Protein synthesis in bacteria
- X-ray Crystal Structures of the E. coli ribosome
- Impact of molecular crowding on translation
- Cellular conditions and protein synthesis: Ribosome dynamics
Regulation of protein synthesis in eukaryotes
- Viral regulation of translation initiation
- Apoptosis and translation regulation
- Role of micro-RNAs in translation regulation
My lab is exploring the molecular basis for protein biosynthesis, a cellular process that is universally conserved. The ribosome, an RNA and protein factory common to all organisms, is responsible for protein synthesis and plays a key role in the conversion of genotype into phenotype in all forms of life. It translates the genetic code in messenger RNA into the corresponding protein chain. Understanding how the ribosome carries out protein synthesis remains a major challenge in molecular biology. In order to determine the mechanisms underlying protein synthesis and its regulation, my lab uses a combination of x-ray crystallography and biochemical assays to probe ribosome function. We are also developing new technologies that will greatly improve our ability to test the dynamics and energetics of translation in conditions closer to the physiology of the cell.
Protein synthesis in bacteria
Since core ribosomal functions are highly conserved, one of the long-term objectives of our research is to better understand the structure and function of the ribosome in the simplest free-living organisms, namely bacteria. An additional motivation for looking at protein synthesis in bacteria lies in the fact that bacterial ribosomes are a prime target for scores of antibiotics. Ribosomes from the bacterium Escherichia coli have been studied genetically, biochemically, and structurally for over four decades. We are therefore using this model organism to examine the central aspects of translation that are likely to be general to all forms of life.
X-ray Crystal Structures of the E. coli ribosome
We are using x-ray crystallography to probe the structural basis for the many aspects of protein biosynthesis that require the intact ribosome. Atomic-resolution structures of the small and large ribosomal subunits have provided an unprecedented glimpse into the inner workings of the ribosome. However, a fully functional ribosome requires both of the subunits to work together as an intact particle. Our goal is to make an atomic-resolution "movie" of a ribosome in the process of making a protein. X-ray crystallography provides the only available means to take atomic-resolution "snapshots" that will serve as frames in this movie. My laboratory has so far solved four x-ray crystal structures of the entire E. coli ribosome (Vila-Sanjurjo et al., 2003, PNAS 100, 8682-7, Vila-Sanjurjo et al., 2004, NSMB, in press). These structures provide new insights into ribosome function, and provide the first steps towards the atomic-resolution images we are striving to determine. In the last few months, we have obtained crystals of the entire E. coli ribosome that diffract x-rays to a resolution close to 3 Angstroms. Thus, we now have the means to determine the first atomic-resolution structure of the intact ribosome, the first frame of the movie. Moreover, these crystals provide an unprecedented opportunity to probe in atomic detail the effects of antibiotics on the full ribosome and mutations in the ribosome that lead to antibiotic resistance or perturb key steps in translation.
Impact of molecular crowding on translation
Under physiological conditions, biochemical reactions occur in a crowded environment. Molecular crowding has its biggest impact on macromolecular interactions, including large conformational changes within a macromolecule. For example, molecular crowding dramatically influences protein folding pathways. In a bacterial cell, the concentration of macromolecules may reach one hundred times that in a typical in vitro biochemical reaction. Since molecular crowding is difficult to emulate outside of the cell, the function of biomolecules in crowded environments is only poorly understood. In protein biosynthesis, a number of conformational rearrangements in the ribosome have been identified from structural and hydrodynamic measurements. The impact of these rearrangements on the energetics of translation, especially in crowded cellular conditions, remains entirely unexplored. We have recently devised new microfluidics devices for probing rapid biochemical kinetics in molecular crowding conditions. Once these devices are fully characterized, we plan to use them to assess the effect of macromolecular crowding on protein synthesis. All steps in translation involve multiple RNA and protein interactions, and are likely to be affected by crowding. We are eager to explore the energetics of the conversion of chemical energy of GTP hydrolysis into mechanical energy that occurs during mRNA and tRNA translocation on the ribosome. Our results will provide an entirely new perspective on the energetics and kinetics of translation that reflect the actual conditions in the cell.
Cellular conditions and protein synthesis: Ribosome dynamics
As noted above, the interior of a cell is densely packed with macromolecules. Confined proteins and supramolecular complexes likely display different thermal structural fluctuations in vivo than those seen in vitro. How, then, do the dynamics of biomolecules in crowded environments affect chemical processes in the cell? How do the rates of enzymatic reactions measured in vivo compare with those measured in vitro? These questions are very difficult to address by ensemble-averaged assays. We anticipate that single-molecule spectroscopy, due to its capability of obtaining the individual dynamics from a distribution, will prove invaluable in efforts to unravel how microscopic, molecular interactions impact macroscopic biological functions. In collaboration with Prof. Haw Yang, we are developing a single-molecule spectrometer with 3D single-particle tracking capabilities in order to image the dynamics of single ribosomes in a bacterial cell. During an experiment, a ribosome to be tracked will be conjugated to a tracer element, a surface-passivated nanoparticle that will report the precise location of the ribosome. In addition to the tracer element, the ribosome will be labeled with fluorescent probes at strategic sites to allow for simultaneous studies of ribosomal dynamics. Although devising these experiments involves significant technical hurdles, we anticipate that the resultant technologies will establish single-molecule spectroscopy as a general approach to study macromolecules in living organisms.
Regulation of protein synthesis in eukaryotes
Regulation of protein synthesis is critical to the viability of eukaryotic cells. A main control point of this regulation is at the level of translation initiation. There is mounting evidence that initiation involves many sequential steps that drive important conformational changes in the ribosome. The molecular mechanism of these conformational changes, and their role in initiation, remain largely unknown. In order to probe the molecular basis and functional roles of these conformational changes in the ribosome, we are examining two aspects of translation initiation regulation, namely the control of viral replication and apoptosis. We are also exploring a newly recognized mode of translation regulation mediated by micro-RNAs.
Viral regulation of translation initiation
Many viruses overcome normal regulatory networks in the cell to drive translation of viral proteins. These viruses contain internal ribosome entry sites (IRESs) in their mRNAs that circumvent conventional ribosomal scanning from the 5' end of the mRNA. The IRESs do not require the full complement of translation initiation factors to function, allowing the viruses to avoid cellular pathways that function to inhibit translation on viral infection. We are working in collaboration with the laboratory of Prof. Jennifer Doudna to map the molecular interactions between the Hepatitis C Virus IRES (HCV IRES) and the ribosome in sequential steps of translation initiation. Other IRESs, such as the Cricket Paralysis Virus intergenic region IRES (CrPV IRES), start translation by directly entering the elongation cycle on the 80S ribosome. The CrPV IRES facilitates translation initiation independently of initiator-tRNAMet in a wide spectrum of eukaryotes, including mammals. Therefore, the underlying mechanism of CrPV IRES initiation may exploit a conserved property of eukaryotic translation. We are collaborating with Prof. Peter Sarnow at Stanford to map the molecular interactions between the CrPV IRES and the ribosome that occur during initiation and the first cycle of elongation. We are using conventional biochemical approaches, and eventually hope to use x-ray crystallography to image CrPV IRES-ribosome complexes at near-atomic resolution.
Apoptosis and translation regulation
Programmed cell death, or apoptosis, plays a key role in the development of metazoans. Apoptosis involves a delicate balance between pro-apoptotic and anti-apoptotic protein regulatory factors, the functions of which are highly conserved across metazoans. For example, the protein Reaper, identified as a potent pro-apoptotic factor in Drosophila, has been shown to induce apoptosis in vertebrate cells. Reaper, a 65 amino acid protein, plays an important role in many pathways, including the inhibition of general translation. However, the molecular basis of Reaper’s inhibition of translation remains unknown. In collaboration with Prof. Sally Kornbluth at Duke University, we are now working to unravel the molecular mechanisms underlying Reaper’s effects on translation. The elements of Reaper that inhibit translation reside in the last 50 amino acids of the protein. Intriguingly, human bunyaviruses responsible for pediatric encephalitis and hemorrhagic fever encode non-structural small (NSs) proteins homologous to the C-terminal 50 amino acids of Reaper. These NSs proteins are important for viral pathogenesis, and like Reaper, are potent inducers of apoptosis, partly through inhibition of translation. We are comparing the effects of NSs proteins on translation with those of Reaper, in order to shed light on how bunyaviral NSs proteins contribute to viral pathogenesis.
Role of micro-RNAs in translation regulation
In the last few years, a new class of small RNAs has been discovered in eukaryotes that regulates gene expression at many different levels. One type of these small regulatory RNAs, called micro-RNAs or miRNAs, has been found to repress protein synthesis by either leading to mRNA degradation or by inhibiting protein synthesis directly. These miRNAs are short RNAs that, when processed, are complementary to sequences within the 3'-untranslated regions of their target mRNAs. Surprisingly, direct inhibition of protein synthesis does not occur during initiation, seemingly the most strategic point to block translation. Rather, inhibition occurs during elongation, or possibly due to increased protein degradation. We are interested in discovering the mechanisms responsible for miRNA function in the direct inhibition of protein synthesis.
