Characterization of functionally active subribosomal particles from Thermus aquaticus

Abstract

Peptidyl transferase activity of Thermus aquaticus ribosomes is resistant to the removal of a significant number of ribosomal proteins by protease digestion, SDS, and phenol extraction. To define the upper limit for the number of macromolecular components required for peptidyl transferase, particles obtained by extraction of T. aquaticus large ribosomal subunits were isolated and their RNA and protein composition was characterized. Active subribosomal particles contained both 23S and 5S rRNA associated with notable amounts of eight ribosomal proteins. N-terminal sequencing of the proteins identified them as L2, L3, L13, L15, L17, L18, L21, and L22. Ribosomal protein L4, which previously was thought to be essential for the reconstitution of particles active in peptide bond formation, was not found. These findings, together with the results of previous reconstitution experiments, reduce the number of possible essential macromolecular components of the peptidyl transferase center to 23S rRNA and ribosomal proteins L2 and L3. Complete removal of ribosomal proteins from T. aquaticus rRNA resulted in loss of tertiary folding of the particles and inactivation of peptidyl transferase. The accessibility of proteins in active subribosomal particles to proteinase hydrolysis was increased significantly after RNase treatment. These results and the observation that 50S ribosomal subunits exhibited much higher resistance to SDS extraction than 30S subunits are compatible with a proposed structural organization of the 50S subunit involving an RNA "cage" surrounding a core of a subset of ribosomal proteins.

Figure 1

Sucrose gradient centrifugation of 50S ribosomal subunits before (A) and after treatment with proteinase K, SDS, and one (B), two (C), or four (D) phenol extractions.

Figure 2

RNA composition of KSP particles. RNA extracted from the gradient peaks was fractionated on a 6% denaturing polyacrylamide gel. Lanes: 1, marker (rRNA from E. coli 70S ribosomes); 2, untreated T. aquaticus 50S subunits; 3, KSP80; 4, KSP50; 5, KSP30.

Figure 3

Sucrose gradient fractionation of ³⁵S-labeled 50S ribosomal subunits (A) and KSP particles (B). Solid circles show optical density (A₂₆₀), and open circles show amount of [³⁵S] radioactivity in gradient fractions.

Figure 4

Peptidyl transferase activity of partially deproteinized large ribosomal subunits. ³⁵S-labeled T. aquaticus 50S subunits were treated with proteinase K in the presence of SDS (proteinase K/SDS) followed by one, two, three, four, or five phenol extractions. Peptidyl transferase activity of the material precipitated from the aqueous phase with ethanol (y axis) was plotted against the amount of remaining proteins (x axis). Activity of the untreated 50S subunits (50S) was taken as 100%.

Figure 5

Two-dimensional electrophoresis of T. aquaticus ribosomal proteins from 50 S subunits (A), KSP50 (B), and KSP80 (C) particles. Proteins present in the KSP particles are marked on total 50S subunit protein gel (A). Correspondence between KSP and 50S subunit proteins was determined by coelectrophoresis of ³⁵S-labeled proteins from KSP50 particles with unlabeled proteins from 50S subunits followed by Coomassie staining of the gel and autoradiography.

Figure 6

N-terminal amino acid sequences of proteins from KSP50 particles and comparison with eubacterial ribosomal proteins. Bsu, Bacillus subtilis; Eco, Escherichia coli; Hin, Hemophilus influencea, Taq, Thermus aquaticus, Tth, Thermus thermophilus.

Figure 7

Effect of RNase treatment on accessibility of proteins in KSP50 particles to proteinase K. SDS-gel electrophoresis of proteins obtained from KSP particles (lane 1); KSP particles treated with proteinase K (lane 2); and KSP particles preincubated with RNase before proteinase K treatment (lane 3).

Figure 8

Extraction of T. aquaticus 30S and 50S ribosomal subunits with SDS. Ribosomal subunits were incubated in the absence (C) or in the presence (SDS) of 0.5% SDS as described in Materials and Methods, purified by sucrose gradient centrifugation, and analyzed by SDS/urea gel electrophoresis. Positions of molecular weight markers are indicated.