Ribosome bypassing at serine codons as a test of the model of selective transfer RNA charging

Abstract

Recently, a model of the flux of amino acids through transfer RNAs (tRNAs) and into protein has been developed. The model predicts that the charging level of different isoacceptors carrying the same amino acid respond very differently to variation in supply of the amino acid or of the rate of charging. It has also been shown that ribosome bypassing is specifically stimulated at 'hungry' codons calling for an aminoacyl-tRNA in short supply. We have constructed two reporters of bypassing, which differ only in the identity of the serine codon subjected to starvation. The stimulation of bypassing as a function of starvation differed greatly between the two serine codons, in good agreement with the quantitative predictions of the model. Keywords: amino acid; bypassing; charging; protein synthesis; tRNA EMBO reports (2005) 6, 147-150. doi:10.1038/sj.embor.7400332 Elf et al (2003) have developed a theoretical model of the response to amino-acid limitation of the different members (isoacceptors) of each family of transfer RNAs (tRNAs) that carry the same amino acid. The theory rests on considerations of supply and demand. The parameters of supply are the rate of synthesis of the limiting amino acid and the abundance of each isoacceptor tRNA in question; the parameters of demand are the frequencies with which the codons read by a given isoacceptor are found in translating ribosomes, as well as the overall capacity of ribosomes to consume the amino acid. The model leads to the counterintuitive prediction that amino-acid limitation can elicit very different degrees of residual aminoacyl charging among the different isoacceptors. Those that have a high concentration relative to the frequency of their cognate codons in messenger RNAs will retain a much higher level of charging than those in which the ratio is lower. We report here a test of this prediction in the case of two serine-tRNA isoacceptors, using ribosome bypassing as a reflection of the starvation experienced by codons calling for the two different isoacceptors. INTRODUCTION RESULTS AND DISCUSSIONIt has been demonstrated elsewhere that 'hungry' codons calling for a tRNA, the acylated form of which is in short supply, are the sites of reading frame errors Peter et al, 1992;Lindsley & Gallant, 1993;Barak et al, 1996;Gallant et al, 2000) and ribosome bypassing (Gallant & Lindsley, 1998;Gallant et al, 2003;Lindsley et al, 2003). Furthermore, minigene experiments have indicated that it is the absence of the aminoacylated tRNA, rather than any positive effect of the deacylated species, that gives rise to these ribosome errors . Therefore, the stimulation of either one of these ribosome gymnastic feats at a hungry codon should reflect the reduced charging level of that codon's cognate tRNA, an analytic approach that has been introduced already in the case of isoleucine tRNAs (Kaplun et al, 2002;Elf et al, 2003).To test the model of selective tRNA charging, we have constructed reporters of bypassing, which differ only at the serine codon subjected to starvation. The repo...

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“…5 Selective charging of two members of an isoacceptor family that read different codons, should therefore map to selective bypassing frequencies for ribosomes with one or the other of these codons in the A site. Lindsley et al 6 demonstrated strongly diverging frequencies of bypassing at two different serine codons for increasingly severe inhibition of the serinyl-tRNA synthetase, in full quantitative agreement with theory. 1 Here, we address another class of theoretical predictions from the theory, which concerns greatly changed charging patterns of a family of tRNA isoacceptors in response to varying concentration of one of its members.…”

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