Identification of molecular interactions between P-site tRNA and the ribosome essential for translocation

Abstract

Translocation of the tRNA-mRNA complex is a fundamental step in the elongation cycle of protein synthesis. Our studies show that the ribosome can translocate a P-site-bound tRNA Met with a break in the phosphodiester backbone between positions 56 and 57 in the T⌿C-loop. We have used this fragmented P-site-bound tRNA Met to identify two 2 -hydroxyl groups at positions 71 and 76 in the 3 -acceptor arm that are essential for translocation. Crystallographic data show that the 2 -hydroxyl group at positions 71 and 76 contacts the backbone of 23S rRNA residues 1892 and 2433-2434, respectively, in the ribosomal E site. These results establish a set of functional interactions between P-site tRNA and 23S rRNA that are essential for translocation.R ibosomes are the macromolecular complexes responsible for protein synthesis. Escherichia coli ribosomes are made up of a large 50S subunit and a small 30S subunit that contain three tRNA-binding sites (A, aminoacyl; P, peptidyl; E, exit). One of the fundamental steps in the elongation cycle of protein synthesis is the step-wise movement-called translocation-of tRNAs from one site to the next within the ribosome. Very little is known about how this highly precise and complex process occurs on the ribosome. Biochemical (1, 2) and biophysical (3) studies revealed that translocation occurs in two steps. In the first step, after peptide-bond formation, the acceptor ends of deacylated tRNA and peptidyl-tRNA move from P to E, and from A to P sites, respectively, in the 50S subunit, whereas their anticodon ends still interact with the 30S subunit P and A sites, respectively. This movement occurs spontaneously after peptide-bond formation, which results in tRNAs occupying A͞P and P͞E hybrid states. In the second step, elongation factor G (EF-G) catalyzes the movement of the anticodon ends of both tRNAs relative to the 30S subunit, translocating deacylated tRNA from P͞E to E state and peptidyl-tRNA from A͞P to P͞P state. Thus, movement of the acceptor end of tRNAs occurs independently of the anticodon ends, relative to the two ribosomal subunits.EF-G uses the chemical energy of GTP hydrolysis to accelerate translocation of the tRNA-mRNA complex (4). Interestingly, ribosomes can perform spontaneous translocation in the absence of EF-G⅐GTP (5, 6), suggesting that the mechanism of translocation is inherent to the ribosome. Additionally, translocation can occur in the absence of mRNA (7,8), demonstrating that neither codon-anticodon interactions nor mRNA-ribosome interactions are essential for translocation. Thus, the only components indispensable for translocation are the A-and P-site tRNAs and the ribosome.The requirement for peptidyl-tRNA in the A site can be satisfied by a 15-nucleotide anticodon stem-loop analog (ASL)-4 of tRNA (9). The ASL, consisting of a seven-nucleotide loop and a four base-pair stem, translocates, indicating that the D stem, T stem, and acceptor arm of the A-site tRNA are not essential for translocation. In contrast to the ribosomal A site, an ASL bound to the ribo...

Functional Importance of the 3′-Terminal Adenosine of tRNA in Ribosomal Translation

“…Feinberg and Joseph have recently identified two 2Ј-OH groups, at positions 71 and 76, which are required for tRNA translocation from the P-to the E-site (42). This result is in agreement with the finding that 2Ј-deoxy-A 76 -substituted tRNA inhibits ribosomal translocation (43).…”

Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

“…Although the minimal requirements for translocation in 80S ribosomes have not been described, on 70S ribosomes there is a strict requirement for a deacylated full-length tRNA in the P site and at least an ASL bound to the A site (44), and there is evidence that a backbone contact between the acceptor stem of the deacylated hybrid P/E state tRNA and helix 68 of 23S rRNA is crucial (45). During IGR IRES-driven initiation, there is no tRNA of any type in the P site and thus critical contacts between the ribosome and P site or P/E hybrid state tRNA are missing.…”

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

“…Structurally, 29-O-methylation stabilizes the 39-endo ribose conformation commonly found in A-form RNA (Kawai et al 1992), although a role such as this in the middle of the acceptor stem might seem unnecessary. Mechanistically, the translocation step of E. coli ribosomes is inhibited by 29-O-methylation of the acceptor stem at positions 66, 70, or 71 (as well as at 76), or by 29-deoxy substitution at positions 71 or 76 (Feinberg and Joseph 2001), suggesting different causes of the inhibition. Thus, it is possible that 29-O-methylation at position 4 of certain tRNAs could be important for one of the steps of translation in yeast.…”