Activities of protein-deficient particles derived from 50-S ribosomal subunits by NH4Cl/ethanol treatment (original) (raw)
Related papers
Cooperative and Antagonistic Interactions of Peptidyl-tRNA and Antibiotics with Bacterial Ribosomes
European Journal of Biochemistry, 1977
There is a single-site interaction of [mrthylene-'4C]thiamphenicol and [methylene-14C]chloramphenicol with run-off ribosomes with dissociation constants Kd = 6.8 pM and Kd = 4.6 yM respectively. Similar affinities for the antibiotics are observed in polysomes totally deprived of nascent peptides, or bearing nascent peptides on the A-site. However, two types of interaction are observed in endogenous polysomes with some ribosomes bearing nascent peptides on the P-site and others in the A-site. The lower-affinity bindings (dissociation constants Kd = 6.4 pM and Kd = 5.9 pM for thiamphenicol and chloramphenicol respectively) are due to the ribosomes bearing nascent peptides on the A-site. The higher-affinity bindings (dissociation constants Kd = 2.3 yM and Kd = 1.5 pM for thiamphenicol and chloramphenicol, respectively) are due to the ribosomes bearing nascent peptides on the P-site. Therefore binding of nascent peptides to the A-site does not affect the affinities of thiamphenicol and chloramphenicol for the ribosome. On the other hand interaction of the nascent peptides with the P-site of the ribosomes increases the affinities of both antibiotics for the ribosome. Thiamphenicol and chloramphenicol are thus good inhibitors of peptide bond formation in ribosomes and polysomes. Their affinities are increased precisely when the peptidyl-tRNA is placed in the P-site preceeding the peptide bond formation step, which is specifically blocked by the antibiotics.
European Journal of Biochemistry, 1977
The turnover of complexes containing elongation factor (EF) G, the ribosome and either G D P plus fusidic acid or guanyl-5'-yl methylene diphosphonate or guanyl-5'-yl imidodiphosphate has been measured by chasing the complexed, radioactive forms of these guanosine nucleotides with either an excess of unlabelled guanosine nucleotide or the antibiotic thiostrepton. With the three different complexes, the decay promoted by thiostrepton was substantially faster than that induced by the guanosine nucleotide and, under both decay conditions, no simple first-order kinetics were observed. Assuming that the dissociation of the complexes proceeds via a guanosinc-nucleotide . EF-G binary complex and a free ribosome, this result suggests that dissociation was reversible and that after it the inactivation of frce ribosomes by thiostrepton was faster than the decay of the newly formed binary complex. Increasing the NH4f concentration from 10 to 180 mM or decreasing the Mg2+ concentration rrom 30 to 10 mM during measurement of turnover accelerated the chase of [3H]GDP from a ['HH]GDP . EF-G . ribosome. fusidic-acid complex. Since these variations did not affect the amount of complex present in solution. they evidently accelerated the reactions leading to formation of the complex. In contrast, increasing the NHZ concentration of the wash buffer used to isolate the complexes on nilrocellulose membranes sharply decreased their recovery.
Functional Roles of 50-S Ribosomal Proteins
European Journal of Biochemistry, 1977
Ribosomal proteins previously inactivated by treatment with fluorescein isothiocyanate have been incorporated into 50-S ribosomal subunits during reconstitution from particles disassembled by 2 M LiCl in the presence of an excess of the modified proteins. The reconstituted particles show alterations in some functional activities resulting from the incorporation of the inactive ribosomal proteins added exogenously. Of the fluorescein-isothiocyanate-treated proteins incorporated, L24 and L25 drastically affect all the activities tested and these proteins possibly play a fundamental role in determining the overall structure of the particle. Proteins L16 and LIO are apparently involved both in the GTP hydrolysis dependent on elongation factor G and in peptidyl transferase activity but the modified protein L11 only affects GTPase activity indirectly and interferes with the ribosome assembly process involving proteins L7 and L12. Protein L1 may be involved with peptidyl transferase activity while proteins L7 and L12, in agreement with many reports in the literature, affect the factor-dependent hydrolysis of GTP.
Acidic Ribosomal Proteins from Eukaryotic Cells. Effect on Ribosomal Functions
European Journal of Biochemistry, 1979
Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles. In the presence of 0.4 M NH4Cl and 50% ethanol only the most acidic proteins from yeast and rat liver ribosomes are released. At 1 M NH4Cl two more non-acidic proteins are lost from the ribosomes. The release of the acidic proteins causes a small inactivation of the polymerizing activity of the particles, additional to that caused by the precipitation itself. The elongation-factor-2-dependent GTP hydrolysis of the ribosomes is, however, more affected by the loss of acidic proteins. These proteins can stimulate the GTPase but not the polymerising activity when added back to the treated particles. Eukaryotic proteins cannot be sustituted for bacterial acidic proteins L7 and L12. We have not detected immunological cross-reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver or between acidic proteins from these eukaryotic ribosomes among themselves.
Modification of the accessibility of ribosomal proteins after
1990
Free- and EF-2-bound 80 S ribosomes, within the high-affinity complex with the non-hydrolysable GTP analog: guanylylmethylenediphosphonate (GuoPP(CH2)P), and the low-affinity complex with GDP, were treated with trypsin under conditions that modified neither their protein synthesis ability nor their sedimentation constant nor the bound EF-2 itself. Proteins extracted from trypsin-digested ribosomes were unambiguously identified using three different two-dimensional gel electrophoresis systems and 5 S RNA release was checked by submitting directly free- and EF-2-bound 80 S ribosomes, incubated with trypsin, to two-dimensional gel electrophoresis. Our results indicate that the binding of (EF-2)-GuoPP[CH2]P to 80 S ribosomes modified the behavior of a cluster of five proteins which were trypsin-resistant within free 80 S ribosomes and trypsin-sensitive within the high-affinity complex (proteins: L3, L10, L13a, L26, L27a). As for the binding of (EF-2)-GDP to 80 S ribosomes, it induced an intermediate conformational change of ribosomes, unshielding only protein L13a and L27a. Quantitative release of free intact 5 S RNA which occurred in the first case but not in the second one, should be related to the trypsinolysis of protein(s) L3 and/or L10 and/or L26. Results were discussed in relation to structural and functional data available on the ribosomal proteins we found to be modified by EF-2 binding.