Antibiotics inhibit prokaryotic ribosomes in a variety of ways. Aminoglycoside antibiotics that bind to the 30s ribosomal A-site RNA cause misreading of the genetic code and inhibit translocation. The aminoglycoside antibiotic paromomycin binds specifically to the RNA oligonucleotide at the 30S A site. This antibiotic binds the major groove on the A-site RNA within a pocket created by an A-A pair and a single bulged adenine. There are several interactions that occur between the aminoglycoside chemical groups and the conserved nucleotides in the RNA. The structure explains binding of diverse aminoglycosides to the ribosome, their specific activity against prokaryotic organisms and various resistance mechanisms.

During translation aminoacyl-tRNA anti-codons and mRNA requires specific interaction on the small 30S ribosomal subunit, more specifically, on a highly conserved ribosomal RNA sequence. The antibiotic binds to this rRNA region and disrupts protein synthesis by codon misreading. The precise mechanism is unknown. Antibiotics decrease the dissociation rate of cognate and near cognate aminoacyl tRNA from the A – site.

This site focuses on the interactions of the 16S ribosomal region A- site in the major loop, with aminoglycoside antibiotic paromomycin. This exhibit is based on the structure solved by NMR by Puglisi et.al. (Science 274, 1367-1371 (1996) The PDB code is 1PBR

A 27nt RNA is used to characterize the structure and antibiotic (usu. 64nt) binding of the small subunit ribosomal A site. Paromomycin binds to this 27nt RNA oligonucleotide because of the critical nucleotides C1407-G1494 base pair, A1408, A1493, U1495. It is also due to the base pairs in the lower stem and asymmetry of the internal loop resulting from the presence of a nucleotide at position 1492. Usually, when there is no antibiotic to bind to, the internal loop of the A site oligonucleotide is closed by formation of base pair U1406-U1495 and C1407-G1494. These additional pairs form a longer oligonucleotide that has 2 GC pairs in the upper stem. The A1408, A1492, and A1493 region is very dynamic as well.

The RNA structure in the complex is essentially two continuos A-form helical stems that closes the asymmetric internal loop, and contains non-canonical pairings. The upper stem is basically left unchanged, and is extended through a non-canonical U1406-U1495 base pair. The N3 and O4 of U1406 plus hydrogen bonding with O2 and N3 of U1495 forms the U1406-U1495 pair. The C1407-G1494 base pair closes the internal loop. (A1408, A1492, and A1493) . The paromomycin is stacked between the two stems and base pair A1408 and A1493. The A1408 and A1493 pair is buckled in the ensemble of conformations with A1493 at a 45 degree angle to the plane of the A1408. The formation of the A1408-A1493 pair is consistent with the protection of the N1 position of A1408 from methylation upon binding of paromomycin to the ribosome and A site oligonucleotide. The reactivities of N1 positions of A 1492 and A1493 are unaffected by antibiotic binding and these groups are solvent accessible on the minor groove side in the antibiotic RNA complex. The RNA backbone is distorted by the presence of the bulged adenine A1492 and non-canonical A1408-A1493 pair. This distortion arises from non-standard values of backbone dihedral torsion angles and leads to formation of a distinct binding pocket.

Paromomycin binds in the major groove of the A site RNA within the internal loop. Paromomycin has a rigid structure except where the glycosidic linkages connects the rings. When the RNA is bound, the antibiotic adopts an L-conformation. Rings II, III, IV form a linear array that lines the major groove from U1406-U1495 base pair to the A1410-A1490.

Ring I of paromomycin lies in the pocket that is opened by the bulged nucleotide A1492 and A1408-A1493 and approx. 90 degrees to ring II, III, IV. Ring I and II adopt chair configurations with amino and hydroxyl substituents in equatorial positions, they make specific contacts that stabilize the antibiotics RNA complex. Ring I stacks base moiety of G1491 and surrounded by phosphate of A 1492 and A1493 . Internal H bonds in paromomycin may help to orient ring I for specific binding with RNA.

Ring II spans U1406-U1495 and C 1407 G1494 base pairs. The amino group at 1 and 3 of ring II make H bonds to O4 of U1495 and N7 of G1494 respectively. The 3 amino group may also interact with the phosphates between A1493 and G1494. The C1407 G1494 base pair is required for specific amino glycoside binding and carboxyethylation of the N7 position of G1497 interferes with paromomycin binding. The H bond acceptor in the major groove at 1495 is essential for specific binding. U1406-U1495 and C1407-G1494 with specific contacts with ring II are universally conserved. They are required for specific binding of these antibiotics to ribosomal RNA.

Antibiotic-RNA is extended on major groove by rings 3 and 4 . Ring 4 hydroxyl group probably causes electrostatic interactions with phosphate backbone at U1406, C1407, U1490 . The relative sequence is independent but does need base pairs in lower stem .

Results and Conclusions:

Antibiotics act on prokaryotes better because they are more sensitive to antibiotic concentration then eukaryotic organisms. Also prokaryotes rRNA have an A at 1408 while in eukaryotes it’s a G. The A1408-A1493 pair is essential for antibiotic binding and leads to formation of specific binding pocket for Ring I. The base pair at 1409-1491 provides extra support and without it, leads to aminoglycoside resistance.

Also methylation of rRNA residues A 1408 or G1405 at N1 and N7 positions also lead to resistance and hinders the formation of A1408-A1493 base pair. G 1405 sterically clashes. The modification of aminoglycosides is the predominat resistance mechansim to this class of antibiotics. Usually rings I and II are targeted because of the specific interactions with A site. If ring I is modified, may not fit in the tight pocket due to sterics.

The paromomycin A site Rna structure suggests origin of induced miscoding. The ribosome contributes to specific reading of the genetic code as Watson and Crick base pairs between anticodon and codon is insufficient to account for the fidelity of translation. Binding of the antibiotic then decreases the dissociation rate of aminoacyl tRNA inhibits translational processivity and favors misreading by affecting proofreading.

When ring I and II locks the A1492 and A1493 the Rna is in a unique conformation. Ribosomes can sense the conformation through the structure specific interaction. Paromomycin RNA complex shows how therapeutic agents can target RNA structures. Antibiotics interfere with ribosome function. Studies on these antibiotic-rRNA interactions increases our understanding.

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