Biomedical subjects
Publications and source records attributed to H A Thompson.
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The binding of the initiator tRNA Met-tRNAf, and of acetylphenylalanyl-tRNA, has been examined with rat liver 40S subunits derived from 80S ribosomes by dissociation with native 40S subunits sedimented from the postmicrosomal fraction and with native 40S subunits extracted with high salt-containing solutions. Binding of Met-tRNAf and acetylphenylalanyl-tRNA to derived and to salt-extracted native 40S subunits is observed in the presence of the appropriate polynucleotide template and a highly purified binding factor obtain from the soluble fraction of rat liver homogenates (R.L. IF-1). Native 40S subunits bind acetylphenylalanyl-tRNA in a reaction that requires poly(U) but not exogenous binding factor; however, Met-tRNAf is not bound to native subunits, even when supplemented with the soluble binding factor, or under conditions where factor-independent, high Mg2+-stimulated binding is observed with the derived and the salt-washed native 40S subunits. The extract obtained from native 40S subunits promotes the binding of acetylphenylalanyl-tRNA but not Met-tRNAf to derived and to salt-extracted native subunits. The addition of native 40S extract to incubations containing R.L. IF-1, Met-tRNAf, and derived 40S subunits, inhibits the formation of 40S-Met-tRNAf complex. These data suggest that the binding activity that is specific for 40S subunits and initiator tRNA, and an activity that inhibits the interaction with Met-tRNAf specifically, are both associated with native 40S subunits, and can be extracted from them by treatment with high salt-containing solutions. Derived 40S subunits react quantitatively with 60S particles to form 80S ribosomes which do not bind acetylphenylalanyl-tRNA with binding factor R.L. IF-1. Native 40S subunits react only partly with 60S subunits; about half of the native 40S subunit population forms 80S ribosomes which do not subsequently bind acetylphenylalanyl-tRNA; the remaining native 40S subunits which do not react with 60S particles bind acetylphenylalanyl-tRNA but to a lesser extent. When preformed native 40S-acetylphenylalanyl-tRNA complex is incubated with 60S subunits, about half of the subunits form an 80S-acetylphenylalanyl-tRNA complex, while the rest remains as 40S-acetylphenylalanyl-tRNA. The addition of native 40S subunit salt extract to incubations containing preformed 80S ribosomes dissociates the particles to subunits. These data suggest that in addition to the initiator tRNA binding activity and the activity that inhibits Met-tRNAf interaction, part of the native 40S subunit population also contains an activity that dissociates 80S ribosomes.
Peptidyltransferase with rat liver 60S subunits can be measured by the reaction between exogenous acylaminoacyl-tRNA and puromycin to form acylaminoacylpuromycin in the presence of 33% methanol, 0.3 M KCl, and 4 mM MgCl2. An assay system has been developed that allows examination of the binding of acetylphenylalanyl-tRNA to the ribosomal subunit "P" site, the transpeptidation of the 60S-bound substrate to puromycin, and the requirements for these individual steps. Binding of acetylphenylalanyl-tRNA to 60S subunits is stimulated several-fold by the addition of methanol, but the extent of binding in alcohol is the same in 60 as in 300 mM KCl containing solutions. Formation of acetylphenylalanyl-puromycin from 60S-acetylphenylalanyl-tRNA complex and puromycin stringently requires alcohol and the initial rate of the reaction is markedly greater at 300 mM KCl than at 60 mM KCl concentrations. Thus, alcohol and high concentrations of monovalent cation affect the reaction of an event subsequent to the binding of substrate to the "P" site. Preincubation of 60S subunits with poly(U), which stimulates the overall peptidyltransferase reaction, does not affect the amount of acetylphenylalanyl-tRNA that is bound to the particles; however, it markedly stimulates the initial rate of the transpeptidation reaction between 60S-acetylphenylalanyl-tRNA complex and puromycin. The codon specificity and the failure to affect binding with poly(U) suggest a role for the polynucleotide in the alignment or stabilization of the acylaminoacyl-tRNA on the "P" site rather than an effect on binding to either of the two particle sites or on the peptidyltransferase "active center." The effect of 40S subunits, which inhibit the overall peptidyltransferase reaction, on the binding of substrate could not be clearly interpreted since all three preparations, 60S subunits, 40S subunits, and combinations of 60S plus 40S particles, appear to bind acetylphenylalanyl-tRNA in the presence of methanol. However, the initial rate of peptide bond formation is several times greater with 60S-acetylphenylalanyl-tRNA complex than with 60S plus 40S particles containing bound acetylphenylalanyl-tRNA and the addition of 40S subunits to preformed 60S-acetylphenylalanyl-tRNA complex during the transpeptidation phase of the reaction in methanol does not affect the rate of peptide bond formation. Thus, 40S subunits seem to inhibit peptidyltransferase by forming less reactive particles in aqueous solutions. Contd.
Lymphocytic choriomeningitis (LCM) virus infection of the mouse is the best-studied model of persistent viral infection. In cell culture, persistent LCM virus infections are associated with the production of large quantities of defective interfering (DI) LCM virus. These defective interfering particles cannot replicate by themselves yet can interfere with the replication of the standard virus and prevent the cytolytic effect caused by the standard virus. It is important to determine the mechanism of interference and to establish whether the DI virus plays a role in vivo. Biological and biochemical properties of the standard and DI virus particles and also virus enzymes are compared. Antigenic analyses reveal that cells releasing only DI virus particles have less cell surface expression of viral antigens than cells releasing the standard virus. In the animal model, the DI virus is shown to have a protective effect against the pathogenesis of the LCM virus disease both in the mouse and in the rat.
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Protein and ribonucleic acid (RNA) syntheses increase in the liver during Q fever, coincident with progressive increments in cortisol levels. Cell-free protein-synthesizing systems indicate that the pH 5 enzyme fraction was chiefly responsible for enhanced activity. Although the polysome profiles from normal and infected livers were similar, the number of liver ribosomes doubled during infection. There was a concomitant increase in orotate incorporation in the 28s, 18s, and 4s RNA species of infected liver.
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