Current Research and Projects in the Williamson Lab @ TSRI
Ribosome Assembly in Bacteria
A major focus in our laboratory is the dynamics of assembly of the ribosome, which is the large macromolecular machine responsible for protein synthesis in all cells. The ribosome is ~2.7 MDa, and is composed of three large ribosomal RNA molecules and 55 ribosomal proteins. Ribosome assembly in bacteria occurs co-transcriptionally at highly specialized loci that are sites of this major metabolic activity that accounts for about 1/3 of the energy budget of a rapidly dividing cell.
Key questions about ribosome assembly focus on the structure of assembly intermediates, the role of > 30 asssembly cofactors, the sequence of RNA conformational changes that occurs, and the coordination of binding of the ribosomal proteins. Ribosome assembly is incredibly efficient, requiring only ~2 minutes, which is only somewhat longer than the transcription time for the ribosomal RNA.
We use quantitative mass spectrometry to determine the protein composition of assembly intermediates in conjunction with electron microscopy to determine the structure of those intermediates. In addition, we use fluorescence methods to monitor protein binding and RNA conformational changes during assembly.
Overall, we are working to define the series of events that will define the ribosome assembly pathway in terms of a molecular movie of transcription, RNA folding, RNA processing, protein binding and cofactor assistance that ultimately produces the mature ribosome.
Quantitative Mass Spectrometry of RNA Modifications
Ribosomal RNAs contain numerous modifications, such as base or ribose methylations and pseudouridine, in both bacteria and eukaryotic cells. Bacterial ribosomes contain over 50 modifications, yeast ribosomes contain over 75 modifications, and human ribosomes contain over 100 modifications. We are developing isotope labeling methods that allow the precise quantification of these modified residues in ribosomal RNAs.
Methylations can be conveniently monitored using metabolic deuteration with the labeled precursor D3-methionine, resulting in a +3 mass shift. Pseudouridylation can be monitored using D2-uracil, which gives a +1 mass shift after exchange with water during the isomerization reaction. Using these methods, we can quantify 29 of 35 modifications in E. coli ribosomal RNA.
Economics of the Bacterial Proteome
Extension of our quantitative mass spectrometry methods to study the entire E. coli proteome allows us to precisely quantify protein levels of > 800 proteins that account for ~95% of the proteome by mass. The ribosomal proteins represent a large sector of the proteome, and the abundance of these proteins is strongly linked to the growth rate of the cell, since the production of ribosomes determines the overall protein synthesis capacity for the cell.
In collaboration with the laboratory of Professor Terry Hwa at UCSD, we have developed a quantitative framework that describes the coarse-grained behavior of the proteome as a function of growth rate, in response to different nutrient limitations. Key sectors are “CARUSO”, which respond to Carbon-limitation, Ammonia-limitation, Ribosome-limitation, Unlimited, S-limitated (both C- and A-limitation), and O (unchanged). Remarkably, these sectors respond in a linear way as the growth rate changes, and these linear responses can be characterized by a small number of adjustable parameters. The slopes of the growth rate dependencies correspond to the “cost” of synthesizing the proteins that carry a particular metabolic flux. Thus, the bulk behavior of the proteome can be accurately modeled based on the change price of limiting nutrients in a macroeconomic model.
Biophysical Studies of HIV virus assembly
Using mass spectroscopy (MS), fluorescence microscopy (FM) and electron microscopy (EM) as biophysical tools, we are characterizing the mechanism of HIV capsid protein Gag assembly in cells. In collaboration with the Bruce Torbett laboratory at Seattle Children’s Hospital we have adapted previously described Gag assembly assays to protocols that are appropriate for these techniques. Using these methods, we are identifying and characterizing cellular components involved in Gag assembly, measuring the kinetic assembly of Gag and its accessory cellular component and directly visualizing Gag assembly.