Protein synthesis in the cotyledons of Pisum sativum L. Protein factors involved in the binding of phenylalanyl-transfer ribonucleic acid to ribosomes

1. Proteinaceous factors contained in a 0.5m-KCl extract of ribosomes from pea cotyledons form a ternary complex at 0 degrees C with [(14)C]phenylalanyl-tRNA and poly(U). The complex is measured by its quantitative retention on Millipore filters. 2. Complex-assembly is optimal at 5mm-Mg(2+) and is independent of GTP and ribosomes. 3. The addition of ribosomes is required to stabilize the complex at 34 degrees C. The complex binds to a puromycin-sensitive site on the ribosome. 4. Soluble factors from the 250000g supernatant of pea cotyledon form a Millipore-retainable complex dependent on GTP and ribosomes. 5. Complex-formation by soluble factors has a Mg(2+) optimum of 10-12mm and forms a puromycin-insensitive complex with ribosomes. 6. The function of the ribosomal protein factors and the supernatant fraction in initiation of protein synthesis is discussed.     

Cross-reactivity of phenylalanyl-transfer ribonucleic acid ligases from different microorganisms.

The cross-reaction of phenylalanyl-transfer ribonucleic acid (tRNA) ligases from different microorganisms with antibodies raised against the purified enzyme from Escherichia coli has been investigated. The results of immunotitration and immunodiffusion experiments and of the sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of immunoprecipitates revealed: (i) a high degree of immunochemical identity of this enzyme only within the family Enterobacteriaceae; (ii) intermediate-to-weak cross-reaction with the phenylalanyl-tRNA ligases from Pseudomonadaceae, Rhodopseudomonas spheroides, and Bacillus stearothermophilus; (iii) no detectable cross-reaction (with the methods employed) with the enzymes from several gram-positive organisms, Euglena gracilis, and several fungi. As revealed by immunochemical analysis, a merodiploid strain of E. coli carrying an episome (F148) that covers the aroD region of the E. coli chromosome possesses at least twice the amount of phenylalanyl-tRNA ligase in comparison with its haploid parent strain. This suggests that the cistrons for both the alpha and beta polypeptides of this enzyme are mapping in this area.     

The binding of spermine to the ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

1. The total intracellular concentrations of Na(+), K(+), Mg(2+), spermine, spermidine and RNA were measured in Bacillus stearothermophilus. 2. The binding of spermine to ribosomes and to ribosomal RNA from B. stearothermophilus was studied under various conditions by using a gel-filtration technique. 3. The affinity of spermine for ribosomes and for ribosomal RNA decreased with increasing ionic strength of the medium in which they were suspended. 4. The extent of spermine binding did not change appreciably in the temperature range 4-60 degrees . 5. Optimum binding occurred at about pH7.0. 6. The number of binding sites for spermine on either ribosomes or ribosomal RNA was 0.10-0.13/RNA phosphate group. 7. A high proportion of the intracellular spermine is likely to be bound to the ribosomes in vivo; spermine competes with Mg(2+) on equal terms for sites on the ribosomes.     

Polyribosomal and particulate distribution of lysyl- and phenylalanyl-transfer ribonucleic acid synthetases

1. Only two aminoacyl-tRNA synthetases from Chinese hamster ovary cells are found associated with ribosomes and polyribosomes. 2. Phenylalanyl-tRNA synthetase activity is found with the 60S subunit, 80S monoribosome and individual polyribosomes. An additional 15S form of the enzyme is also seen. 3. Lysyl-tRNA synthetase activity is found in a form of about 20S and associated with ribosomal subunits and polyribosomes. The ribosomal subunits having lysyl-tRNA synthetase activity are about 6S larger than the bulk of the ribosomal subunits. 4. The lysyl- and phenylalanyl-tRNA synthetases found in different complexes have differential sensitivity to EDTA and centrifugation properties.     

Stability of ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

After heating at 65 C, ribosomes isolated from Bacillus stearothermophilus were strikingly more heat-stable than comparable preparations from Escherichia coli when tested for ability to support polyuridylic acid-directed phenylalanine incorporation at 37 C. The stability of ribosomes was also determined by measurements of hyperchromicity at 259 mmu while heating them from 25 to 90 C. In standard buffer containing 0.01 m Mg(++), the T(m) (temperature at the midpoint of total hyperchromicity) of E. coli and B. stearothermophilus ribosomes was 71 and 81 C, respectively. In a magnesium-free buffer, the T(m) of E. coli and B. stearothermophilus ribosomes was 44 and 64 C, respectively. Putrescine (0.01 m) was more effective in stabilizing ribosomes from B. stearothermophilus than those from E. coli. Spermidine (0.001 m), on the other hand, was more effective in stabilizing ribosomes from E. coli than those from B. stearothermophilus. Melting curves of total ribosomal ribonucleic acid (rRNA) from E. coli and B. stearothermophilus revealed T(m) values of 50 and 60 C, respectively. Putrescine stabilized thermophile rRNA, but had no effect on E. coli rRNA. Sucrose density gradients demonstrated that thermophile 23S ribonucleic acid was degraded during storage at -20 C; the 23S component from E. coli was stable under these conditions. The results are discussed in terms of the mechanism of ribosome heat stability and the role of the ribosome in governing the temperature limits for bacterial growth.     

Sedimentation of ribosomal ribonucleic acid from ribonuclease treated and untreated ribosomes

The formation of a slowly sedimenting form of 23-S ribosomal RNA from E. coli has been investigated by analytical ultracentrifugation and thermal denaturation in aqueous solution and in formamide. Evidence is presented that the slow form of 23-S arises as a result of nucleate damage to the RNA in the 50-S ribosome. The 30-S ribosome (and 16-S RNA), is unaffected. The slow form of 23-S RNA cannot be demonstrated under conditions of complete denaturation in formamide, but only by partial denaturation in aqueous solution of low ionic strength (< 0.01M Na). Apparent maintenance of the integrity of 23-S RNA in formamide after nuclease treatment suggests that this may not be a simple linear molecule. An alternative model is suggested containing a circular element in the structure.     

Properties of 5-fluorouracil-containing ribonucleic acid and ribosomes from Bacillus subtilis

Growth of a strain of Bacillus subtilis that requires uracil, thymine, adenine, and tryptophan in the presence of 5-fluorouracil (FU) results in the synthesis of ribonucleic acid (RNA) and ribosomes in which 55 to 65% of the RNA uracil has been replaced by the fluorine derivative. Examination of analogue-containing ribosomes by sucrose density gradient centrifugation and thermal denaturation studies suggests that, as far as the size, shape, and packing structure are concerned, extensive FU substitution has little or no effect. FU appears to replace uracil in RNA without selectivity for one RNA class over another, as determined by methylated albumin-kieselguhr column chromatography and sucrose density gradient centrifugation. The total amino acid content of the cells is markedly affected by growth in the presence of FU. The possibility of an FU effect on genetic translation is discussed.