tRNA-Neomycin Complex
Created by Hinda Mezaache
Although phenylalanine is the least common amino acid in proteins, the phenylalanine tRNA (tRNAPhe), specifically from Saccharomyces cerevisiae, is one of the most comprehensively studied. Neomycin B, also known as framycetin, is a five-ring aminoglycoside antibiotic derived from Streptomyces decaris. Neomycin B, along with other aminoglycosides, potently inhibits prokaryotic protein synthesis1. This non-specific inhibition is likely due to the aminoglycoside’s ability to recognize similar structural elements and domains within folded RNA2. Its PDB ID is 1I9V.
Even though the employment of different tRNAs gives rise to the addition of different amino acids to a growing polypeptide chain, they all share very similar primary, secondary, and tertiary structures. The primary structure includes several invariant pyrimidine (Y) and purine (R) residues, as well as specific invariant guanine (G) and thymine-pseudouridine-cytosine sites (TψC)3.
The secondary structure consists of hairpin turns that bring complementary stretches of bases in the chain together, forming double helical regions, thus giving rise to stem-loop structures via cloverleaf base pairing that is achieved through hydrogen bonding. Four base-paired segments make up each cloverleaf—three loops and the region where the 3’- and 5’-ends of the tRNA meet. These segments are the acceptor stem, the D loop, the anticodon loop, and the TψC loop. The acceptor stem is where the amino acid— in this case, phenylalanine— is linked to the invariable 3’-cytosine-cytosine-adenine-hydroxyl group (3’ CCA-OH) via phenylalanine’s (or the corresponding amino acid’s) carboxyl group, forming an ester linkage. This reaction is catalyzed by phenylalanine-tRNA-synthetase (PheRS), and the entire molecule is called an aminoacyl-tRNA4. The D loop typically contains dihyrdouridine (D) residues. However, this is not the only unusual base found in tRNA; the others include inosine, thiouridine, pseudouridine, and hypermethylated purines. The anticodon loop is the means through which tRNAs recognize mRNA codons that correspond to the amino acid carried by a specific tRNA. It contains a double helical portion and seven unpaired bases, three of which make up the anticodon that is complementary to the mRNA’s codon. Just beyond the anticodon stem loop, moving in the 5’ to 3’ direction, is the TψC loop stem loop, which contains seven unpaired bases, including TψC. Variable loops are found between the anticodon and TψC loops; as their name suggests, their base compositions contrast between different tRNA molecules. These secondary structures are invariable due to the absolute requirement of base pairing between them in order to achieve the correct tertiary structure3.
Tertiary structure of the tRNA is achieved via base pairing between the D loop, the variable loop, and TψC loop. Interestingly, many of the base pairings are different from the standard A:T and G:C coupling found in deoxyribonucleic acid (DNA). The D and TψC loops are brought together, forming the corner of an “inverted, backward L-shaped” tertiary structure. The acceptor stem is at one end of this L-shape and the anticodon loop is found at the opposite end of the L. This L-shape is stabilized by hydrophobic stacking interactions between flat faces of the bases3.
There are eleven divalent cation-binding sites, designated M1 through M11, in tRNAPhe5. Usually magnesium (Mg2+) is bound at these sites, but some sites show preference for other divalent cations. The main interactions between tRNAPhe and the divalent metal cations (specifically magnesium) are found at M3, M7, and M85. At the M3 site, Mg2+ is fully hydrated with six water molecules and is located at the peak of the tight loop formed by residues 7-135. M7 and M8 interact with the tRNA at the junction of the acceptor stem and the D loop5. Together, M3, M7, and M8 neutralize the electrostatic repulsion of backbone phosphate groups, thus acting to stabilize the turn with which they are associated5.
Neomycin B interacts with tRNAPhe via six hydrogen bonds: two to the sixth oxygen (O6) of guanine at position 45 (G45), one to the 2’-hydroxyl group of G20, and three to oxygens in the phosphodiester backbone4. Specifically, ring I of neomycin B is positioned such that its amino protons form the hydrogen bonds between O6 of G45 and the phosphate group of adenine at position 23 (OP1 of A23)4. Substitution of this amino group with a hydroxyl group causes a significant increase in KI, demonstrating the necessity of the amino group’s presence and specific conformation for the powerful inhibitory capacity of neomycin B4. The tRNAPhe –neomycin B structure harbors three magnesium ions which correspond to the three main Mg2+ identified in yeast tRNAPhe4. Superimposition of the tRNAPhe-neomycin B complex and tRNAPhe with bound Mg2+ shows a direct overlap of neomycin-B and Mg2+, suggesting that neomycin B acts to displace divalent cations bound to the tRNA at sites other than M3, M7, and M84. Neomycin B further interacts with the tRNA at G20 and adenosine at position 44 (A44), which are the key players in charging the tRNA with phenylalanine4. Neomycin B is thus a potent inhibitor of aminoacylation because it disturbs the interaction with PheRS and tRNAPhe through its binding to the major charging determinants4.
A tRNAPhe homolog of particular interest is the insertion domain within mammalian mitochondrial initiation factor 2 (IF2; PDB ID: 3IZY), which is found in Bos Taurus6. The two share the exact same nucleotide sequence. The insertion domain of IF2 blocks the ribosomal A site during translation initiation7.