[A modified method of isolation of "tight" 70S ribosomes from Escherichia coli highly active at different stages of the elongation cycle]. / Modifitsirovannyi sposob polucheniia "tight" 70S ribosom iz Escherichia coli, vysokoaktivnykh v otdel'nykh stadiiakh tsikla élongatsii.

The functional activity of the wide-spread "tight" 70S ribosomes is usually equal to 55-80%. We show here that the inactive fraction of this type of ribosomes is virtually blocked by residual endogenous RNA's. These RNA's are shown to be removable by introducing an additional stage in the isolation procedure including: 1. short heating (15 min, 37 degrees C) of "tight" 70S under dissociation conditions, i. e. in a buffer containing 3 mM MgCl2 and 200 mM NH4Cl; 2. washing off endogenous RNA's on a sucrose density gradient in the same buffer; 3. final selection of purified "tight" 70S on the sucrose gradient containing 5 mM MgCl2 and 50 mM NH4Cl. "Tight" 70S ribosomes isolated by such a procedure are 90-100% active with respect to tRNA binding (including the factor-dependent one), peptide bond synthesis and translocation.

Correlation between biological activity of 30 S subunits of Escherichia coli ribosomes and their conformation changes revealed by optical mixing spectroscopy.

Spectral analysis of light scattered from solutions of 30 S subunits was performed by the method of regularization of the inverse spectral problem. The subunits observed under ionic conditions which preserved their biological activity (200 mM NH4Cl at 1 mM MgCl2) revealed a monodisperse pattern of scattering with diffusion constant D = (1.83 +/- 0.10) X 10(-7) cm2/s. The polydispersity and compaction of 30 S subunits were observed under inactivation ionic conditions (30 mM NH4Cl at 1 mM MgCl2). The number of compacted particles correlates with the irreversible loss of biological activity, the ability of 30 S subunits to bind specific tRNA.

[Correlation between the biological activity of 30S subunits of Escherichia coli ribosomes and their conformational changes revealed by mixing optical spectroscopy]. / Korreliatsiia mezhdu konformatsionnym izmenenniem, nabliudaemym metodom spektroskopii opticheskogo smesheniia, i biologicheskoi aktivnost'iu 30S subchastits ribosom Escherichia coli.

The analysis of light spectra scattered from solution of 30S subunits were performed by the method of regularization of inverse spectral problem. The subunits observed at ionic conditions which preserved their biological activity (200 mM NH4Cl at 1 mM MgCl2) revealed a monodisperse pattern of scattering with diffusion constant D = (1,83 +/- 0.10) . 10(-7) cm2/sec. The polydispersity and compactization of 30S subunits was observed in inactivating ionic conditions (30 mM NH4Cl at 1 mM MgCl2). The number of compactized particles correlates with the irreversible loss of biological activity, the capability of 30S subunits to bind specific tRNA.

[Nature of the heterogeneity of the 30S ribosomal subunits in vitro. II. Two types of inactivation of the 30S subunits of Escherichia coli ribosomes]. / Priroda geterogennosti 30S ribosomnykh subchastits in vitro II. DVA tipa inaktivatsii 30S subchastits ribosom Escherichia coli.

The influence of concentration of monovalent cations on the binding constant of Phe-tRNAPhe to 30S.poly(U) complex was studied. Two types of inactivation of the 30S subunits by ammonium ions at the low magnesium concentration (1 mM) were found. The first type of inactivation was observed at high concentrations of NH4+ ions (from 0.5 to 1.5 M) and due to the dissociation of ribosomal proteins from 30S subunits. This inactivation only decreased the binding constant of Phe-tRNAPhe to 30S.poly(U) complex up to 50 times but all 30S subunits were equally achieved in Phe-tRNAPhe binding. This type of inactivation was reversible, addition of S-proteins restored the association constant to the original value. At low concentration of NH4+ ions (below 100 mM) about half of the 30S subunits is irreversibly inactivated (the binding constant of Phe-tRNAPhe decreased below detectable level) probably as a result of conformational changes in ribosomal RNA. Both types of inactivation of the 30S subunits can take place during the preparation of isolated subunits of ribosomes.

Separation of ribosomal subunits of Escherichia coli by Sepharose chromatography using reverse salt gradient.

A mixture of 30 S and 50 S subunits quantitatively absorbs on a column of Sepharose--4B from the buffer: 0.02 M Tris--HCl, pH 7.5, containing 1.5 M (NH4)2SO4. During elution by reverse gradient of ammonium sulphate (1.5--0.05 M) the subunits are eluted at different salt concentrations. Complete separation of subunits is attained in the absence of Mg2+ ions. The 30 S subunits prepared from 70 S ribosomes according to this procedure are fully active in the codon--dependent binding of a specific aminoacyl--tRNA. After their reassociation with 50 S subunits isolated by zonal centrifugation, the resulting 70 S ribosomes are active in polypeptide synthesis at the same degree as control 70 S ribosomes in which both types of subunits were prepared by zonal centrifugation. The initial 70 S ribosomes for the chromatographic separation into subunits can be obtained by their pelleting from a crude extract with subsequent washing with concentrated solutions of NH4Cl in the ultracentrifuge, or by salt fractionation of the crude extract according to a slightly modified procedure of Kurland.

[Nature of the heterogeneity of 30S ribosomal subparticles in vitro. I. Effect of large centrifugal fields during 30S subparticle isolation on their capacity for codon-dependent tRNA binding]. / Priroda geterogennosti 30S ribosomnykh subchastits in vitro. I. Vliianie bol'shikh tsentrobezhnykh polei pri vydelenii 30S subchastits na ikh sposobnost' k kodonzavisimomu sviazyvaniiu tRNK.

Measurements of association constants (Ka) of specific [14C]Phe-tRNAPhe with a 30S..poly(U) complex revealed that values of these constants vary from 0.5.10(7) up to 1.5.10(8) M--1 when different 30S subunit preparations were used at the same medium conditions (20 mM Mg2+, 200 mM NH4, 0 degrees C). Analysis of these data showed that the higher the rotor speeds were used during separation of 70S ribosomes into subunits, the less Ka values were measured. In special experiments on sedimentation of pure 30S subunits at different rotor speeds it was found that the decrease of Ka values was caused due to the additional reversible dissocation of ribosomal proteins from 30S subunits at high (the order of 100 000.g) centrifugal fields. As a possible mechanism of such dissociation we suggest the influence of high hydrostatic pressure on the association constants of S-proteins with 30S subunits. Data presented in this paper demonstrate that at least one of the reasons for the physical and functional heterogeneity of 30S subunits in vitro derives from the application of high centrifugal fields during isolation of ribosomal subunits.