Partial reaction of peptide initiation inhibited by the reticulocyte hemin-controlled repressor

The hemin-controlled repressor from rabbit reticulocytes inhibits binding of Met-tRNA_f to reticulocyte 40S ribosomal subunits in a partial reaction containing these components, two initiation factor fractions and GTP. The inhibitor does not interfere with the formation of the Met-tRNA_f· initiation factor IF-E₂·GTP complex.     

Inhibition of globin chain initiation in reticulocyte lysates by pactamycin: accumulation of methionyl-valine

In a globin synthesizing system containing reticulocyte lysate and [³⁵S] met-tRNA_f, low levels of pactamycin cause an accumulation of radioactivity on the monosomes and small oligosomes concomitant with the breakdown of polysomes. About 50% of the ribosome-bound radioactivity corresponds to methionyl-valine, the initial dipeptide of globin chains, with negligible amounts of tri- or other oligopeptides. This suggests that the site of its action is after the formation of the first peptide bond. The ribosome-bound radioactivity in the presence of sparsomycin, an inhibitor of chain elongation, is in di-, tri- and oligopeptides. Sparsomycin levels (10^?5M) that cause almost complete inhibition of polypeptide synthesis have only a small inhibitory effect on the pactamycin-induced accumulation of methionyl-valine. Fusidic acid and chlortetracycline do not cause accumulation of any significant amounts of methionyl-valine.     

The attachment of polyuridylic acid to reticulocyte ribosomes

The attachment of polyuridylic acid to reticulocyte ribosomes was studied by using polyadenylic acid, which inhibits the attachment reaction only, while permitting translation of polyuridylic acid bound to ribosomes. After addition of polyadenylic acid the amount of polyphenylalanine synthesized under standard conditions was taken as a measure of the bound polyuridylic acid. In this way certain parameters of the attachment reaction and the subsequent translation of attached polyuridylic acid were defined: (1) polyuridylic acid-ribosome interaction at 37 degrees requires only Mg(2+) at an optimum concentration of 8mm; (2) K(+) (required for translation) is a non-competitive inhibitor of the attachment reaction; (3) optimum polyphenylalanine synthesis directed by attached polyuridylic acid occurs at 5mm-Mg(2+) concentration; (4) from kinetic studies single ribosomes appear to participate in the attachment reaction.     

Binding of inosine-substituted mRNA to reticulocyte ribosomes and eukaryotic initiation factors 4A and 4B requires ATP

We examined the hypothesis that initiation of eukaryotic protein synthesis involves ATP-dependent melting of 5'-cap-proximal secondary structure in mRNA by eukaryotic initiation factors 4A and 4B. In reticulocyte lysate depleted of ribonucleoside triphosphates by pretreatment with hexokinase/glucose, initiation complex formation by native reovirus mRNA showed a strict requirement for ATP. The corresponding mRNA synthesized with ITP in place of GTP to minimize secondary structure also required ATP for binding to 40 S ribosomal subunits in complexes characteristic of initiation. In a partial reaction without ribosomes, purified eukaryotic initiation factors 4A and 4B bound and cross-linked to the capped 5'-end of oxidized mRNA. This interaction was ATP-dependent with inosine-substituted or bromouridine-containing reovirus RNAs as observed previously with native mRNA. The results indicate that if initiation involves ATP-dependent denaturation of mRNA, the effect must occur after initiation factor-mediated attachment of mRNA to the 40 S ribosomal subunit.     

Inhibition of peptide initiation by a low molecular weight RNA from rabbit reticulocytes

A heat-stable inhibitor of protein synthesis has been isolated from the postribosomal supernatant of rabbit reticulocytes. Its activity is not susceptible to protease treatment but is destroyed by incubation with alkali. Inhibitory activity can be quantitatively recovered in the aqueous phase after phenol extraction and has the ultraviolet absorption spectrum of a nucleic acid. It is concluded that the inhibitor is RNA. The inhibitory activity sediments in the range of 3 S, but it has not been demonstrated whether the inhibitor RNA is a single molecular species. The inhibitory RNA does not affect peptide elongation but rather blocks a step of peptide initiation. It does not interfere with the formation of the ternary complex between initiation factor 2, GTP, and methionyl-tRNAMetf and does not activate a protein kinase phosphorylating initiation factor 2. The inhibitory RNA appears to be a novel type of RNA that inhibits polypeptide initiation at a step involving ribosomal subunits.