Actions of the anticodon arm in translation on the phenotypes of RNA mutants

Abstract

In previous publications, we have shown that it is practical to study the translational activity of tRNAs by replacement and alteration of the anticodon arm sequence of the genus on a plasmid clone. Experiments in which the anticodon arm sequence is transplanted between tRNA genes suggest that the translational activity is determined by these sequences. We have therefore made every variant of the anticodon loop and the three base-pairs of the stem proximal to the loop, in order to resolve the relation between the structure of Su7 Am tRNA Trp, and its function. All derivatives conserved the normal secondary structure of the molecule, which was known to be essential for translational activity. The probability of translation of the amber codon by these suppressors is measured in this work. This translational activity in vivo is rationalized in terms of data on the copy numbers of the plasmid clones, the nucleotide modifications of the tRNAs, the steady-state level of the mature tRNA, and the aminoacylation of these molecules. Nucleotide modification levels vary among these tRNAs, giving information about the specificities of modification systems that make O-methylribose, pseudouridine, and modified A in the anticodon arm. However, for this series of tRNAs, none of these modifications has a strong effect on translational efficiency of the tRNAs. A few of the substitutions reduce aminoacylation of the tRNAs with glutamine, as determined by comparison of suppression in normal strains and related strains, which have 25-fold elevated levels of the glutaminyl-tRNA synthetase (GlnRS). The substitutions that have the largest effect on GlnRS action are, unexpectedly, purines for conserved pyrimidines on the 5′ side of the anticodon loop. Data on the concentrations of tRNA in vivo suggest that the anticodon loop and helix contribute similarly to the determination of the steady-state level of the tRNAs. This level varies sevenfold, though all tRNAs are processed from a homologous precursor made from the same transcription unit. Effects on levels appear to be mediated by changes in anticodon arm structure. A robust equation that relates aminoacyl-tRNA levels to suppressor efficiency is developed in order to resolve effects on tRNA levels and on ribosomal steps: E = A (K + A) , where E is efficiency, A is aminoacyl-tRNA concentration, and K is the effective concentration, or cellular tRNA content required for an individual tRNA to have an efficiency of 0.50. The tRNAs vary in their intrinsic ability to function on the ribosome (represented by K), after other influences have been normalized. Loop sequences vary more than 1000-fold in translational activity, and smaller, twofold effects may be attributed to variation of the helix sequence and structure. Loop positions 37 and 38 are the most important determinations of the efficiency of the ribosomal steps. This loop pattern is consistent with the action of a 3′ stacked loop on the ribosome, like the stack in the crystallographic structure. The sequence of the natural anticodon loop region is optimal for translational efficiency. All variants are less active than that present in the parental Su7 tRNA, which most resembles a natural elongator tRNA that translates codon UNN. The natural loop sequence optimizes the translational function of the tRNA molecule in spite of the fact that it minimizes the tRNA level. Sequences frequently found in tRNAs for other codons, when introduced into Su7, substantially reduce the efficiency of this tRNA. This behavior is consistent with the extended anticodon hypothesis. In contrast to loop nucleotides, helix nucleotide pairs influence translational efficiency mainly through tRNA levels. The natural helix sequence yields the highest levels of tRNA. The most influential substitutions are always transversions from the natural consensus, and changes become more influential as they become most distant from the loop end of the anticodon arm. For example, the base-pair adjacent to the loop must exist, but varied pairs produce the minimal effects for any nucleotides examined.