Protein synthesis in rabbit reticulocytes: control of protein synthesis initiation by a met-tRNA Met f deacylase and peptide chain initiation factors

The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNA_f^Met. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$). Other aminoacyl tRNAs tested including fMet-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$), Phe-tRNA ($\text{E.}$$\text{coli}$), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNA_f^Met forms the initiation complex Met-tRNA_f^Met:IF1:GTP (2), and in this ternary complex Met-tRNA_f^Met is not degraded by the deacylase. $\text{E.}$$\text{coli}$ Met-tRNA_f^Met binds to IF1 independent of GTP, and in this complex, this Met-tRNA_f^Met is degraded by the deacylase.Prior incubation of f1 with Met-tRNA_f^Met (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNA_f^Met (retic.) was pre-incubated with peptide chain initiation factors.     

Hybridization of low molecular weight nuclear RNA with pre-mRNA from erythroid bone marrow cells and rabbit mRNA

Hybridization of labeled low molecular weight (LMW) nuclear RNA's to pre-mRNA from rabbit non-matured erythroid bone marrow cells or globin mRNA from reticulocytes revealed three RNA species having approximately 90, 100 and 160 nucleotides which are were specifically hybridized with purified cytoplasmic globin messenger RNA, while one (100 nucleotides) was also hybridized with rabbit 18S rRNA. The identity of these rabbit RNAs to LMW RNAs described for other animal species, as well as their possible hybridization sites and function are discussed.     

The initiation of haemoglobin synthesis in rabbit reticulocytes

1. The incorporation of labelled valine by rabbit reticulocytes into the N-terminal position of nascent haemoglobin was investigated by deaminating the nascent peptides with nitrous acid and isolating labelled alpha-hydroxyisovaleric acid and valine after acid hydrolysis. 2. The amount of radioactivity in alpha-hydroxyisovaleric acid relative to that in valine indicated the presence of 12.3% N-terminal valine having a free amino group. This high value suggests that most if not all nascent peptides contain valine in the N-terminal position. 3. Cell-free preparations containing reticulocyte ribosomes and pH5 enzymes incorporated alpha-hydroxy-[(14)C]isovaleryl-tRNA (where tRNA refers to transfer RNA), which was obtained by deamination of [(14)C]valyl-tRNA from yeast or liver with nitrous acid, into both soluble and nascent protein. 4. When the soluble protein was chromatographed on CM-cellulose, radioactivity was found to be associated with both the alpha-and beta-globin chains. 5. The kinetics of hydrolysis of [(14)C]valine, was also investigated. Most of the material was hydrolysed rapidly at pH10, but a minor component that was relatively stable appeared to be present to the extent of about 10% of the total valyl-tRNA. Valine was, however, the only hydrolysis product detected by paper chromatography. 6. It is concluded that chain initiation in haemoglobin synthesis involves valine as the N-terminal amino acid and that the amino group of nascent protein is probably not substituted.     

Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes.

Initiation factor IF-E2 was isolated from rabbit reticulocytes and purified 120-fold to near homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and phosphocellulose, and, when suitable, by sucrose density gradient centrifugation. The factor is a complex protein containing three nonidentical polypeptides of molecular weight 57,000, 52,000, and 36,000. It behaves as a complex throughout its purification and during polyacrylamide gel electrophoresis in nondenaturing buffer but its thress components are readily separated by electrophoresis in denaturing buffers. None of its components corresponds to any of the polypeptides of the other initiation factors or to any proteins of ribosomes washed in buffers containing a high salf concentration. A stoichiometric ratio of 1:1:1 was determined for the three polypeptides; based on the assumption of one copy each per complex, the calculated factor molecular weight is 145,000, a value in agreement with the measured value of 160,000. Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase also isolated from rabbit reticulocytes. Neither procedure causes a measurable change in the ability of the factor to form a ternary complex with GTP and the initiator methionyl-tRNA. 5'-Guanylyl-methylenediphosphonate may substitute for GTP, but only at relatively high concentrations. The binding of labeled initiation factor IF-E2 and methionyl-tRNA to the 40 S ribosomal subunit was studied by sucrose density gradient centrifugation. Appreciable binding of the factor is seen only when all three components of the ternary complex are included in the reaction mixture. The binding of either the factor or methionyl-tRNA was not stimulated by the addition of globin messenger RNA and initiation factor IF-E3. It was shown that all three polypeptide components of initiation factor IF-E2 are bound to these nascent initiation complexes.     

Stabilization of glucocorticoid receptor association with RNA by a low molecular weight factor from rat liver cytosol

A small (Mr less than 500) anionic, heat-stable molecule has been identified in rat liver cytosol which prevents the RNase-induced decrease in the glucocorticoid receptor sedimentation properties which we described previously. This factor, which can be removed by dialysis, molecular exclusion chromatography, or ultrafiltration, functions as a true stabilizer of the RNA-glucocorticoid receptor association, and not as a RNase inhibitor. Preliminary characterization shows that the factor is not a protein, nucleic acid, or nucleotide, is not absorbed by activated dextran-charcoal, and is unaffected by extraction with organic solvents. This factor prevents activation of the glucocorticoid receptor by dilution. The relationship of this stabilization factor to a low molecular weight activation inhibitor described by others is discussed.     

Bacteriophage phi 80-induced low molecular weight RNA

A species of low molecular weight RNA (M₃) which is produced in bacteriophage φ80-infected Escherichia coli has been isolated and characterized. M₃ appears immediately after infection and its synthesis continues until lysis. This RNA is unstable; its mean half-life is 13.5 min. The structural gene for M₃ is localized on the φ80 genome to the right of the exonuclease locus and to the left of gene Q.     

Identification of bacteria by low molecular weight RNA profiles: a new chemotaxonomic approach

A new chemotaxonomic method is presented for the identification of eubacteria. This method is based on one-dimensinal gel electrophoresis of total RNA extracts from eubacteria. Only low molecular weight (<150 nucleotides) RNA, comprising 5S ribosomal and transfer RNA, was used for the identification. The high resolution of the electrophoresis, better than half a nucleotide, allowed construction of low molecular weight (LMW) RNA profiles that contained 10 to 20 bands per strain. LMW RNA profiles of a set of eubacterial reference strains showed on variation in dependence on culture conditions or physiological state of the cells. Computer-assisted data evaluation, including six molecular weight markers, enabled the calculation of relative nucleotide units (RNU) for every band. The resulting normalized band pattern allowed the identification of identical strains on different gels. The relative position of the single bands from the different groups of RNAs made an identification of bacterial strains to genus and often species level possible.Especially valuable for the identification were the large, class 2 tRNAs that showed certain variation among species of the same genus and varied considerably among different genera. RNA profiles can provide a rapid and inexpensive screening technique for the taxonomic classification of single bacterial strains. Potential fields of application for this technique might be bacterial taxonomy, biotechnology and ecology.