David Kyner

Inhibition of protein chain initiation in eukaryotes by deacylated transfer RNA and its reversibility by spermine

Abstract The eukaryotic 80-S initiation complex, (80-S ribosome complexed with Met-tRNA f and the trinucleotide AUG, 80-S-Met-tRNA f -AUG) is readily assembled at 5 mM MgCl 2 from mouse fibroblast 40-S and 60-S ribosomal subunits, rat liver Met-tRNA f , and AUG. The subunits were prepared and separated in 0.5 M KCl and required no exogenous initiation factors or GTP. Formation of the (80-S-Met-tRNA f -AUG) complex as measured by the incorporation of [ 35 S]Met-tRNA f was inhibited by uncharged tRNA. The extent of inhibition was analyzed by three assays: (a) binding of the complex on nitrocellulose membranes, (b) [ 35 S]Met-puromycin synthesis, and (c) sedimentation of the complex in sucrose density gradients. All of the eukaryotic species of tRNA examined were strongly inhibitory; these included partially purified tRNA f Met , tRNA M Met , tRNA Leu , tRNA Phe , and unfractionated tRNA. No selective inhibition was displayed by tRNA f Met . By contrast, amino acyl-tRNA was a poor inhibitor and 18-S rRNA was not inhibitory. Inhibition was proportional to the concentration of uncharged tRNA, but could be reversed by increasing the concentration of Met-tRNA f or ribosomes relative to tRNA. The order of addition was of importance. Preincubation of the ribosomes with uncharged tRNA produced extensive inhibition; conversely, preincubation with Met-tRNA f greatly reduced inhibition. Mg 2+ did not affect the extent of inhibition from 3 to 10 mM. Spermine displayed two functions in this system: (a) it could partially replace Mg 2+ during 80-S complex formation, 3 mM Mg 2+ plus 0.2 mM spermine being as effective as 5 mM Mg 2+ ; and (b) at higher spermine concentrations, inhibition by tRNA was reversed. The addition of Met-tRNA f caused extrusion of uncharged tRNA from ribosomes. The results suggest that tRNA may have a regulatory function of protein chain initiation by interfering with Met-tRNA f binding to the ribosomal P site.

Messenger activity in mammalian cell-free extracts of reovirus single-stranded RNA prepared in vitro

Abstract Reovirus ssRNA synthesized in vitro by a viral-specific RNA transcriptase displays messenger activity when incubated with supplemented S150 extracts and purified ribosomes prepared from L-cells. Pre-treatment of the ssRNA with HCHO enhances amino acid incorporation several-fold. 80S ribosomes and 60S and 40S ribosomal subunits are equally effective in supporting polypeptide synthesis.

Effect of l-ethionine on macromolecular synthesis in mitogen-stimulated lymphocytes

2--4 mM L-ethionine completely inhibits DNA synthesis in phytohaemagglutinin- or concanavalin A-stimulated lymphocytes even though it does not prevent the morphological changes characteristic of blast formation. Evidence is presented which indicates that complete commitment to DNA synthesis as well as a substantial increase in the rates of RNA and protein synthesis can occur in the presence of ethionine. Ethionine, however, does inhibit methylation of tRNA and prevents mitogen-induced increase in the activity of histone-modifying enzymes. All of these effects of exposure to ethionine are completely reversible. Removal of ethionine after 24 h or more of exposure results in a rapid, synchronous wave of DNA synthesis, an increase in the rate of methylation of RNA and an increase in activity of histone-modifying enzymes.

Characterization of a viral messenger ribonucleoprotein particle accumulated during inhibition of polypeptide chain initiation in reovirus-infected L cells

Abstract A prior study has shown that L cells deprived of essential amino acids in isotonic Tris-buffered KCl show a marked and rapid inhibition of protein synthesis which is due to a decrease in the rate of initiation of new peptide chains. The inhibition, which is manifested by a disaggregation of polysomes with apparent conservation of mRNA, is rapidly reversed when the cells are resuspended in complete growth medium. This phenomenon has been applied in studies on the mechanism of mRNA storage and reutilization in reovirus-infected L cells. When reovirus-infected L cells are incubated in KCl-Tris medium, the viral messengers accumulate as ribonucleoprotein particles which sediment at approximately 50 S in sucrose density gradients and have a density in CsCl of 1.40. These 50-S messenger-containing ribonucleoprotein particles do not appear to be complexed with ribosomal subunits. The particles contain all 10 species of viral mRNA and have no demonstrable viral proteins. The mRNA from these particles is readily and rapidly mobilized into functional polysomes when the infected cells are restored to normal growth medium. Moreover, the mRNA moiety of partially purified 50-S ribonucleoprotein particles can direct the incorporation of amino acids into protein in a cell-free system.

Reassociation of eukaryotic ribosomal subunits by a factor from mouse fibroblast ribosomes

Abstract A factor contained in a 0.8 M KCl extract of L-cell ribosomes promotes the reassociation of highly purified L-cell 40S and 60S ribosomal subunits. No amino acyl-tRNA or mRNA is required. Only subunits stripped of initiation factors by differential treatment with 1 M KCl are responsive to the factor. Factor-dependent reassociation is optimal at 1.5 mM MgCl 2 ; above that concentration, there is increasing spontaneous reassociation in the absence of factor. During chromatography on DEAE-cellulose, the reassociation factor elutes at 50 mM KCl. Factor-dependent reassociation is considerably enhanced by GDPCP, and partially stimulated by GTP. Subunit reassociation is completely inhibited by 0.05 mM aurin tricarboxylic acid.

Factor and GTP requirements in a eukaryotic protein initiation system with reovirus messenger RNA, Met-tRNAF, and ribosomal subunits

A previous report described the formation of a eukaryotic initiation complex composed of Reovirus mRNA, rat liver Met-tR.NA,, and 40 S and 60 S ribo somal subunits from mouse L-929 cells [ 11. All of the three size classes of Reovirus mRNA were able to induce the formation of 80 S initiation complexes at a puromycin-sensitive site on the ribosome. Complex assembly was mRNA-dependent and was specific for Met-tRNAF [ 11. However, no requirement for GTP or ribosomal factors could be demonstrated. The 40 S and 60 S subunits retained their initiation factors and sufficient GTP to support complex formation even after treatment with detergent and 0.5 M KCl. This phenomenon restricted detailed mechanism studies in this system. In the present investigation, we report that differential treatment of L-cell 40 S and 60 S subunits with 1 M KC1 is essential in order to establish a GI’P dependency and a requirement for exogenous ribosomal factors during initiation. Although the 40 S subunit is relatively stable during such treatment, the 60 S subunit is rapidly inactivated. Treatment of either subunit with 1 M KC1 elicits both the GTP and factor dependencies. Utilization of the treated subunits has facilitated the stepwise analysis of the initiation process.

Age-dependent expression of a novel protein in mouse liver immunologically and functionally homologous with dihydrofolate reductase

Dihydrofolate reductase with a molecular weight of 22,000 has been purified by salt precipitation and methotrexate-Sepharose affinity chromatography from mouse livers having a mean weight of 2.4 g each. When the same purification procedure was followed using livers with a mean weight of 1.4 g or less, a protein with a molecular weight of 27,500 co-purified with dihydrofolate reductase. This 27.5 kDa species was recovered with dihydrofolate reductase following a second passage through the affinity column and it reacted by Western immunoblotting with a monospecific polyclonal antiserum raised to the purified 22 kDa enzyme. The two proteins could not be separated in a native state to compare their functional activity, but the 27.5 kDa protein appeared to have catalytic reductase activity when assayed directly on the polyacrylamide gel following non-denaturing electrophoresis. The catalytic activity of the mixture of the purified proteins was stoichiometrically inhibited by a molar equivalent of methotrexate.

Effect of tumor promoters on induction of aryl hydrocarbon hydroxylase in human lymphocytes.

The induction of aryl hydrocarbon hydroxylases in lymphocytes is dependent on their activation. The tumor-promoting phorbol esters which induce blast formation and DNA synthesis in lymphocytes enable polycyclic aromatic hydrocarbons to induce aryl hydrocarbon hydroxylases. Melittin, the major constituent of bee venom, acts synergistically with these phorbol esters in enhancing both lymphocyte activation and hydroxylase synthesis. Since aryl hydrocarbon hydroxylases convert procarcinogens to carcinogens these results suggest that tumor promotion by phorbol esters may be associated with their ability to affect the induction of these enzymes. This hypothesis is supported by the finding that phorbol and phorbol esters which lack tumor-promoting activity fail to enhance induction of aryl hydrocarbon hydroxylases in lymphocytes. However mezerein, although rather ineffective in promoting tumors, activates lymphocytes and permits polycyclic aromatic hydrocarbons to induce their hydroxylases effectively.