Boyd Hardesty

Structure, function, and genetics of ribosomes

The papers in this book cover the Ribosome conference held at the University of Texas Marine Science Institute. The topics covered include: Structure of ribosomes; Self-organization of ribosomal RNA; Structural dynamics of the translating ribosome; and Mechanism of Ribosome Translocation.

The mechanism of sodium fluoride and cycloheximide inhibition of hemoglobin biosynthesis in the cell-free reticulocyte system.

Abstract The mechanism by which sodium fluoride and cycloheximide inhibit protein synthesis in the rabbit reticulocyte complete, cell-free system has been investigated. Both compounds have been found to inhibit the initiation of new chains on ribosomes. Studies of the rate of amino acid incorporation indicate that cycloheximide also inhibits incorporation into nascent chains that were initiated in intact cells and remain attached to ribosomes during their isolation. Sodium fluoride was found to have no apparent effect on the rate of incorporation into these previously initiated nascent chains, but to cause an apparent breakdown of polysomes to single ribosomes in intact cells. These 80 s ribosomes isolated from sodium fluoride-treated reticulocytes actively incorporate amino acids in the cell-free system and exhibit other properties that indicate they are similar or identical to monomeric ribosomes isolated from untreated cells. These observations are interpreted to indicate a sodium fluoride-sensitive reaction related to the initiation of new peptide chains on ribosomes. The sodium fluoride inhibition of this reaction may bring about the increase in the proportion of monomeric ribosomes because of a failure to re-form polysomes.

The mechanism of cycloheximide inhibition of protein synthesis in rabbit reticulocytes

Abstract Cycloheximide blocks the movement of peptidyl-tRNA from acceptor (aminoacyl) site to the donor (peptidyl) site on reticulocyte ribosomes. This translocation reaction is dependent upon the transfer enzyme, TF-II, and GTP hydrolysis. Cycloheximide has no effect on the ribosome dependent GTPase activity of TF-II or on the peptidyl transferase reaction by which peptides on tRNA in the donor ribosomal site are transferred to an amino acid on tRNA in the acceptor site.

Folding of a nascent peptide on the ribosome

Even though very significant progress has been made recently in elucidating the structure of the bacterial ribosome and topological assignments of its functional parts, the molecular mechanism of how a peptide is formed and how the nascent peptides is folded on the ribosomes remains uncertain. Here, the current progress and remaining problems are considered from the standpoint of the authors. Topics considered include formation of peptide bonds and models that represent this process, the vicinity of RNA to the nascent peptide, the cotranslational folding hypothesis, evidence that some but not all nascent peptides pass through a region within the 50S ribosomal subunit, presumably the tunnel, in which they are folded and sheltered, pause-site peptides, and the involvement of chaperones in folding of nascent proteins on ribosomes. The chaperone-like activity of the large ribosomal subunit in renaturation of denatured proteins is reviewed. It is concluded that cotranslational folding of some but not all nascent peptides occurs in the large ribosomal subunit. It is suggested that this folding is facilitated by changes in the conformation of the ribosome that are related to the reaction cycle of peptide elongation.

The effect of an antiviral peptide on the ribosomal reactions of the peptide elongation enzymes, EF-I and EF-II.

Abstract A basic peptide with antiviral properties isolated from pokeweed is shown to inhibit the synthesis of globin and phenylalanine peptides on ribosomes isolated from rabbit reticulocytes. The inhibition appears to involve a specific effect of the peptide inhibitor on the larger ribosomal subunit that can be produced at a ratio of inhibitor to ribosomes of less than one to one. Ribosomes treated with the inhibitor have a reduced capacity to support enzymatic binding of Phe-tRNA to ribosomes and GTP hydrolysis caused by the elongation enzyme, EF-I. Treated ribosomes exhibit a concomitant capacity for increased GTP hydrolysis by EF-II but do not efficiently support EF-II-dependent binding of [ 3 H]GTP. Such binding appears to involve the formation of an EF-II·GDP·ribosome complex. Thus, the inhibitor has an effect on GTP-dependent reaction carried out by both of the peptide elongation enzymes. The relation between these effects in the reticulocyte system is discussed in relation to the effects of siomycin or thiostrepton in blocking GTP hydrolysis by EF-T and EF-G on prokaryotic ribosomes.

Ribosomes and ribosomal RNA as chaperones for folding of proteins

BACKGROUND Provocative recent reports indicate that the large subunits of either prokaryotic or eukaryotic ribosomes have the capacity to promote refolding of denatured enzymes. RESULTS Salt-washed Escherichia coli ribosomes are shown to promote refolding of denatured rhodanese. The ability of the ribosomes to carry out renaturation is a property of the 50S ribosomal subunit, specifically the 23S rRNA. Refolding and release of enzymatically active rhodanese leaves the ribosomes in an inactive state or conformation for subsequent rounds refolding. Inactive ribosomes can be activated by elongation factor G (EF-G) plus GTP or by cleavage of their 23S rRNA by alpha-sarcin. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex. CONCLUSIONS Large subunits of E. coli ribosomes, specifically 23S rRNA, have the capacity to mediate refolding of denatured rhodanese. Refolding activity is related to the state or conformation of ribosomes that is promoted by EF-G. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex.

Polyamines are necessary for maximum in vitro synthesis of globin peptides and play a role in chain initiation

Abstract The salt wash fraction removed from rabbit reticulocyte ribosomes with 0.5 m KCl contains dialyzable components required for maximum in vitro synthesis of globin peptides. The active substances were identified as spermidine and spermine. Rabbit reticulocyte ribosomes contain spermine and spermidine in a 1:3 ratio of which about 75% is removed in the 0.5 m KCl wash fraction. Dialyzed salt wash can be reactivated for in vitro protein synthesis by addition of either spermine, spermidine, or Mg 2+ ion. A twofold higher leucine incorporation into protein was obtained with the optimum concentration of either polyamine than with Mg 2+ . Spermidine is effective in lowering the Mg 2+ requirement for initiation of phenylalanine peptides in the poly(U)-directed system, apparently by formation of an initiation complex. Also, spermidine competitively interferes with edeine inhibition of globin chain initiation. These results indicate that spermidine may play a special role in peptide initiation.

Renaturation of Rhodanese by Translational Elongation Factor (EF) Tu PROTEIN REFOLDING BY EF-Tu FLEXING

The translation elongation factor (EF) Tu has chaperone-like capacity to promote renaturation of denatured rhodanese. This renaturation activity is greatly increased under conditions in which the factor can oscillate between the open and closed conformations that are induced by GDP and GTP, respectively. Oscillation occurs during GTP hydrolysis and subsequent replacement of GDP by EF-Ts which is then displaced by GTP. Renaturation of rhodanese and GTP hydrolysis by EF-Tu are greatly enhanced by the guanine nucleotide exchange factor EF-Ts. However, renaturation is reduced under conditions that stabilize EF-Tu in either the open or closed conformation. Both GDP and the nonhydrolyzable analog of GTP, GMP-PCP, inhibit renaturation. Kirromycin and pulvomycin, antibiotics that specifically bind to EF-Tu and inhibit its activity in peptide elongation, also strongly inhibit EF-Tu-mediated renaturation of denatured rhodanese to levels near those observed for spontaneous, unassisted refolding. Kirromycin locks EF-Tu in the open conformation in the presence of either GTP or GDP, whereas pulvomycin locks the factor in the closed conformation. The results lead to the conclusion that flexing of EF-Tu, especially as occurs between its open and closed conformations, is a major factor in its chaperone-like refolding activity.

INHIBITION OF POLIOVIRUS REPLICATION BY A PLANT ANTIVIRAL PEPTIDE

Treatment of poliovirus with the antiviral protein isolated from Phytolacca americana resulted in as much as 96% inhibition of subsequent release of infectious virus from HeLa cells. The inhibitor can be quantitatively removed from poliovirus by centrifugation through a sucrose solution. These washed viruses were not inactivated by their prior treatment with the peptide nor does the peptide block attachment of labeled poliovirus to HeLa cells. In the absence of virus the peptide was not cytotoxic for HeLa cells and had no detectable effect on the rate of protein synthesis. However, protein synthesis was reduced in cells infected with virus treated with the peptide compared with cells infected with untreated virus. It is suggested that the mechanism of the antiviral activity of the peptide involves its transport into intact cells by the virus followed by inactivation of host cell ribosomes.

Isolation and partial characterization of an aminoacyl-tRNA synthetase complex from rabbit reticulocytes

Abstract A procedure is described for isolation of an aminoacyl-tRNA synthetase complex from the soluble fraction of rabbit reticulocyte lysates. The product of the isolation procedure appears to be a physically homogeneous structure of about 16S and 550,000 daltons. Successive preparations contain aminoacyl-tRNA synthetase activity for arginine, lysine, isoleucine, leucine, and methionine in a constant ratio of activity. The enzymatic activities of the complex are protected during storage and from heat inactivation by glycerol. The multienzyme structure has been named the reticulocyte 16S aminoacyl-tRNA synthetase complex.