Effect of replacing uridine 33 in yeast tRNAPhe on the reaction with ribosomes.

We have determined several kinetic parameters for the reaction of poly(U)-programmed ribosomes with ternary complexes of elongation factor Tu, GTP, and yeast Phe-tRNA analogs with different bases substituted for uridine in position 33. These analogs test whether disruption of the hydrogen bonds normally formed by uridine 33 and steric crowding in the anticodon loop are detrimental to tRNA function on the ribosome. Single-turnover kinetic studies of the reaction of these ternary complexes with ribosomes show that these Phe-tRNA analogs decrease the apparent rate of GTP hydrolysis (kGTP) and the ratio of peptide formed to GTP hydrolyzed. Thus, the substitution of uridine 33 affects not only the selection of a ternary complex by the ribosome but also the selection of an aminoacyl-tRNA in the proofreading reaction. The effects become greater as first one, and then the other, H-bond is disrupted. Steric crowding in the anticodon loop is also important, but does not have as great an effect on the rate constants. An analysis of the elementary rate constants which comprise the rate constant, kGTP, demonstrates that the reduction in kGTP results from a decreased rate of ternary complex association with the ribosome (k1) and that there is little or no effect on the rate of GTP cleavage (k2). An analysis of the rate constants involved in proofreading shows that all the modified (tRNAs have increased rates of aminoacyl-tRNA rejection (k4) but that the rate of peptide bond formation (k3) is unaffected.

Specific replacement of functional groups of uridine-33 in yeast phenylalanine transfer ribonucleic acid.

Functional groups of the highly conserved uridine at position 33 in the anticodon loop of yeast tRNAPhe were altered by a synthetic protocol that replaces U-33 with any desired nucleotide and leaves all other nucleotides of the tRNA intact. The U-33-substituted tRNAs were prepared in an eight-step protocol that begins with partial cleavage of tRNAPhe at U-33 by ribonuclease A. By use of the combined half-molecules as substrate, U-33 was removed from the 5' half-molecule in three steps and then replaced by using RNA ligase to add the desired nucleoside 3',5'-bisphosphate. Each position 33 substituted 5' half-molecule was isolated and annealed to the original 3' half-molecule from the ribonuclease A digestion. The two halves were then rejoined in three steps to give a full-size tRNAPhe variant. This protocol should be applicable to other RNA molecules where a nucleotide substitution is desired at the 5' side of an available unique cleavage site. Seven substituted tRNAPheS containing uridine, pseudouridine, 3-methyluridine, 2'-O-methyluridine, cytidine, deoxycytidine, and purine riboside at position 33 were assayed for aminoacylation with yeast phenylalanyl-tRNA synthetase. Each of the seven tRNAs aminoacylated normally. Thus, unlike the adjacent guanine residue at position 34, U-33 is not involved in the interaction between yeast tRNAPhe and yeast phenylalanyl-tRNA synthetase.

Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes.

Twenty-two anticodon arm analogues were prepared by joining different tetra, penta, and hexaribonucleotides to a nine nucleotide fragment of yeast tRNAPhe with T4 RNA ligase. The oligomer with the same sequence as the anticodon arm of tRNAPhe bind poly U programmed 30S ribosomes with affinity similar to intact tRNAPhe. Analogues with an additional nucleotide in the loop bind ribosomes with a weaker affinity whereas analogues with one less nucleotide in the loop do not bind ribosomes at all. Reasonably tight binding of anticodon arms with different nucleotides on the 5' side of the anticodon suggest that positions 32 and 33 in the tRNAPhe sequence are not essential for ribosome binding. However, differences in the binding constants for anticodon arms containing modified uridine residues in the "constant uridine" position suggest that both of the internal "U turn" hydrogen bonds predicted by the X-ray crystal structure are necessary for maximal ribosome binding.