Joel G. Flaks

Magnesium dependence and equilibrium of the Escherichia coli ribosomal subunit association

Abstract The well-known, magnesium ion-dependent, reversible association of Escherichia coli ribosomal subunits: 30 s + 50 s ⇌ 70 s has been quantitatively studied with a simple light-scattering approach which appears to be the most convenient means for the assay of this reaction in both directions. The results with this technique correlated exactly with those obtained by zone sedimentation. The reaction has been found to be a true equilibrium that is dependent upon both the concentrations of ribosomes and Mg 2+ . Expressions have been formulated to describe the equilibrium, and the equilibrium constant and the number of Mg 2+ involved has been evaluated. The critical value of the latter is small relative to the total Mg 2+ bound per ribosome. Increased temperature enhances dissociation of the 70 s particle and leads to a lowering in the critical number of Mg 2+ . From the temperature and equilibrium studies the following thermodynamic constants have been determined: ΔG ° = −35·3 kcal./mole (at 25°C), ΔH ° = − 70 kcal./mole and ΔS ° = 120 e.u. (all determined in 50 mu-Tris buffer (pH 7·8) and 50 mM-KCl). The enthalpic change is the dominant term and the magnitude of the parameters are suggestive of a conformational change in structure in the course of the reaction. A variety of cations (potassium, calcium and polyamines) which have previously been shown to influence the reaction were studied. Potassium, similar to increased temperature, favors dissociation of the 70 s particle, with decreases in the magnitude of the thermodynamic parameters. Polyamines, on the other hand, favor association of the two subunits, with spermidine being 100-fold more effective on a molar basis than putrescine. Both potassium and the polyamines reduced the critical number of Mg 2+ as reflected in a broadening of the Mg 2+ concentration range over which the transition takes place. This is due to either a direct competition of the cations with Mg 2+ , or an indirect induced conformational change in the ribosome. Calcium could be substituted for Mg 2+ , but was much less effective in enhancing association. The dissociation pattern of native 70 s ribosomes is multiphasic and differs from the dissociation of purified particles. However, reassociation of these subunits was identical to the pattern with purified subunits.

The specific cross-linking of two proteins from the Escherichia coli 30 s ribosomal subunit☆

Abstract The reaction of Escherichia coli 30 s ribosomal subunits with any one of the three different N,N′ -phenylenedimaleimides (1,2-, 1,3- or 1,4- ) leads to the rapid and nearly quantitative cross-linking of two specific proteins, S18 and S21, of this subunit. Both of these proteins contain exposed sulfhydryl groups through which the cross-linking is presumed to occur. With all three of the reagents the reaction is irreversible and is 80% complete in one minute at 37 °C, and about 95% or more in ten minutes. The cross-linked product has been identified by two-dimensional polyacrylamide gel electrophoresis and its molecular weight estimated to be very nearly equal to the sum of the molecular weights of proteins S18 and S21. Based on an examination by one-dimensional polyacrylamide gel electrophoresis no other proteins on either the 30 s or 50 s subunits, or the 70 s ribosome, are crosslinked to any detectable degree with any of these reagents. The results suggest that proteins S18 and S21 are perhaps located adjacent to each other and at, or near, the surface of the 30 s subunit.

Some physiological and genetic properties of a strain of Escherichia coli requiring thymine, arginine and uracil

A polyauxotrophic mutant of E. coli strain 15 has been studied. The organism requires thymine, uracil and arganine for normal growth and multiplication. The formation of citrulline and ureidosuccinate is blocked almost completely. Nevertheless, significant although depressed amounts of both ornithine and aspartate transcarbamylases are readily detectable in cell-free extracts of the organism. The bacterium can be induced to synthesize xybose isomerase in the presence of exogenous arginine and in the absence of exogenous uracil, as in strain 15T−U−. Relatively extensive peptide synthesis is obtained under these conditions. Analysis of the observed RNA metabolism suggests a turnover of part of the RNA and a greater conservation of the bases than of the ribose. Most of the turnover appears to depend on an exogenous supply of arginine, as does the function of enzyme formation. Reversion to arginine and uracil independence occurs in a single genetic step. Thymine starvation increase the percentage of revertants in the culture before the bacteria die as a result of unbalanced growth.

Topography of the Escherichia coli ribosome: II. Preliminary sequence of 50 s subunit protein attack by trypsin and its correlation with functional activities☆☆☆

Abstract Roughly 50% of the proteins on the intact 50 s ribosomal subunit are attacked by trypsin in sequential fashion, similar to findings made previously with the 30 s subunit. However, the over-all sensitivity of this subunit to trypsin is lower than that found for the 30 s subunit. There is little change in the sedimentation rate of the 50 s particle even at the highest concentration of trypsin used, and very few of the trypsin-generated peptide fragments are spontaneously released from the subunit. With the 30 s subunit the change in sedimentation rate after trypsin attack is somewhat greater and from 50 to 90% of the peptide fragments are released from the trypsin-treated particle. Peptidyl transferase activity and the ability to bind chloramphenicol, both properties of the 50 s subunit, are lost in parallel and at the relatively early stages of trypsin attack. The present findings, when considered along with the previous results of others, suggest the possibility that one of two proteins in electrophoretic band P13 is the peptidyl transferase activity itself or a major contributor to the transferase site.

Unequal contribution to ribosomal assembly of both str alleles in Escherichia coli merodiploids and its relationship to the dominance phenomenon

Abstract Two Escherichia coli strains were constructed which are reciprocal diploids for the str locus and isogenic for this region of the chromosome ( str s /′F str r and str r /′F str 3 ). During exponential growth the steady-state ribosomal pools of both merodiploids are comprised of about 90%, or more, of streptomycin-sensitive ribosomes. Little effect of allele position was found. A similar preponderance of sensitive 30 S subunits over those that are resistant has been found during limited subunit reconstitution when an equimolar mixture of ribosomal proteins from both phenotypes was initially present. The results indicate that the rates of 30 S subunit assembly of both phenotypes are different, and that the sensitive sub-units predominate over the resistant subunits, suggesting that the difference in biosynthetic rate may be the basis for the dominance of this phenotype in vivo . An explanation for some aspects of the physiology of str diploids have been suggested in terms of these findings.

Alanine or pyruvate is required for the development of myotubes from myoblasts and cortisol satisfies this requirement

Development of myotubes from chick embryo breast muscle myoblasts has been shown to occur in the combined presence of insulin, and both the low and high molecular weight components (LMW, HMW) of chick embryo extract. We report here that alanine, or pyruvate, will replace the LMW component with serine enhancing this effect. In addition, extracts of embryonic kidneys were the most active of five embryonic tissues tested in replacing the LMW component, which led to the finding that cortisol is active in promoting both the development of myotubes and the expression of creatine phosphokinase activity, a muscle-specific indicator of myogenesis. Since it was observed that myoblasts are highly glycolytic, it may be that pyruvate is limiting, suggesting that some aspect of oxidative metabolism is also limiting in these cells.