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Biomedical subjects

A Parmeggiani

Publications and source records attributed to A Parmeggiani.

At least 145 records · Page 8Linked to original sources
PubMed1977

Stabilization by the 30S ribosomal subunit of the interaction of 50S subunits with elongation factor G and guanine nucleotide.

The role of the 30S ribosomal subunit in the formation of the complex ribosome-guanine nucleotide-elongation factor G (EF-G) has been examined in a great variety of experimental conditions. Our results show that at a large molar excess of EF-G or high concentrations of GTP or GDP, 50S ribosomal subunits are as active alone as with 30S subunits in the formation of the complex, while at lower concentrations of nucleotide or lower amounts of EF-G, addition of the 30S subunit stimulates greatly the reaction. The presence of the 30S ribosomal subunit can also moderate the inhibition of the 50S subunit activity that occurs by increasing moderately the concentrations of K+ and NH4+, and extends upward the concentration range of these monovalent cations in which complex formation is at maximum. The Mg2+ requirement for complex formation with the 50S subunit appears to be slightly less than that needed for association of the 30S and 50S ribosomal subunits. Measurement of the reaction rate constants of the complex formation shows that the 30S ribosomal subunit has only little effect on the initial association of EF-G and guanine nucleotide with the 50S subunit; but once this complex is formed, the 30S subunit increases its stability from 10- to 18-fold. It is concluded that stabilization of the interaction between EF-G and ribosome is a major function of the 30S subunit in the ribosome-EF-G GTPase reaction.

Ammonia↗
PubMed1977

Elongation factor Tu resistant to kirromycin in an Escherichia coli mutant altered in both tuf genes.

A mutant of Escherichia coli is described that displays kirromycin resistance in a cell-free system by virtue of an altered elongation factor Tu (EF-Tu). In poly(U)-directed poly(Phe) synthesis the kirromycin resistance of the crystallized enzyme ranged between a factor of 80 and 700, depending on temperature. Similarly, kirromycin-induced EF-Tu GTPase activity uncoupled from ribosomes and aminoacyl-tRNA required correspondingly higher concentrations of the antibiotic. Resistance of EF-Tu to kirromycin is a consequence of a modified enzyme structure as indicated by its altered fingerprint pattern.P1 transduction experiments showed that the kirromycin-resistant EF-Tu is coded by an altered tufB gene (tufB1). The known existence of two genes coding for EF-Tu would interfere with the recognition of a mutant altered in only one of those genes, if the mutation were recessive. Because kirromycin blocks EF-Tu release from the ribosome, kirromycin sensitivity is dominant, as shown by the failure of a mixed EF-Tu population to express resistance in vitro. Therefore, phenotypic expression of kirromycin resistance in vivo appears to be only possible if the EF-Tu mutant lacks an active tufA gene, a property likely to be inherited from the parental D22 strain. The observations that introduction of a tufA(+) region makes the resistant strain sensitive to the antibiotic and that transduction of tufB1 into a recipient other than E. coli D22 yields kirromycin-sensitive progeny support these conclusions.

Anti-Bacterial Agents↗
PubMed1976

Activity of the 30-S CsCl core in elongation-factor-dependent GTP hydrolysis.

The activity of a 30-S CsCl core lacking proteins S1, S2, S3, S5, S9, S10, S14, S20 and S21 has been studied in the ribosome-dependent FTPase reactions in the presence of the 50-S subunit with and without methanol. Without methanol, the 30-S CsCl core was unable to sustain GTPase activity dependent on elongation factor G (EF-G), while it was only slightly active in the presence of elongation factor T (EF-T). With EF-T, addition of methanol induced in the presence of either 30-S subunits or 30-S CsCl cores an activity which was more than 10-fold higher than that observed with the 30-S subunit in the absence of methanol. Methanol lowered the Mg2+ optimum of the EF-T-dependent GTPase reaction from approximately 20 mM to approximately 10 mM. In the absence of methanol the EF-G-dependent (GTPase reaction at low concentration of monovalent cations depends on the 50-S subunit alone (30-S-uncoupled EF-G GTPase). Addition of the intact 30-S subunit but not of its CsCl core abolished inhibition of the 30-S-uncoupled EF-G-GTPase by NH4+. The 30-S CsCl core caused the same effect as the 30-S subunit when methanol was present. 30-S-uncoupled EF-G GTPase activity was lower than the GTPase activity dependent on 30-S plus 50-S subunits at [EF-G]/[50-S] below 5 but was considerably higher in the presence of a large excess of EF-G. In the presence of methanol the 30-S CsCl core behaved similarly to the 30-S subunit. Our results indicate that the action of the 30-S subunit in elongation-factor-dependent GTPases is supported by structural features that are preserved in the 30-S CsCl core. The 30-S split proteins are therefore not essential for EF-G and EF-T activities in the hydrolysis of GTP. With EF-T, in all conditions tested association of the ribosomal subunits appeared to accompany GTPase activity. Association seems also to be a prerequisite of the EF-G GTPase activity that depends on both ribosomal subunits.

Ammonia↗
PubMed1975

Synthesis of guanosine 5'-di- and -triphosphate derivatives with modified terminal phosphates: effect on ribosome-elongation factor G-dependent reactions.

A series of GTP and GDP analogues modified in the terminal phosphate has been synthesized and their activities were investigated in elongation factor G dependent reactions. All of the analogues, with the exception of guanosine 5'-O-(3-thiotriphosphate), were not hydrolyzed by EF-G and ribosomes, but were competitive inhibitors of the ribosome-dependent EF-G GTPase. The most active inhibitors were P3-fluoro P1-5'-guanosine triphosphate and P3-methyl P1-5'-guanosine triphosphate with a Ki of 1.0 X 10(-6) and 2.5 X 10(-6) M, respectively. The activity of the GTP alkyl ester derivatives decreased with increasing number of carbon atoms in the side chain. GTP analogues were much more effective inhibitors than the corresponding GDP derivatives. This points out the necessity of the presence of at least three negative charges in the phosphate chain of the nucleotide for an effective interaction with the active site of the ribosomal EF-G GTPase. Guanosine 5'-O-(3-thiotriphosphate), which was hydrolyzed at one-third the rate of GTP, was able to support poly(U)-directed poly(phenylalanine) polymerization. Possible mechanisms of ribosome-EF-G GTP hydrolysis that arise from our results are discussed. Activity of the nucleotide analogues in EF-G-ribosome complex formation compared well with their ability to inhibit ribosome-dependent EF-G GTPase, P3-fluoro P1-5'-guanosine triphosphate and P3-methyl P1-5'-guanosine triphosphate being again the most effective ones. The stabilizing action of fusidic acid on the EF-G-ribosome complex formation induced by the various nucleotides could not be correlated to any of the structural modifications of the substrate. Guanylyl methylene diphosphonate was displaced more readily than GDP from the EF-G-ribosome complex by GTP analogues insensitive to fusidic acid.

Escherichia coli↗
PubMed1975

Function of sulfhydryl groups in ribosome-elongation factor G reactions. Assignment of guanine nucleotide binding site to elongation factor G.

Titration of elongation factor G (EF-G) with the thiol reagents 5,5'-dithiobis(2-nitrobenzoate) (DNTB), p-hydroxymercuribenzoate (HMB), and N-ethylmaleimide and analysis of cysteic acid after performic acid oxidation revealed a total of four sulfhydryl groups per EF-G molecule. One of these is exposed in the native state and could be used to distinguish between two different conformations of EF-G in our preparations according to its rate of reaction with DTNB and HMB. No evidence for disulfide bridges was obtained. Among the different nucleotides tested, GTP, GDP, and GMP were able to protect the native sulfhydryl group against reaction with DTNB in the absence of ribosomes. Their Kd values with the faster reacting EF-G were 3.4 x 10(-4) M, 0.3 X 10(-4)M, and 2.0 x 10(-4) M, respectively. Because of the specificity of protection by guanine nucleotides and the correspondence of the Kd values with Ki values for GDP and GMP in the ribosome-EF-G GTPase reaction, their binding site on EF-G should be closely related to the active center for ribosome-dependent GTP hydrolysis. Blockage of the native sulfhydryl group of EF-G with a variety of irreversible thiol reagents reduced its activity from one to two-thirds in ribosome-dependent complex formation, GTP hydrolysis, and poly(U)-directed poly(phenylalanine) synthesis. A test of the N-ethylmaleimide-treated EF-G showed both the Km and Vmax of the GTPase reaction to be affected. Thus, the native sulfhydryl group, although important, appears not to be located in the GTPase active center. Denaturation of EF-G with guanidine-HCl and random blockage of any of the three masked sulfhydryl groups caused inactivation, likely due to steric interference with proper chain folding upon renaturation. Treatment of ribosomes or ribosomal subunits with six different thiol reagents at a concentration of 0.27 mM had little or no effect on the ribosome-EF-G GTPase, except for the case with HMB which inactivated the 30 S subunit. An interaction of EF-G with the 30 S subunit in addition to that known to occur with the 50 S subunit is suggested by a rapid and preferential exchange of HMB from the native sulfhydryl group of EF-G to the 30 S subunit of 70 S ribosomes.

Binding Sites↗
PubMed1975

A comparative study of the 50S ribosomal subunit and several 50S subparticles in EF-T-and EF-G-dependent activities.

A series of ribosomal subparticles derived from the 50S subunit has been studied and compared in EF-T- and EF-G-dependent reactions. Three different 50S cores were prepared by CsC1 isophycnic centrifugation and one by NH(4)Cl-ethanol extractionm the 50S CsCl core a had lost proteins L1, L7, L8, L10, L12, L16, L25, L33, and some L6 and L11. The 50S CsCl core b additionally lacked protein L6, and 50S CsCl core c also lacked protein L5, L15, L18, L27, L28, L30, and most of L9, L14, L19, and L21. The 50S NH(4)Cl-ethanol core had lost up to 90 percent of proteins L7, L12 and 30-60 percent of proteins L8, L10, and L29. The 50S CsCl core a had much reduced activity in EF-G and none in EF-T GTPase reactions while 50S CsCl cores b and c were inactive. Addition of proteins L7, L12 restored the activity for both the EF-T- and EF-G-dependent GTPase with all of the three 50S CsCl cores, increasing stepwise from core c to core a; The 50S NH(4)Cl-ethanol core was partially active in the EF-G GTPase over the 2-30 mM MG-2+ range tested, while EF-T only showed some activity inthe upper portion of this range...

Bacterial Proteins↗
PubMed1974

Kirromycin, an inhibitor of protein biosynthesis that acts on elongation factor Tu.

Kirromycin, a new inhibitor of protein synthesis, is shown to interfere with the peptide transfer reaction by acting on elongation factor Tu (EF-Tu). All the reactions associated with this elongation factor are affected. Formation of the EF-Tu.GTP complex is strongly stimulated. Peptide bond formation is prevented only when Phe-tRNA(Phe) is bound enzymatically to ribosomes, presumably because GTP hydrolysis associated with enzymatic binding of Phe-tRNA(Phe) is not followed by release of EF-Tu.GDP from the ribosome. This antibiotic also enables EF-Tu to catalyze the binding of Phe-tRNA(Phe) to the poly(U).ribosome complex even in the absence of GTP. EF-Tu activity in the GTPase reaction is dramatically affected by kirromycin: GTP hydrolysis, which normally requires ribosomes and aminoacyl-tRNA, takes place with the elongation factor alone. This GTPase shows the same K(m) for GTP as the one dependent on Phe-tRNA(Phe) and ribosomes in the absence of the antibiotic. Ribosomes and Phe-tRNA(Phe), but not tRNA(Phe) or Ac-Phe-tRNA(Phe), stimulate the kirromycin-induced EF-Tu GTPase. These results indicate that the catalytic center of EF-Tu GTPase that is dependent upon aminoacyl-tRNA and ribosomes is primarily located on the elongation factor. In conclusion, kirromycin can substitute for GTP, aminoacyl-tRNA, or ribosomes in various reactions involving EF-Tu, apparently by affecting the allosteric controls between the sites on the EF-Tu molecule interacting with these components.

Anti-Bacterial Agents↗
PubMed1973

Requirement of proteins S5 and S9 from 30S subunits for the ribosome-dependent GTPase activity of elongation factor G.

During CsCl isopycnic centrifugation at 20 mM Mg(++), Escherichia coli 30S ribosomal subunits specifically lose proteins S1, S2, S3, S5, S9, S10, and S14. The resultant 30S core is unable to stimulate the GTPase activity of EF-G in the presence of 50S subunits. Activity could be restored to a small extent by adding back S2, S5, or S9. However, when S5 and S9 were added together, they cooperatively produced 30S particles 1.5 times more active than the original native 30S subunits. The small amount of activity restored by S2 was simply additive to that restored by S5 or S9. None of the other split proteins showed any restoring capability. Ability of the various protein-deficient 30S particles to couple with 50S subunits corresponded closely to their activity in the EF-G GTPase reaction. It is concluded that S5 and S9 together enable the 30S subunit to participate in the formation of a GTPase-active 30S-50S-EF-G complex.

Centrifugation, Density Gradient↗