the P-site and E-site. For instance, tetracyclines and glycylcyclines, derivatives of tetracycline that include tigecycline, bind near the A-site and prevent aminoacyl-tRNA from binding. Hygromycin B and aminocyclitol antibiotics inhibit translocation by binding near the A-site. Edeine A inhibits initiation by binding at the E-site, with its spermidine moiety docked in the P-site blocking the initiator tRNA. Negamycin inhibits protein synthesis termination and induces miscoding.
Creative Biolabs' professional group has the ability to determine the MOA for various antibacterial drugs. For more detailed information, please feel free to contact us or directly sent us an inquiry.
Reference
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