Scientific Report 2005

Molecular Biology

Assembly Landscape of the 30S Ribosome

J.R. Williamson, F. Agnelli, A. Beck, A. Bunner, A. Carmel, J. Chao, S. Edgcomb, M. Hennig, E. Johnson, D. Kerkow, E. Kompfner, K. Lehmann, H. Reynolds, W. Ridgeway, S.P. Ryder, L.G. Scott, E. Sperling, B. Szymczyna, M. Trevathan

The 30S ribosome is 1 of 2 subunits of the 70S ribosome, which is responsible for the synthesis of all proteins in bacterial cells. The 30S ribosome is responsible for decoding the mRNA for protein synthesis. It is composed of a large 16S RNA of approximately 1500 nucleotides and 20 small proteins (S2–S21). The biogenesis of ribosomes consumes approximately half of the energy of the cell in bacteria, and about 20% of the mass of a bacterium is composed of ribosomes. Thus, the assembly of ribosomes must be rapid and efficient.

We are using a wide variety of biophysical techniques to study the mechanism of assembly of the 30S ribosome in vitro. We have used nuclear magnetic resonance, x-ray crystallography, isothermal titration calorimetry, single-molecule fluorescence, and transient electric birefringence to probe the details of the mechanism.

Pioneering work by Nomura led to the in vitro assembly map for the 30S ribosome: some proteins bind independently to the 16S rRNA, and some require prior binding of other proteins. Using this map as a framework, we used 30S components from Escherichia coli, Thermus thermophilus, and Aquifex aeolicus to do detailed studies. We have constructed an updated and revised assembly map for the 30S subunit (Fig. 1) that contains all of the currently available information about the assembly pathway.

Fig. 1. The revised assembly map of the 30S subunit. The 16S ribosomal RNA is shown at the top, oriented from 5´ to 3´ direction. Each of the arrows indicates an observed dependency of binding for each ribosomal protein. The primary binding proteins depend solely on interactions with 16S rRNA (top row); the secondary and tertiary binding proteins depend on prior binding of other proteins.

The 30S unit has 3 structural domains, the 5´, central, and 3´, and each of these has one or more primary binding proteins that will bind independently to RNA. This binding is followed by a wave of secondary binding proteins for each domain and a third wave of tertiary binding proteins. Most of the proteins have dependencies solely within their domain; a few of the later binding proteins have interdomain dependencies. The assembly proceeds in a parallel manner, although each domain has a defined hierarchy of binding order.

To probe the kinetics of the assembly of the 30S subunit, we developed a novel assay that allows binding of all 20 ribosomal proteins simultaneously. To achieve this simultaneous binding, we initiate assembly of the 30S subunit by combining 16S rRNA with a mixture of all 20 ribosomal proteins uniformly labeled with the stable isotope nitrogen 15. The isotopic label does not perturb the system, but it does result in a mass change of approximately 150 units for each protein. After assembly proceeds for a brief period, we add an excess of unlabeled ribosomal proteins that contain the natural stable isotope nitrogen 14. We can readily determine the amount of the 2 isotopes for each protein by using mass spectrometry. By measuring this fraction as a function of the assembly time, we can monitor the kinetics of all proteins; we term this assay isotope pulse-chase kinetics.

Using this approach, we did an extensive analysis of the assembly kinetics of the 30S ribosome under a variety of conditions. We systematically varied the concentration of the reaction, the temperature, and the magnesium ion concentration during assembly. Using the temperature dependence of the binding rates, we characterized the activation energy of binding for all of the proteins. We found that the rates of binding are not correlated to the activation energies, and we can monitor many different assembly steps in this complex parallel process.

To combine all of the mechanistic information, we have cast the assembly mechanism in terms of an assembly landscape, which has been recently developed in research on protein folding. The assembly landscape of the 30S subunit (Fig. 2) shows the many possible conformations of 16S rRNA in the horizontal plane, and the energy of those conformations is the height of the surface. The 30S final conformation is located at the lower corner of the landscape, but in the absence of ribosomal proteins, it is not the lowest energy conformation.

Fig. 2. The assembly landscape of the 30S subunit. The conformations of the 16S rRNA are represented in the horizontal plane, and the energy of the conformations is the height of the plane. Folding of parallel pathways is indicated by the arrows. The effects of protein binding are schematically illustrated by the 2 successive changes in the landscape. After protein binding (circles), new downhill folding directions are created. All parallel pathways converge on the native 30S conformation at the bottom corner of the landscape.

The assembly proceeds in many parallel directions, heading downhill on the landscape, and the energy of the RNA is lowered by RNA-folding reactions that create more RNA structure. RNA folding creates the binding sites for the ribosomal proteins, which can then bind, and this binding has an important consequence: new downhill directions are created for more RNA folding. The assembly reaction proceeds by a series of alternating RNA conformational changes and protein-binding events that eventually result in the complete assembly of the 30S subunit by the convergence of many parallel pathways.

Publications

Chao, J.A., Williamson, J.R. Joint x-ray and NMR refinement of the yeast L30e-mRNA complex. Structure (Camb.) 12:1165, 2004.

Klostermeier, D., Sears, P., Wong, C.-H., Millar, D.P., Williamson, J.R. A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly. Nucleic Acids Res. 32:2707, 2004.

Lehmann-Blount, K.A., Williamson, J.R. Shape-specific recognition of single-stranded RNA by the GLD-1 STAR domain. J. Mol. Biol. 346:91, 2005.

Recht, M.I., Williamson, J.R. RNA tertiary structure and cooperative assembly of a large ribonucleoprotein complex. J. Mol. Biol. 344:395, 2004.

Ryder, S.P., Williamson, J.R. Specificity of the STAR/GSG domain protein Qk1: implications for the regulation of myelination. RNA 10:1449, 2004.

Scott, L.G., Geierstanger, B.H., Williamson, J.R., Hennig, M. Enzymatic synthesis and ¹⁹F-NMR studies of 2-fluoroadenine substituted RNA. J. Am. Chem. Soc. 26:11776, 2004.

Torres, F.E., Kuhn, P., De Bruyker, D., Bell, A.G., Wolkin, M.V., Peeters, E., Williamson, J.R., Anderson, G.B., Schmitz, G.P., Recht, M.I., Schweizer, S., Scott, L.G., Ho, J.H., Elrod, S.A., Schultz, P.G., Lerner, R.A., Bruce, R.H. Enthalpy arrays. Proc. Natl. Acad. Sci. U. S. A. 101:9517, 2004.