Scientific Report 2006

Molecular Biology

Assembly Landscape of the 30S Ribosome

J.R. Williamson, F. Agnelli, A. Beck, C. Beuck, A. Bunner, A. Carmel, J. Chao, S. Edgcomb, M. Hennig, E. Johnson, D. Kerkow, E. Kompfner, S. Kwan, P. Mikulecky, W. Ridgeway, H. Schultheisz, L.G. Scott, E. Sperling, B. Szymczyna

The ribosome is a large molecular machine that is responsible for synthesis of all proteins in the cell. It is composed of 2 multicomponent subunits that bind mRNA, tRNAs, and other factors to carry out translation of the genetic code from RNA into protein product. In bacteria, the large, or 50S, subunit is responsible for catalyzing the formation of peptide bonds, whereas the small, or 30S, subunit is responsible for reading out the genetic code. An elaborate process exists for biogenesis of the ribosome machinery in cells to assemble the ribosome from individual components. We using a wide variety of biophysical techniques to study the mechanism of assembly of the 30S ribosome in vitro.

The 30S ribosome can be reconstituted from purified components in vitro with extremely high efficiency, a characteristic that has enabled detailed mechanistic studies. The 30S ribosome is composed of a single RNA chain of approximately 1500 nucleotides and 20 small proteins of 8–20 kD. Pioneering work by Nomura more than 30 years ago led to the development of an assembly map that outlines the basic order of protein binding. However, the mechanistic basis for these early observations was unknown. Using nuclear magnetic resonance, x-ray crystallography, calorimetry, and fluorescence methods, we have studied the details of assembly of small RNA-protein complexes derived from the 30S subunit to elucidate these molecular events.

A guiding principle for ribosome assembly is that each protein recognizes a small local region of the RNA as its binding site. The protein cannot bind until the RNA structure in its binding site is properly folded. We showed that the assembly reaction can be considered an alternating series of RNA conformational changes and protein binding events (Fig. 1). RNA helices must be properly arranged to create the binding site for the first protein, which is protein S15 in Figure 1. Binding of S15 effectively consolidates the gains from RNA folding in the previous step. Furthermore, after S15 binding, the next RNA conformational change is facilitated; this change sets up the binding site for the next proteins, which are S6 and S18 in Figure 1.

Fig. 1. A model for ribosome assembly. The RNA chain is shown as cylinders representing helical regions of RNA structure. In the first step, the RNA changes conformation, which creates a protein-binding site for the protein S15. Next, a subsequent RNA folding event occurs, which in turn creates a binding site for the proteins S6 and S18. Assembly appears to proceed as an alternating series of folding and binding events.

Thus, each protein serves as a local reporter for RNA folding in a specific region of the 30S subunit. The overall assembly reaction can be schematically illustrated as shown in Figure 2, where an unfolded RNA chain is combined with 20 different proteins, a change that after a complex series of RNA conformational changes and protein-binding events results in the structured 30S subunit. Our previous analyses involved fragments of the overall structure, and we were interested in monitoring the kinetics of binding and assembly of the intact 30S subunits.

Fig. 2. The 30S ribosome assembly reaction. The RNA chain is represented as a thin line that is disordered at the beginning of the experiment. The 20 small proteins that bind to the RNA are represented as circles. The final assembled subunit is composed of highly folded RNA with each protein bound at a specific location.

Monitoring the simultaneous binding of 20 different proteins to an RNA molecule is a serious technical challenge. To surmount this challenge, we developed an isotope-pulse chase assay in which mass spectrometry is used to indicate binding of the proteins to the RNA. Assembly is initiated by using a pulse of a mixture of the 20 ¹⁵N-labeled proteins; after a short assembly time, a mixture of ¹⁴N-proteins is added as the chase. The fully assembled subunits are isolated, and the fraction of ¹⁵N for each protein is measured as a function of the pulse time by using quantitative mass spectrometry. In this way, the time course of binding for each protein can be measured. The strength of the method is that the binding rates can all be measured simultaneously.

Using this method, we can perform mechanistic experiments on 30S ribosome assembly by using the kinetic tools of physical chemistry. We have varied the protein concentration to show that the binding rates correspond to a bimolecular association, not to a rate-limiting RNA conformational change. We have varied the magnesium ion concentration to show that ions can play 2 opposing roles during assembly. Some parts of the 30S subunit speed up at lower magnesium concentrations, and different parts slow down. Perhaps most important, we have measured the rates as a function of temperature and performed Arrhenius analysis of the activation energies for binding.

The main conclusion from these studies is that 30S assembly has no single global rate-limiting step. Rather, a number of parallel pathways exist by which the ribosome can assemble. In addition to the functional restrictions placed on the sequence of the RNA, most likely the sequence is also selected under evolutionary pressure to fold efficiently under a variety of conditions encountered by bacteria in the environment.

Publications

Chao, J.A.., Lee, J.H., Chapados, B.R., Debler, E.W., Schneemann, A., Williamson, J.R. Dual modes of RNA-silencing suppression by Flock House virus protein B2. Nat. Struct. Mol. Biol. 12:952, 2005.

Davis, J.H., Tonelli, M., Scott, L.G., Jaeger, L., Williamson, J.R., Butcher, S.E. RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex [published correction appears in J. Mol. Biol. 760:742, 2006]. J. Mol. Biol. 351:371, 2005.

Hennig, M., Munzarova, M.L., Bermel, W., Scott, L.G., Sklenar, V., Williamson, J.R. Measurement of long-range ¹H-¹⁹F scalar coupling constants and their glycosidic torsion dependence in 5-fluoropyrimidine-substituted RNA. J. Am. Chem. Soc. 128:5851, 2006.

Scott, L.G., Williamson, J.R. The binding interface between Bacillus stearothermophilus ribosomal protein S15 and its 5′-translational operator mRNA. J. Mol. Biol. 351:280, 2005.

Talkington, M.T., Siuzdak, G., Williamson, J.R. An assembly landscape for the 30S ribosomal subunit. Nature 438:628, 2005.