RESUMO
A novel method was developed to estimate the translational fidelity of mammalian ribosomes in vitro with protamine mRNA of rainbow trout as template. Protamines are mixtures of basic proteins consisting of only seven types of amino acids (Arg, Ile, Val, Ser, Pro, Ala, and Gly), arginine (codon, AGR and CGN) being abundant. Taking advantage of the absence of lysine (codon, AAG) in the proteins, we determined the misincorporation of this amino acid into protamines in a cell-free translation system consisting of mouse liver ribosomes, protamine mRNA, [3H]lysine, [14C]arginine, and seven unlabeled amino acids: Ile, Val, Ser, Pro, Ala, Gly, and Met. After the reaction, translation products were analyzed by either sucrose gradient centrifugation or polyacrylamide gel electrophoresis. In the former method, radioactive protamines are mostly found on monosomes, but not on polysomes, probably because of the basic nature of the proteins. The error frequency was calculated from the molar ratio of [3H]lysine to [14C]arginine incorporated into protamines with an appropriate correction. The frequency was found to be 0.0006-0.002. This method enabled us to determine the frequency of misrecognition of purine bases at the second position of arginine codons in mRNA.
Assuntos
Protaminas/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Códon , Globinas/genética , Histonas/isolamento & purificação , Cinética , Protaminas/isolamento & purificação , RNA de Transferência/genética , RNA de Transferência/isolamento & purificação , Moldes Genéticos , TrutaRESUMO
We have developed a novel method to examine the translational fidelity of mammalian ribosomes in vitro, where protamine mRNA was used as a template. This method enabled us to determine frequency of misrecognition of purine bases at the second position of arginine codons (AGR/AAR) in the mRNA. Using this method the fidelity of translation of ribosomes derived from mouse livers was found to remain unchanged from 2 to 29 months, the maximum life span of the animal. This conclusion is not consistent with the "error catastrophe" theory of aging. This is the first report in which translational fidelity of ribosomes of animals of various ages has been compared by an in vitro translation of a natural mRNA.
