Molecular Morphology of Ribosomes

Abstract

Using either soluble or solid-state lactoperoxidase, a comparison was made between the enzymic iodination of ribosomal proteins iodinated as 3 0 4 and 50-S subunits or as 7 0 4 monosomes. Proteins S7, S11 and S12 of the 30-S subunit and proteins L2, L11, L26 and L28 of the 50-S subunit were labelled to a greater extent in isolated particles than in the 70-S ribosome. In contrast, proteins S4, S6, S19 and S20 were labelled to a lesser extent in the isolated subunit. No significant differences were observed in the iodination patterns of ribosomes iodinated in the presence of soluble lactoperoxidase and those iodinated in the presence of lactoperoxidase bound to Sepharose 4B. It is suggested that the 30-S subunit undergoes a conformational change during its association with the 50-S subunit to form a 70-S monosome. Implications from results obtained with solid-state lactoperoxidase-catalyzed iodination of ribosomal proteins are also discussed.Enzymic iodination of ribosomal proteins has been employed as a probe for the identification of surface proteins and detection of conformational changes in ribosome structure [l -41. Although a greater majority of 30-S proteins are extensively modified as compared to those of the 5 0 4 subunit, these studies reveal that essentially all of the 30-S and 50-S proteins become labelled, to some extent, with iodide. To eliminate the possibility that the accessibility of the individual ribosomal proteins to iodination is related to the ability of the lactoperoxidase to penetrate the structure of the ribosome, use was made of lactoperoxidase immobilized by covalent coupling to Sepharose 4B. Employing both soluble and immobilized lactoperoxidase, we have compared the ability of these two preparations to iodinate ribosomal proteins of isolated 3 0 3 and 5 0 4 subunits as well as subunit proteins derived from iodinated 70-S monosomes. Differences in iodination patterns between ribosomal proteins derived from isolated subunits and from 70-S ribosomes are also discussed with respect to structural alterations. MATERIALS AND METHODSBacterial strains, ribosome preparations, soluble lactoperoxidase-catalyzed iodinations and extraction of ribosomal proteins were as previously described [I, 51. 70-S monosomes were maintained at 10 mM Mg + throughout these experiments. Preparation of Solid-state LactoperoxidaseSolid-state lactoperoxidase was prepared according to the method described by David [6]. Sepharose 4B (5.0ml packed volume) was mixed with an equal volume of deionized water and activated with 2.5 g of cyanogen bromide; the reaction mixture, maintained at 20 "C, was immediately brought to pH 11 .O by the addition of 2 M NaOH. This pH was maintained by the further addition of 2 M NaOH until completion of the reaction as indicated by stabilization of pH. The temperature then was lowered to 4°C and the reaction mixture transferred to a Buchner funnel and washed under suction with phosphatebuffered saline. Washed Sepharose beads were then combined with purified lactoperoxidase [8] at a concentr...

Streptomycin‐ and viomycin‐induced conformational changes of ribosomes detected by iodination

“…In spite of this difference, the distribution of 125 1 among the ribosomal proteins (table 1) was reproducible in 7 different experiments performed with 2 independent preparations of ribosomes. However, it differs from iodination patterns reported [9,10,16], probably due to our different experimental conditions. Streptomycin and viomycin did not appreciably affect the total incorporation of 125 I by 70 S ribosomes (not shown).…”

Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

“…P and T correspond to the puromycin and thiostrepton binding sites, respectively [see Wittmann (1983)], oleophilic reagents, make us fairly confident of the specificity of SI8 labeling. Another point in favor of this specificity is the fact that SI8 is considerably less labeled in the 70S ribosome than in the small subunit, since the labeling of SI8 by unspecific reagents does not seem to be affected by subunit association (Michalski & Sells, 1975).…”

Identification of components of the streptomycin-binding center ofE. coli MRE 600 ribosomes by photo-affinity labelling

“…It has been reported that upon formation of the 70S ribosome a conformational change of the subparticles can take place (see, e.g. Michalski and Sells, 1975 ;Litman et al, 1974;Chang, 1973 ;etc.). However, in our opinion it seems unlikely that this conformational change could significantly influence the effectivity of subparticle labelling because we used a chemically non-specific reagent.…”