Relationship between histidyl‐tRNA level and protein synthesis rate in wild‐type and mutant chinese hamster ovary cells

Don J. Lofgren; Larry H. Thompson

摘要

AbstractA preliminary investigation was carried out to determine how conditional lethal mutants affected in particular aminoacyl‐tRNA synthetases may be used to study the role of tRNA charging levels in protein synthesis. The relationship between rate of protein synthesis and level of histidyl‐tRNA in wild‐type cultured Chinese hamster ovary cells was determined using the analogue histidinol to inhibit histidyl‐tRNA synthetase activity. This response was compared with that obtained using a mutant strain with a defective histidyl‐tRNA synthetase that phenotypically shows decreased rates of protein synthesis at reduced concentrations of histidine in the growth medium. The approach used was based on measuring the histidyl‐tRNA levels in live cells. The percentage charging was estimated by comparing 14Chistidine incorporated into alkali‐labile material in paired samples, one of which was treated with cycloheximide, five minutes before terminating during the incubation, to produce maximal aminoacylation. Wild‐type cells under histidinol inhibition exhibited a sensitive, sigmoidal relationship between the level of histidyl‐tRNA and the rate of protein synthesis. A decrease in the relative percentage of acylated tRNAHisfrom 46 to 35 elicited a large reduction in the rate of protein synthesis from 90 to 30 relative to untreated cells. An unpredicted result was that the relationship between protein synthesis and histidyl‐tRNA in the mutant was essentially linear. High acylation values for tRNAHiswere associated with rates of protein synthesis that were not nearly as high as in wild‐type cells. These findings suggest that the charging levels of tRNAHisisoacceptors could play a regulatory role in determining the rate of protein synthesis under conditions of histidine starvation in normal cells. The mutant appears to be a potentially useful system for studying the pivotal role of tRNA charging in protein synthesis, assuming that the altered response in the mutant is caused by i

Relationship between histidyl‐tRNA level and protein synthesis rate in wild‐type and mutant chinese hamster ovary cells

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