Inhibition of hemoglobin synthesis by pancreatic deoxyribonuclease in rabbit reticulocyte lysates.

A pancreatic deoxyribonuclease preparation, shown to be free of significant ribonuclease activity, inhibits hemoglobin synthesis in a rabbit reticulocytelysate. Preincubation with DNAase (15 min at 25 degrees C) is required to obtain a marked inhibitory effect (nearly 70%). It has been shown that DNAase does not significantly interfere with the elongation or termination steps of translation, but it seems to prevent reinitiation of globin polypeptide chains allowing polysome run-off. In particular, the formation of the 40S/met-tRNAmetf complex is greatly reduced in the DNAase-treated lysate. At the moment the mechanism by which DNAase inhibits initiation is not clear.     

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A⁺) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.     

Regulation of protein synthesis in rabbit reticulocyte lysates by 2,3-bisphosphoglycerate.

2,3-Bisphosphoglycerate was the most potent effector of glycolytic intermediates tested for their effects on protein synthesis in gel-filtered lysates from rabbit reticulocytes. 2,3-Bisphosphoglycerate at low levels was stimulatory but became inhibitory at high levels. Both effects were dependent on Mg²⁺ concentrations. The higher the concentration of Mg²⁺, the higher the concentration of 2,3-bisphosphoglycerate required for maximal activation. 2,3-Bisphosphoglycerate concentrations required to exhibit an inhibitory effect increased as Mg²⁺ concentration increased. Both effects of 2,3-bisphosphoglycerate are discussed in terms of regulation of hemoglobin synthesis during maturation of erythroid cells.     

Inhibition of protein synthesis by double-stranded RNA in reticulocyte lysates: evidence for activation of an endoribonuclease.

Double-stranded RNA is a potent inhibitor of protein synthesis in rabbit reticulocyte lysates. Three lines of evidence suggest that at least part of this inhibitory activity is due to activation of a nuclease which degrades mRNA: (1) In the presence of emetine reticulocyte polysomes are partially degraded to structures containing 1–3 ribosomes; (2) 34S Mengo-virus RNA is degraded to fragments sedimenting at less than 18S; (3) The template activity of globin mRNA extracted from the lysates is reduced by 90% when compared to appropriate controls. The ability of double-stranded RNA to activate a nuclease in the reticulocyte system is very similar to that observed in extracts from interferon treated cells and probably involves formation of the unusual oligonucleotide pppA^2′ p^5′ A^2′ p^5′ A.     

Regulation of protein synthesis in rabbit reticulocyte lysates by guanosine triphosphate

GTP (2 mM) promotes protein synthesis in rabbit reticulocyte lysates in which protein chain initiation is inhibited by the activation of specific adenosine 3′:5′ cyclic monophosphate independent protein kinases in: 1) heme deficiency; or 2) in hemin-supplemented lysates by the addition of the purified heme-regulated protein kinase (HRI); or 3) oxidized glutathione; or 4) by low levels of double stranded RNA. The molecular basis for the promotion of protein synthesis by GTP under these various conditions was investigated by examining the $\text{in}\text{,}\text{situ}$ state of eIF-2 phosphorylation. The results show that GTP (2 mM) blocks eIF-2 phosphorylation and also promotes the dephosphorylation of phosphorylated eIF-2. These findings suggest that GTP restores protein synthesis by a common mechanism that involves the relief of eIF-2 from phosphorylation. The nonphosphorylated eIF-2 is, therefore, available for the maintenance and the restoration of protin chain initiation cycle.     

No effect of cAMP on protein synthesis in reticulocyte lysates.

The results of a series of experiments are interpreted to indicate that protein synthesis in reticulocyte lysates is not affected by the reticulocyte cAMP-dependent protein kinase. The catalytic subunit of this enzyme was isolated to apparent homogeneity. Also, the protein inhibitor of this protein kinase was isolated from muscle. Neither physiological concentrations of cAMP nor any of these protein components had a detectable effect on protein synthesis in reticulocyte lysates in the presence or absence of exogenous heme. Phosphorylation of the smallest subunit of eukaryotic initiation factor 2 or the 90,000 to 100,000-dalton peptide associated with eukaryotic initiation factor 2 kinase activity were not affected by the activity of the cAMP-dependent protein kinase under conditions in which exogenous heme has a pronounced effect on these reactions.     

Optimisation of protein synthesis rates in reticulocyte lysates and reconstituted systems

Means of increasing the very low activity of a reconstituted protein synthesising system from rabbit reticulocytes were investigated. Increasing the concentration of labelled amino acid, addition of polyamines, use of Sepharose-filtered as opposed to centrifuged ribosomes, use of untreated as opposed to gel-filtered cytosol and an increase in ratio of cytosol to ribosomes all contributed to the increase in activity of the system to the point where activity was clearly consistent with initiation taking place. Similar activities could not be attained with rat liver cytosol though rat liver ribosomes incorporated well in reticulocyte cytosol. Incorporation by lysates was also found to be dependent on the concentration of the labelled amino acid added.     

The accuracy of protein synthesis in reticulocyte and HeLa cell lysates

The accuracy of translation in protein synthesis is measured as the rate of misincorporation of a particular amino acid, different from that specified by an mRNA codon, into protein. The cowpea variant of tobacco mosaic virus, CcTMV, contains no cysteine or methionine in its coat protein. Translation in vitro of purified CcTMV coat protein mRNA by rabbit reticulocyte and HeLa cell lysates has been performed. The coat protein product was purified by immunoprecipitation with specific antisera, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The error rate was measured by comparing the incorporation of [35S]cysteine with incorporation of [3H]leucine, and the total CcTMV coat protein synthesized was calculated from its known leucine content. An error rate of (1-2) X 10(-3) cysteines/CcTMV coat protein was obtained with reticulocyte lysates. If errors were cysteine incorporation in place of arginine, this number is converted to 3 X 10(-4) cysteine/codon. If cysteine was incorporated anywhere in the polypeptide, the rate is 9 X 10(-6) cysteines/amino acid. The error frequencies with HeLa cell lysates were 6-fold higher. Paromomycin, a eukaryotic misreading antibiotic, increased error rates 10-fold in both lysates. These data compare well with in vivo measurements and suggest that some transformed cells may survive with higher mistranslation rates.     

Regulation of protein synthesis in rabbit reticulocyte lysates. Thiophosphorylation of initiation factor eIF-2 by heme-regulated protein kinase

The heme-regulated protein kinase, which specifically phosphorylates the 38-kDa subunit of initiation factor eIF-2, can utilize adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma S]) as a substrate. The rate of thiophosphorylation is 5-6-times slower than that observed with ATP. It is of special interest that thiophosphorylated derivatives of eIF-2 are resistant to dephosphorylation catalyzed by eIF-2 phosphoprotein phosphatase. The thiophosphorylated eIF-2 is less effective in promoting protein synthesis in hemin-deficient lysates under physiological conditions. In addition, ATP[gamma S] could also be utilized by the self-phosphorylation activity intrinsically associated with HRI.     

Role of NAD+ in the stimulation of protein synthesis in rabbit reticulocyte lysates.

The stimulation of protein synthesis by NAD⁺ in rabbit reticulocyte lysates has been reported. [Lennon M. B., Wu, J., and Suhadolnik, R. J., (1976) Biochem. Biophys. Res. Commun. 72, 530–538]. NAD⁺ can replace the creatine phosphate-creatine phosphokinase ($\text{CP}\text{CPK}$) energy regenerating system normally used in in vitro protein synthesizing systems. The replacement of $\text{CP}\text{CPK}$ by NAD⁺ is optimal at 37 °C. A significant lag in the rate of protein synthesis with NAD⁺ is observed with decreasing temperatures. Analysis of the adenylate energy charge with NAD⁺ shows an initial rapid decrease. This decrease in the energy charge recovers with increasing NAD⁺ concentrations. The energy level correlates with the rates of incorporation of d,l-[4,5-³H(N)]leucine into protein. ATP production via NAD⁺ pyrophosphorylase or oxidative phosphorylation does not explain the stimulation by NAD⁺. Rather, the stimulation is correlated with the activation of glycolysis. Glycolysis is not active in lysate preparations because NAD⁺ is absent. Additional possible roles of NAD⁺ in protein synthesis are discussed.     

The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates.

Besides heme deficiency, protein synthesis in rabbit reticulocyte lysates becomes inhibited upon exposure to a variety of agents that mimic conditions which induce the heat shock response in cells. This inhibition has been demonstrated to be due primarily to the activation of the heme-regulated eIF-2 alpha kinase (HRI) which causes an arrest in the initiation of translation. In this report, the sensitivity of protein synthesis in hemin-supplemented lysates to inhibition by Hg2+, GSSG, methylene blue, and heat shock was examined in six different reticulocyte lysate preparations. The extent to which translation was inhibited in response to Hg2+, GSSG, methylene blue, and heat shock correlated inversely with the relative levels of the 70-kDa heat shock proteins (hsp 70) and a 56-kDa protein (p56) present in the lysates determined by Western blotting. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates was also examined. While the restoration of protein synthesis correlated roughly with the levels of hsp 90 present, the results also suggest that the heme regulation of HRI probably involves the interaction of HRI with several factors present in the lysate besides hsp 90. A comparison of two lysate preparations, which had a 2-fold difference in their protein synthesis rates, indicated that the slower translational rate of the one lysate could be accounted for by its low level of constitutive eIF-2 alpha phosphorylation, with its accompanying decrease in the eIF-2B activity and lower level of polyribosome loading. The present study supports the notion that the previously demonstrated interaction of HRI with hsp 90, hsp 70, and p56 in reticulocyte lysates may play a direct role in regulating HRI activation or activity. We hypothesize that the competition of denatured protein and HRI for the binding of hsp 70 may be a molecular signal that triggers the activation of HRI in reticulocyte lysates in response to stress. Possible functions for p56 in the regulation of HRI activity are also discussed.     

Differential inhibition of protein synthesis in reticulocyte lysates and wheat germ extracts by a protein isolated from wheat germ extracts.

A protein with a molecular mass of 35-37 kDa has been isolated and partially purified from the postribosomal supernatant of wheat germ by ammonium sulfate precipitation (60-90%), Sephadex G-75, and DEAE-cellulose chromatography. It inhibited endogenous protein synthesis in rabbit reticulocyte lysates but had no effect on translation in wheat germ extracts. At low concentrations (0.34-1.36 ng/15 microliter assay), inhibition was limited to initiation of peptide synthesis. At higher concentrations (13.6 ng/15 microliter assay), elongation was also suppressed.