Control of protein synthesis in brine shrimp embryos by repression of ribosomal activity

Undeveloped encysted embryos of the brine shrimp, Artemia salina, contain a large quantity of metabolically repressed 80S ribosomes. These ribosomes appear to be inactive or nonfunctional due to the presence of an inhibitor protein on their 60S subunit. During development the inhibitor is released or inactivated and the 80S ribosomes and their constituent subunits become fully functional in a poly(U)-directed protein-synthesizing system. The inefficiency of most 80S ribosomes from undeveloped Artemia embryos appears to be due to their inability to form stable complexes with poly(U) and phe-tRNA in the presence of elongation factor, EF-1. A potent inhibitor of protein synthesis has also been found in the 105,000g supernatant fraction from undeveloped Artemia embryos. The exact nature of this inhibitor has not been ascertained but it appears to be a heat-labile protein devoid of RNase and protease activity. It is not known whether this inhibitor is the same as that associated with 60S ribosomal subunits of undeveloped cyst ribosomes.     

Hierarchy of elements regulating synthesis of ribosomal proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells respond to a heat shock by temporarily slowing the synthesis of ribosomal proteins (C. Gorenstein and J. R. Warner, Proc. Natl. Acad. Sci. U.S.A. 73:1574-1551, 1976). When cultures growing oxidatively on ethanol as the sole carbon source were shifted from 23 to 36 degrees C, the synthesis of ribosomal proteins was coordinately inhibited twice as rapidly and 45% more severely than in comparable cultures growing fermentatively on glucose. Within 15 min, the relative rates of synthesis of at least 30 ribosomal proteins declined to less than one-sixth their initial values, whereas the overall rate of protein synthesis increased at least threefold. We suggest that this is due primarily to controls at the level of synthesis of messenger ribonucleic acid for ribosomal proteins but may also involve changes in messenger ribonucleic acid stability. In contrast, a nutritional shift-up causes a stimulation of the synthesis of ribosomal proteins. Experiments designed to determine the hierarchy of stimuli affecting the synthesis of these proteins demonstrated that temperature shock was dominant to glucose stimulation. When a culture growing on ethanol was shifted from 23 to 36 degrees C and glucose was added shortly afterward, the decline in ribosomal protein synthesis continued unabated. However, in wild-type cells ribosomal protein synthesis began to recover within 15 min. In mutants temperature sensitive for ribosome synthesis, e.g., rna2, there was no recovery in the synthesis of most ribosomal proteins, suggesting that the product of rna2 is essential for the production of these proteins under all vegetative conditions.     

Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions.     

Structural elements of rps0 mRNA involved in the modulation of translational initiation and regulation of E. coli ribosomal protein S15.

Previous experiments showed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops and to trap the ribosome on its mRNA loading site in a transitory state. In this study, we investigated the effect of mutations in the translational operator on: the binding of protein S15, the formation of the 30S/mRNA/tRNA(fMet) ternary initiation complex, the ability of S15 to inhibit the formation of this ternary complex. The results were compared to in vivo expression and repression rates. The results show that (1) the pseudoknot is required for S15 recognition and translational control; (2) mRNA and 16S rRNA efficiently compete for S15 binding and 16S rRNA suppresses the ability of S15 to inhibit the formation of the active ternary complex; (3) the ribosome binds more efficiently to the pseudoknot than to the stem-loop; (4) sequences located between nucleotides 12 to 47 of the S15 coding phase enhances the efficiency of ribosome binding in vitro; this is correlated with enhanced in vivo expression and regulation rates.     

Changes in the half-life of ribosomal protein messenger RNA caused by translational repression

The half-life of ribosomal protein operon L11 mRNA in vivo was measured during exponential growth by following the kinetics of incorporation of radioactive precursors into L11 mRNA transcribed from multi-copy plasmids. The degree of translational feedback regulation by L1, the L11 operon-specific translational repressor protein, was changed by altering the site on the "L11 mRNA" where L1 interacts. The half-life of the overproduced L11 mRNA increased by about fivefold when translational repression was abolished, while the half-life of mRNA from the spc ribosomal protein operon, which is not translationally regulated by L1, stayed constant. Furthermore, the half-life of L11 operon mRNA carrying an additional mutation in the ribosome binding site that abolishes translation remains short. This indicates that the change in half-life observed during increased gene dosage is due to translational repression by L1 and is probably a consequence of L1 blocking translation of L11 mRNA and not due to some nucleolytic activity mediated by L1.