Moshe Tal

Polyuridylic acid binding and translating by Escherichia coli ribosomes: Stimulation by Protein 1, inhibition by aurintricarboxylic acid

Abstract Prolonged dialysis of Escherichia coli ribosomes against low ionic strength buffer results in release of several acidic proteins, one of which is capable of interacting with poly(U) as well as augmenting its binding and translation by ribosomes. Its acidic nature, amino acid composition and molecular weight (65 000), identified it as one of the 30-S ribosomal proteins, namely Kurlands Protein 1, Wittmans Protein S1, Moores Protein 13 or Nomuras P1. Aurintricarboxylic acid, which inhibits binding of poly(U) to ribosomes, has the same effect on binding of poly(U) to the pure Protein 1 preparation at a similar concentration. The possibility that Protein 1 comprises the active site for mRNA binding on the 30-S ribosome, or at least contributes to it, is discussed.

Metal ions and ribosomal conformation.

Abstract Divalent metal ions play an important role in preserving the compact structure of ribosomes. This is reflected in the specific viscosity ( η sp ) of the ribosomal solution which is increased at elevated temperatures in the presence of EDTA. Dialysis of EDTA-treated ribosomes (unfolded particles) against 1 mM Zn 2+ , Ni 2+ , Mn 2+ , Co 2+ and Fe 2+ (present in natural ribosomes) results in a sedimentation profile closely similar to that of natural ribosomes, indicating reversibility of the unfolding process caused by EDTA. On the other hand, addition of Mg 2+ to the unfolded particles results in intermediate s values only. An extended thermal denaturation scheme of ribosomes, taking into consideration the role of metal ions, is proposed.

Novel method for measuring polyuridylic acid binding to ribosomes

Abstract Nitrocellulose membranes, mildly pretreated with alkali, lose their ability to adsorb poly U, but retain their potentiality for binding ribosomes. According to this observation, a new method for measuring poly U binding to ribosomes is proposed. Results indicate that both the secondary structure of poly U and the chemical nature of the nitrocellulose filter influence the interaction of the polymer and the membrane.

A protein factor stimulating binding and translating of polyuridylic acid by Escherichia coli ribosomes

Abstract Exposure of Escherichia coli MRE-600 ribosomes to low-ionic-strength (1 mM) Tris-acetate causes release of about 10 % of their proteins. Removal of this fraction results in a decrease in binding of poly U and in incorporation of phenylalanine by the ribosomes. Adding this fraction to the depleted ribosomes restores both activities. DEAE-cellulose chromatography reveals that this fraction contains a few acidic proteins. Only the one, which elutes at 0.175 M NaCl enhances binding and translation of poly U by the ribosomes. It also has the ability to bind poly U in the absence of ribosomes. Use of [3H]leucine-labeled factor shows that it interacts with the depleted 30-S ribosomal subunit. The binding activity of depleted ribosomes supplemented with the protein factor is higher than the binding activities of either the ribosomes or the factor alone. This increase suggests cooperative effect in the reconstituted system. Polyphenylalanine synthesis shows that the binding of poly U took place at the mRNA site on the supplemented ribosomes.

Isolation and partial characterization of retinol-binding protein from chicken plasma

Abstract A simple rapid three-step procedure for the isolation of pure retinol-binding protein from chicken plasma is described. The method comprises DEAE-cellulose chromatography followed by preparative disc gel electrophoresis and final purification by Sephadex G-100 gel filtration. Amino acid analysis showed that there is only a general similiarity between this retinol-binding protein and that obtained from human or rat sources. The molecular weight, which was estimated to be 19 000, is similar to that of rat retinol-binding protein.

A new method for stoichiometric analysis of proteins in complex mixture — reevaluation of the stoichiometry of E. coli ribosomal proteins

A novel way is presented for determination of the stoichiometry of ribosomal proteins in the ribosome. The 70S E. coli r-proteins, completely separated on a two-dimensional gel system, were used throughout our experiments. The method is based on our previous observation that the amount of Coomassie R bound to a protein molecule is directly proportional to the number of positive charges on that protein. By plotting the amount of bound Coomassie as a function of the number of positive charges of each r-protein, and relating the experimental amount of the dye bound to each r-protein to the value obtained from the linear regression line based on all (a total of some 50 proteins), one can obtain the molar concentration of every protein in the ribosome. A parallel experiment can be carried out, which relates the radioactivity contributed by 3H-labeled amino acid in each r-protein to its amino acid content in that molecule. The two sets of data, which are completely independent of each other, are well correlated. Further verification of the validity of our procedure is provided by the fact that we found the known proportions of four copies of L7/L12 and one copy of S6 per ribosome. The rationale behind the present study was our finding that recalculation of Hardys data (Hardy, S.J.S. (1975) Mol. Gen. Genet. 140, 253-274), with the accurate molecular weight value of the r-proteins provided by Giri et al. (Adv. Protein Chem. (1984) 36, 1-78), raises some doubt with regard to his experimental results, although we agree with his final conclusion that E. coli ribosome is homogeneous with respect to its proteins.

Synthesis and degradation of lac mRNA in E. coli depleted of 30S ribosomal subunits

SummaryEscherichia coli was depleted of active ribosomes by a thermal shock at 47°C which quantitatively destroyed the 30S ribosomal subunits. During recovery, RNA is synthesized while protein synthesis resumes only after about 90 minutes. It is shown that lac mRNA is synthesized in the complete absence of ribosomal activity and hence RNA synthesis is not coupled to protein synthesis. Transcription time and average transcript length were slightly less than in untreated cells. lac mRNA was degraded much more slowly in bacteria depleted of ribosomes. In E. coli W both functional half life (T1/2=28 min vs. 2.25 in untreated cells) and chemical stability (T1/2=32 min vs. 7 in untreated cells) was increased. The analysis of rna and pnp mutants showed that polynucleotide phosphorylase is involved in lac mRNA degradation in heat treated cells but that RNase I is not. The functional T1/2 was increased in pnp mutants and was 95 min during the recovery period. The rate of chemical decay is so slow that the half-life cannot be accurately determined.

Circular dichroism of Escherichia coli ribosomes: Effect of heating and metal ions

Abstract 1. Ribosomes and rRNA from a low-nuclease strain of Escherichia coli were examined by means of circular dichroism (CD) in 0.001 M Tris-acetate buffer (pH 7.2). 2. The ribosome CD spectrum was altered when metal ions were chelated with EDTA. The following changes occurred: disappearance of the small negative RNA band at 297 mμ, a red shift of the positive RNA band at 264 mμ and a slight decrease of the protein band at 222 mμ. Addition of Mg 2+ caused only partial reversal of these differences, and other metal ions including Ni 2+ had even smaller effects. 3. Similar irreversible CD changes were noted after heating and recooling the ribosomes. Hysteresis was evident in the heating and cooling curves. EDTA caused a lowering of the melting temperature. 4. The CD spectrum of isolated 30-S and 50-S ribosomes showed similar temperature effects. The CD spectra of the subunits differ in the protein region. 5. The CD spectrum of rRNA is dependent only upon metal ion concentration and not upon prior EDTA or heat treatment.

In vivo reassembly of 30-S ribosomal subunits following their specific destruction by thermal shock

Abstract When Escherichia coli cells suspended in 0.25 M potassium phosphate, pH 7.2 are heated at 48°C for 25 min the 30-S ribosomal subunits are completely destroyed while the 50-S subunits remain intact and active. On transfer to Davis minimal medium with shaking at 30°C, the bacteria recover from the shock and reassemble new 30-S subunits which consist of newly synthesized 16 S rRNA and preexisting 30-S ribosomal proteins. Kinetic studies show that the RNA synthesis capability of the recovering bacteria attains a level similar to untreated cells within 30 min of incubation; by contrast, their protein synthesis capability in terms of l -[ 3 H]leucine incorporation is neglible for as long as 90 min, at which time it begins to increase sharply and reaches the control levels after an additional 60 min. Sedimentation analyses of cell extracts show that the new subunits begin reforming after about 45 min of incubation and the sedimentation profile acquires a normal appearance at 90 min. Various kinds of evidence indicate that the recovery process involves at least 45% of the original bacteria, including studies of the various types of rRNA species, the composition of ribosomal proteins in the new subunits, and the nature of the extended lag in recovery before the onset of protein synthesis. Both 16 S and 23 S rRNA are accumulated, albeit with a predominance of the 16 S species. After 90 min the ribosomal proteins S1 and S21 are still unpresented in the reassembled 30 S particle while S2 and S10 are absent altogether. This is in agreement with the reconstitution experiments of Nomura and Held (Nomura, M. and Held, W.A. (1974) in Ribosomes (Nomura, M., Tissieres, A. and Lengyel, P., eds.), pp. 193–223, Cold Spring Harbor Laboratory, New York), in which S10 and S21 are among the last to be attached. Since protein synthesis begins after 90 min, it is likely that a small fraction of the reassembled 30-S subunits already contain the whole set of proteins while most of them still do not. The gross time required for in vivo reassembly can be estimated by the onset of l -[ 3 H]leucine incorporation. In the presence of methionine this incorporation begins about 30 min earlier than in its absence. The effect of methionine is due to the thermolability of homoserine trans-succinylase. The net time required for the reassembly is estimated to be about 30 min.

A sensitive spectrophotometric method for determination of tris

Abstract The paper describes a sensitive spectrophotometric method for qualitative and quantitative determination of Tris, in the 0.1–1.0 μmole range. The procedure is almost identical with that used for histidine. The green-colored reaction product has two absorption peaks with maxima at 422 nm and 640 nm, in contrast to the single peak (540 nm) in the case of histidine. Absorbance and Tris concentration are mutually linear at both wavelengths, but reading conditions are more favorable at 640 nm.