Lack of stable initiation factor 3 (IF‐3) binding to dimers of the 30 S ribosomal subunits

Gualerzi, Claudio O.; Wabl, Matthias; Pon, Cynthia L.

doi:10.1016/0014-5793(73)80312-6

Cited by 33 publications

(2 citation statements)

References 13 publications

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“…In all photolytic experiments the mixture (0.48 pM ribosomes and 2 pM antibiotic) was incubated for 10 min at 37°C in burfer B, then irradiated for 10 min between 30°C and 37°C in pyrex tubes placed 8 cm away from the light source as previously described [4]. Subunits and RNA separation The separation of ribosomal subunits was performed by analytical centrifugation on 5 -20% sucrose gradients according to Gualerzi [16] in buffer A with 1 mM Mg(OAc), and 10 mM NH4CI. The rRNA analysis was performed by ultracentrifugation on a 5 -20% sucrose gradient in LiCl/ sodium dodecyl sulfate as previously described [4].…”

Section: Photolysis Experimcntsmentioning

confidence: 99%

About the specificity of photoinduced affinity labeling of Escherichia coli ribosomes by dihydrorosaramicin, a macrolide related to erythromycin

Siegrist

Moreau

1985

European Journal of Biochemistry

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Photoactivation of the [3H]dihydrorosaramicin chromophore at a wavelength above 300 nm allows the covalent attachment of the macrolide antibiotic to the bacterial ribosome. Bidimensional electrophoresis shows that the radioactivityisniainly associated with proteins L1, L5, L6, L15, L18, L19, S1, S3, S4, S 5 and S9. When photoincorporation of the drug is conducted in the presence of puromycin as effector of ['H]dihydrorosaramicinbinding sites, a decrease in the labeling of most proteins is observed, except for LIX and Ll9, which are radiolabeled to a larger extent. These results allow us to speculate that L 18 and L 19 belong to the high-affinity binding site of rosaramicin antibiotic.Photoaffinity labeling has become an important method for identifying functional sites on the Escherichia coli ribosome [l] and antibiotics such as puromycin and chloramphenicol have been used as tools for providing information on the peptidyltransferase center [2 -41.Although it has been a quarter century since macrolide antibiotics were reported to block protein synthesis [S] the mechanism by which they accomplish this inhibition has remained unknown. Erythromycin, the major representative of the macrolide group, binds on the large subunit ofprokaryotic ribosomes [6] and, among other assumptions, could be considered as an inhibitor of the peptidyl residue near the peptidyltransferase [7]. In contrast to the large number of studies concerning the mode of action of the macrolides, very little is known about the macromolecules involved in the structure of their binding site.Rosaramicin (Fig. 1) is a 16-membered macrolide, whose activity is similar to that of erythromycin. This antibiotic possesses an aldehyde function, easily reducible to an alcohol group without loss of antibiotic activity; this led to the obtaining of a tritiated molecule, which was used to determine rosaramicin-binding parameters onto E. coli ribosomes, showing one high-affinity binding site (Kd = 0.16 x M) and multiple low-affinity binding sites [8, 91. Moreover, the antibiotic has an a,p-ethylenic y,S-epoxyketo system that offers a promising approach to photoactivated affinity labeling. This method, which eliminated the need for preparing reactive derivatives, has been recently used to label steroid-binding proteins and receptors [lo]. In a preliminary report [Ill we have shown that photoexcitement of ['H]dihydrorosaramicin allows its covalent attachment to E. coli ribosome proteins, mainly on L1, L5, L6 and S1. Recently components of the macrolide-binding site on the ribosome have been identified [ 121 using dihydrocarbomycin under different labeling conditions and showing L27 as the major labeled protein.

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Section: Photolysis Experimcntsmentioning

confidence: 99%

About the specificity of photoinduced affinity labeling of Escherichia coli ribosomes by dihydrorosaramicin, a macrolide related to erythromycin

Siegrist

Moreau

1985

European Journal of Biochemistry

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“…IF-3 was labeled via reductive methylation, using [14C] formaldehyde [New England Nuclear, 44 Ci/mol] and sodium borohydride [6]. Typically, labeling of 3000-5000 cpm/pg was achieved.…”

Section: Methodsmentioning

confidence: 99%