Electrophoretic Separation of Escherichia coli Ribosomal Particles on Polyacrylamide Gels

Abstract

Escherichia wli ribosomes display a remarkable heterogeneity when submitted to polyacrylamide-gel electrophoresis (4O/, acrylamide). Both the 70-S ribosomes and 5 0 3 ribosomal subunits are resolved into a t least four and the 30-S ribosomal subunits into three particle subclasses.After reversal of the current, all particles refocus into one single band a t the origin. Ribosomes recovered from the gel after electrophoresis have retained some 40--100°/, of their capacity to synthesize peptide bonds. Their sedimentation behaviour has remained unaltered. Upon reelectrophoresis, no new peaks are detectable although the relative proportions of the various subclasses are frequently changed. The ratio in the occurrence of these subclasses is readily affected by a variety of factors, however, some of which are not directly related to the electrophoretic technique itself.The three classes of ribosomes and subunits (70-8, 5 0 3 and 30-S) display approximately equal electrophoretic mobilities when the sieving action is reduced to a minimum. This conclusion follows from experiments in which the ribosomes are submitted to electrophoresis in a sucrose gradient or in 0.4O/, agarose gels. I n contrast, electrophoresis in polyacrylamide gels with a gradient in pore size reveals the same ribosomal diversity as noted in gels with a fixed acrylamide concentration of 4O/,. I n these pore-gradient gels (3 -So/, acrylamide), the migration rate of each ribosomal subclass decreases continuously when the time of electrophoresis is prolonged. After about 30 h, further penetration seems to be blocked.All three classes consist of particles with low electrophoretic mobility (designated I particles) and those which move faster (designated I1 particles). The I particles are partially converted to I1 particles by incubation a t 37 "C and/or pelleting prior to elcctrophoresis. The I1 particles are converted to I particles upon raising the ribosome concentration. The electrophoretic profiles displayed by the various 50-S subclasses arc not strongly affected by variations in Mg2+ concentration in the range 0.5-10.0 mM. The electropherograms of 30-8 subunits become more diffuse when the Mg2+ concentration is raised. The possible occurrence of ribosomal dimers and/or conformation changes is discussed.Polyacrylamide-gel electrophoresis has proved to be a power-ful tool for fractionating various kinds of biologically important macromolecules like proteins [1,2], RNAs [3--51 and DNAs [el. Hjerten et al. [7] were the first to describe an electrophoretic separation of Escherichia coli ribosomal subunits in polyacrylamide gels. Later on, Dahlberg et al. [S] used composite gels of agarose and polyacrylamide to characterize bacterial polyribosornes, ribosomes and ribosomal subunits. Agarose can be omitted when only 70-S ribosomes and subunits are to be separated and we have used 40/, polyacrylamide gels to analyse ribosomal mixtures of E . coli after their interaction with the initiation factor IF-3 [9]. Dahlberg et al. [S] already noticed an unanti...

Binding of the Initiation Factor IF‐3 to Escherichia coli Ribosomes and MS2 RNA

“…A disadvantage of the IF-3 assay is that the large amounts of IF-3 which bind to polyacrylamide gels has been described by Talens et al [28,30]. It reveals a remarkable heterogeneity of both 70-53 ribosomes and the two types of subunits.…”

“…The samples applied to the gels contained 0.4pg [36S]IF-3 to which either 15pg 70-S ribosomes (Fig.7A) or 10 pg 30-5 subunits (Fig.7B) were added. The 70-S ribosomes were partly contaminated with 50-S subunits and due to the incubation with IF-3 some 30-5 and 50-5 subunits have arisen by dissociation (Fig.7B). [36S]IF-3 is detectable in the 70-S, 50-S and 30-5 peaks (for a characterization of the multiple ribosomal peaks see Talens et al [30]) but the specific activity of the 30-S particles exceeds that of the other ribosomes by far. From the available IF-3 both 70-5 ribosomes and 50-5 subunits in Materials and Methods.…”

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

“…As stated above for viruses, biological nanoparticles demand large pore gels to enable entering into the matrix and for reasonable separation. Preparative electrophoresis in 4% acrylamide gels provided ribosome particles for the study of their biosynthetic activity [277]. Also structural studies like electron microscopy can be performed with ribosomes eluted from gels [278].…”

Role of 100S ribosomes in bacterial decay period

“…The S10-GFP or S2-GFP proteins in the form of ribosomes and aggregates Polyacrylamide and its composite gels were used to separate various forms of ribosomes (Dahlberg et al 1969;Talens et al 1973). In a 2% agarose gel, the resolution is too low to separate the 70S and 100S forms, and as a result, both forms migrate as a single major band.…”