thiostrepton and alpha-sarcin

Provocative recent reports indicate that the large subunits of either prokaryotic or eukaryotic ribosomes have the capacity to promote refolding of denatured enzymes.. Salt-washed Escherichia coli ribosomes are shown to promote refolding of denatured rhodanese. The ability of the ribosomes to carry out renaturation is a property of the 50S ribosomal subunit, specifically the 23S rRNA. Refolding and release of enzymatically active rhodanese leaves the ribosomes in an inactive state or conformation for subsequent rounds refolding. Inactive ribosomes can be activated by elongation factor G (EF-G) plus GTP or by cleavage of their 23S rRNA by alpha-sarcin. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex.. Large subunits of E. coli ribosomes, specifically 23S rRNA, have the capacity to mediate refolding of denatured rhodanese. Refolding activity is related to the state or conformation of ribosomes that is promoted by EF-G. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex.

Topics: Endoribonucleases; Enzyme Activation; Escherichia coli; Fungal Proteins; Fusidic Acid; Guanosine Triphosphate; Molecular Chaperones; Peptide Elongation Factor G; Peptide Elongation Factors; Protein Folding; Protein Synthesis Inhibitors; Ribosomes; RNA, Ribosomal; Thiostrepton; Thiosulfate Sulfurtransferase

The Escherichia coli ribosomal protein (r-protein) L11 and its binding site on 23 S ribosomal RNA (rRNA) are associated with ribosomal hydrolysis of guanosine 5'-triphosphate (GTP). We have used hydroxyl radical footprinting to map the contacts between L11 and the backbone riboses in 23 S rRNA, and to investigate how this interaction is influenced by other ribosomal components. Complexes were characterized in both naked 23 S rRNA and ribosomes from an E. coli L11-minus strain, before and after reconstitution with L11. The protein protects 17 riboses between positions 1058 and 1085 in the naked 23 S rRNA. Within the ribosome, L11 also interacts with this rRNA region, although the protection effects are subtly different and extend to nucleotide 1098. The pentameric r-protein complex L10.(L12)4 binds to an adjacent site on the rRNA, protecting riboses at positions 1043, 1046 to 1049, 1053 to 1055 and increasing the accessibility of position 1068. The overlap in the positions affected by r-proteins L11 and L10.(L12)4, and the increase in protection between positions 1078 and 1084 when they are bound at the same time, reflect the mutually cooperative nature of their interaction with the rRNA. The data support a model for the tertiary configuration of the rRNA region, in which two stem-loop structures fold so that the loops lie in close proximity, with the main ribose interactions of L11 within the minor groove of one of the stems. The conformation of the rRNA-L11 interaction is modulated by L10.(L12)4 and other proteins within the ribosome. The antibiotics thiostrepton and micrococcin inhibit the catalytic functions of this region by slotting in between the accessible loops and interacting with nucleotides there.

Topics: Base Sequence; Binding Sites; Endoribonucleases; Escherichia coli; Fungal Proteins; GTP Phosphohydrolases; Molecular Sequence Data; Nucleic Acid Conformation; Protein Binding; Ribosomal Protein L10; Ribosomal Proteins; Ribosomes; RNA, Ribosomal, 23S; Thiostrepton

The translocation reaction catalyzed by elongation factor G (EF-G) is inhibited either by alpha-sarcin cleavage of 23S rRNA or by the binding of thiostrepton to the E. coli ribosome. Here we show that the transitory binding of EF-G and GDP to the ribosome inhibited the rate of alpha-sarcin cleavage and that stabilization of this binding with fusidic acid completely prevented alpha-sarcin cleavage. A similar pattern of inhibition was seen upon the binding of elongation factor 2 to the S. cerevisiae ribosome. The irreversible binding of the antibiotic thiostrepton to the E. coli ribosome, on the other hand, decreased the rate of cleavage by alpha-sarcin approximately 2-fold. These results suggest that the alpha-sarcin site is located within the ribosomal domain for EF-G binding and that the conformation of this site is affected by the binding of thiostrepton.

Topics: Aspergillus; Binding Sites; Electrophoresis, Polyacrylamide Gel; Endoribonucleases; Escherichia coli; Fungal Proteins; Peptide Elongation Factor G; Peptide Elongation Factors; Ribosomes; RNA, Ribosomal, 23S; Thiostrepton