Recognition of Cognate Transfer RNA by the 30 S Ribosomal Subunit

Abstract

Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or "wobble," position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs.During protein synthesis, the ribosome catalyzes the sequential addition of amino acids to a growing polypeptide chain, using mRNA as a template and aminoacylated tRNAs (aatRNAs) as substrates. Correct base pairing between the three bases of the codon on mRNA and those of the anticodon of the cognate aatRNA dictates the sequence of the polypeptide chain. Discrimination against noncognate tRNA, which generally has two or three mismatches in the base pairing, can be accounted for by the difference in the free energy of base pairing to the codon compared with cognate tRNA. For near-cognate tRNA, which usually involves a single mismatch, the free-energy difference in base pairing compared with cognate tRNA would predict an error rate that is one to two orders of magnitude higher than the actual error rate of protein synthesis (1), and it has long been recognized that the ribosome must improve the accuracy of protein synthesis by discriminating against near-cognate tRNAs (2). This discrimination involves the 30S subunit, which binds mRNA and the anticodon stem-loop (ASL) of tRNA.At the beginning of the elongation cycle, which involves the addition of a new amino acid to a growing polypeptide chain, the aatRNA is presented to the ribosome as a ternary complex with elongation factor Tu (EF-Tu) and guanosine triphosphate (GTP). The selection of cognate tRNA is believed to occur in two stages-an initial recognition step and a proofreading step-that are separated by the irreversible hydrolysis of GTP by EF-Tu (3-6). In this scheme, the discrimination energy inherent in codon-anticodon base pairing is exploited twice to achieve the necessary accuracy. Recent experiments suggest that the binding of cognate rather than near-cognate tRNA results in higher rates of both GTP hydrolysis by EF-Tu, and accommodation, a process in which the acceptor arm of the aa-tRNA swings into the peptidyl transferase site after its release from EF-Tu (7,8). In both steps, the higher rate is proposed to be the result of structural changes in the ribosome induced by cognate tRNA. In the context of proofreading mechanisms alone, it is unclear whether additional structural discrimination by the ribosome, ...

Targeting Antibiotic Resistance

“…Co‐crystal structures of a variety of bound aminoglycosides have been published, including with streptomycin ( 31 , PDB ID: 1FJG),110 spectinomycin ( 32 , PDB ID: 1FJG),110 hygromycin B ( 33 , PDB ID: 1HNZ),97 neomycin B ( 34 , PDB ID: 4V52),111 paromomycin ( 35 , PDB ID: 1FJG, 1IBK),110, 112 tobramycin ( 36 , PDB ID: 1LC4),113 and kanamycin A ( 37 , PDB ID: 2ESI) 109…”

Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity

“…Three universally conserved nucleotides in the E. coli decoding site-G530, A1492, and A1493-interact with the codon-anticodon helix to monitor the accuracy of base pairing (Cate et al 1999;Ogle et al 2001;Yusupov et al 2001). The prokaryotic decoding site is also the target for many aminoglycosides antibiotics, which interfere with translational fidelity (Carter et al 2000).…”

“…Structural and chemical protection studies have shown that aminoglycosides bind to specific nucleotides in the decoding site of 16S rRNA in bacteria (Fig. 1A), where they induce a conformational change similar to the transition that occurs upon cognate tRNA binding (Purohit and Stern 1994;Ogle et al 2001;Ogle and Ramakrishnan 2005). As a result, aminoglycosides interfere with bacterial protein synthesis by facilitating amino acid misincorporation.…”

“…1A). When a cognate codon-anticodon interaction occurs, G530 transitions from a syn to an anti conformation, while A1492 and A1493 flip out into the minor groove of the codon-anticodon helix (Carter et al 2000;Ogle et al 2001). These conformational changes allow the decoding site to monitor the proper codon-anticodon interaction by hydrogen bonding to 29 hydroxyl groups on either side of the cognate codon-anticodon helix, providing a sensitive test of codon-anticodon pairing.…”

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

“…(B) The SPF specific region of RF2 was mapped exclusively to rRNA from one of the two ribosomal subunits. The cleavages in E. coli rRNA derived from hydroxyl radicals generated from near 205 SPF 207 (Cys 204 or 209 on modified RF2s with unique cysteines have been mapped here on the rRNA structures derived from X-ray structures of 16S rRNA from Thermus thermophilus [36]. The release factor at the ribosomal active centre 596 npg bromoacetamidobenzyl)-EDTA (BABE) was attached to the specific cysteine, newly engineered into the protein so that Fenton chemistry could be used to activate the generation of free hydroxyl radicals at the site.…”