Jamie H.D. Cate - US grants

The ribosome plays a central role in the conversion of genotype into phenotype in all forms of life. Since core ribosomal functions are highly conserved, the broad, long-term objective of the proposed research is to better understand the structure and function of the ribosome in the simplest free-living organisms, bacteria. Ribosomes from the bacterium Escherichia coli have been studied genetically, biochemically, and structurally for over four decades. Thus, although preliminary x-ray crystal structures have been solved of the bacterial ribosome from a poorly characterized thermophile, structures of the E. coli ribosome will bring significant advances to our understanding of protein synthesis. The ribosome works by means of specific initiation, elongation, and termination steps. During elongation, the ribosome must read the genetic code in messenger RNA (mRNA) for each successive amino acid that needs to be added to the growing polypeptide chain, a process termed decoding. Ribosomal decoding of mRNA will be probed by both structural and biochemical approaches. The specific aims are: 1) to improve the resolution of diffraction from E. coli ribosome crystals obtained in my laboratory by forming a ribosome complex that mimics decoding, 2) to solve the structure of the ribosome in the decoding complex, and 3) as a model for decoding defects, to examine the mechanism of stop codon read-through, or nonsense suppression, caused by mutations in tRNA. These experiments will advance our knowledge of how genetic information is converted into the working parts of the cell. They will provide a structural and biochemical foundation for modeling the molecular basis for ribosome function in protein synthesis. Insights from these studies will also lead to a clearer view of steps in protein synthesis that are inhibited by many classes of antibiotics.

The environment of a cell is highly crowded with proteins and nucleic acids. Unfortunately, the study of biochemical reaction kinetics in vitro has been limited to very dilute conditions. Microfluidics may provide a platform to investigate biochemical reactions in crowded conditions like those of the cell. The laboratories of Jamie Cate and Arun Majumdar will optimize a Microfluidics mixer with the goal to achieve millisecond time resolution in high concentrations of a crowding agent such as Bovine Serum Albumin (BSA). Once developed, such a mixing platform will be of general use to biochemists studying a wide range of biochemical phenomena, under conditions more closely matching those in the cell. During the first year of funding, the laboratories will characterize numerous mixer geometries to better understand mixing timescales and characteristics. Association of the small and large ribosomal subunits from Escherichia coli will be used as a test case for the effectiveness of the Microfluidics platform. The rate and degree of ribosomal subunit association will be measured under different levels of molecular crowding. Furthermore, the ionic conditions will be varied to reflect either in vitro biochemical conditions, or conditions that more closely match those thought to exist in E. coli. Graduate students and postdoctoral associates will receive interdisciplinary training in mechanical engineering and biochemistry.

DESCRIPTION (provided by applicant): The ribosome plays a key role in the conversion of genotype into phenotype in all forms of life. It is the large RNA and protein factory responsible for protein synthesis, and translates the genetic code in messenger RNA into the corresponding protein. Recent biochemical and structural studies of the ribosome have started to reveal the molecular basis of translation. Images of the two ribosomal subunits have been determined individually at atomic resolution by x-ray crystallography. However, the individual ribosomal subunits lack many functions of the ribosome necessary for protein synthesis. The ribosomal subunits must work together as an intact ribosome, in order for translation to occur. In order to provide a complete picture of the protein synthesis cycle at the molecular level, an atomic resolution "movie" of the intact ribosome, adding an amino acid to a growing polypeptide chain, will be necessary. We propose to make the first "frames" of this movie, by determining the atomic-resolution structures of two intact ribosomes from the model organism, Escherichia coli. By focusing on the E. coli ribosome, we will be able to compare our structural results directly to decades of biochemical and genetic research on translation, that has been carried out with this model system. During the first four years of this grant, we determined x-ray crystal structures of the entire [unreadable] coli ribosome at resolutions of 9-12 A, that shed some light on the structural basis of translation. These structures provide a first step in our goal of making "snapshots" of the ribosome at atomic resolution during the protein elongation cycle. We have now obtained crystals of the intact E. coli ribosome that diffract x-rays to atomic resolution. These crystals will allow us, for the first time, to image the ribosome at atomic resolution, and to explore, in atomic detail, the impact of ribosomal mutations that confer antibiotic resistance. Our results will significantly impact our understanding of translation, and will be widely useful in many biological fields where translation plays an important role. The specific aims of the proposal are as follows: 1. Solve and refine atomic-resolution structures of two intact 70S ribosomes from E. coli. 2. Determine the functional states of the two [unreadable] coli ribosome structures. 3. Probe the structural and functional consequences of mutations in the ribosome that cause antibiotic resistance.

The viability of eukaryotic cells relies upon the tight regulation of the initiation of protein synthesis.[unreadable] There is mounting evidence that this regulation involves important conformational changes within the[unreadable] small (40S) subunit of the ribosome. In order to probe the molecular basis and functional roles of[unreadable] these conformational changes in the 40S subunit, we will examine aspects of translation initiation[unreadable] regulation controlled by viral gene expression. Many viruses such as hepatitis C contain internal[unreadable] ribosome entry sites (IRESs) in their messenger RNAs that circumvent conventional initiation. These[unreadable] IRESs do not require the full complement of translation initiation factors to function. This enables[unreadable] viruses to bypass cellular pathways that inhibit translation upon viral infection. Understanding the[unreadable] molecular mechanisms used by viral IRESs will greatly aid in the development of antiviral strategies to[unreadable] combat devastating human viral diseases. The Cricket Paralysis Virus contains and IRES (CrPV IRES)[unreadable] that requires none of the initiation factors, and even circumvents the need for initiator tRNA.[unreadable] Interestingly, the CrPV IRES functions on all classes of eukaryotic ribosome, including human[unreadable] ribosomes. Elucidating its underlying mechanism of action will therefore reveal general features of[unreadable] eukaryotic ribosomes important for translation initiation.[unreadable] The specific aims of the proposal are the following. 1) Map conformational changes in the 40S subunit[unreadable] induced by the hepatitis C Virus IRES during translation initiation, and 2) map conformational changes[unreadable] in the 40S subunit induced by the Cricket Paralysis Virus IRES during translation initiation.

The viability of eukaryotic cells relies upon the tight regulation of the initiation of protein synthesis.[unreadable] There is mounting evidence that this regulation involves important conformational changes within the[unreadable] small (40S) subunit of the ribosome. In order to probe the molecular basis and functional roles of[unreadable] these conformational changes in the 40S subunit, we will examine aspects of translation initiation[unreadable] regulation controlled by viral gene expression. Many viruses such as hepatitis C contain internal[unreadable] ribosome entry sites (IRESs) in their messenger RNAs that circumvent conventional initiation. These[unreadable] IRESs do not require the full complement of translation initiation factors to function. This enables[unreadable] viruses to bypass cellular pathways that inhibit translation upon viral infection. Understanding the[unreadable] molecular mechanisms used by viral IRESs will greatly aid in the development of antiviral strategies to[unreadable] combat devastating human viral diseases. The Cricket Paralysis Virus contains and IRES (CrPV IRES)[unreadable] that requires none of the initiation factors, and even circumvents the need for initiator tRNA.[unreadable] Interestingly, the CrPV IRES functions on all classes of eukaryotic ribosome, including human[unreadable] ribosomes. Elucidating its underlying mechanism of action will therefore reveal general features of[unreadable] eukaryotic ribosomes important for translation initiation.[unreadable] The specific aims of the proposal are the following. 1) Map conformational changes in the 40S subunit[unreadable] induced by the hepatitis C Virus IRES during translation initiation, and 2) map conformational changes[unreadable] in the 40S subunit induced by the Cricket Paralysis Virus IRES during translation initiation.

[unreadable] DESCRIPTION (provided by applicant): The development of novel and effective treatments for viral infections requires a fundamental understanding of how viruses recruit the host cell translational machinery for viral protein synthesis during the early stages of infection. Understanding how large multi-subunit translation initiation factors and viral RNAs control the binding and activity of ribosomes poses a formidable challenge that forms the basis of this grant proposal. To tackle the difficult mechanistic and structural problems inherent in studying translation, we propose to launch a coordinated and interdisciplinary effort to determine the compositions, intermolecular interactions and structures of key initiation factor complexes and unravel the mechanisms that regulate protein synthesis in human cells and viruses. A key aspect of this P01 Program Project Grant will be to establish highly interactive collaborations across disciplines and research institutions to study the chemical, structural and mechanistic properties of key complexes controlling translation initiation. By combining the expertise of several investigators we propose to implement a battery of complementary biophysical and biochemical approaches including: Projects by Doudna and Cate, cryo-electron microscopy and X-ray crystallography of translation initiation factors and complexes; Project by Hershey, in vitro and in vivo translation assays with viral and cellular translation components; and Project by Sarnow, purification and analysis of viral translation complexes. A major component of this proposal will be the establishment of a core laboratory for mass spectrometry of large multi-subunit complexes that will serve as the nerve center and clearinghouse for analyzing the many protein and protein-RNA complexes involved in translation initiation. This core laboratory will be used extensively by all five Research Projects to identify the macromolecules and post-translational modifications that, respectively, comprise and control translation activity in human cells and viruses. Thus, our goal is to develop a highly synergistic and concerted effort to carry out a pioneering set of interdependent experiments by using overlapping molecular reagents but applying distinct and complementary research strategies to dissect the core machinery responsible for protein synthesis in normal and virally-infected mammalian cells. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Protein synthesis, or translation, directly couples genotype to phenotype in the cell. Translation occurs on the ribosome, an RNA-protein complex conserved in all forms of life. The ribosome is composed of two subunits that must work together as an intact complex in order to function. A complete understanding of protein synthesis will require an atomic-resolution "movie" of the intact ribosome during one cycle of amino acid addition to a growing polypeptide chain. In this regard, the ribosome is no different from other molecular machines, such as RNA and DNA polymerases, helicases, myosins and kinesins. All of these molecular machines use conformational changes as a basis for function. And in each case, many x-ray crystal structures at atomic resolution have been necessary to obtain clear mechanistic insights into function. Cryo-EM reconstructions and x-ray crystal structures have identified many moving parts within the ribosome that remain to be probed at atomic resolution due to a lack of high-resolution structural information. One functional state of the ribosome that is critical to many steps of translation, the "ratcheted" state, remains poorly understood at the molecular level. The ratcheted state of the ribosome is central to the process of moving messenger RNA (mRNA) and transfer RNA (tRNA) by one codon after each peptide bond is made, a process called translocation. The ratcheted state also plays an important role in ribosome recycling, the step in which ribosomes that have completed synthesis of one protein are dissociated into the two ribosomal subunits to promote reinitiation of translation. The ratcheted state is also a key target of many antibiotics. In the past four years of this grant, we have successfully used the model organism Escherichia coli for x-ray crystallographic and biochemical studies of the intact ribosome, bridging structural data with functional insights at atomic resolution. We obtained two crystal forms of the intact ribosome that diffract to atomic resolution, which resulted in several groundbreaking structural studies of the ribosome. Our focus on the E. coli ribosome allows us to compare our results directly to decades of biochemical and genetic data obtained with the E. coli translational machinery. We have now obtained atomic-resolution diffraction from crystals of the E. coli 70S ribosome in the ratcheted state. This proposal describes three strategies for probing both the structure and function of the intact E. coli ribosome based on this new breakthrough. 1. Determine the atomic-resolution structure of the intact 70S ribosome in the ratcheted state. 2. Determine atomic-resolution structures of the ratcheted ribosome in complexes with tRNAs, ribosome recycling factor (RRF), elongation factor G (EF-G), or antibiotics that target the ratcheted state. 3. Probe the effects of macromolecular crowding on the kinetics and thermodynamics of ribosome recycling. These experiments will build on the structural insights gained in the present funding period and in Specific Aims 1 and 2. PUBLIC HEALTH RELEVANCE: Protein biosynthesis depends on the ribosome, which translates the genetic code into protein sequences. This proposal aims to probe the structure and function of an essential structural state of the ribosome, the ratcheted state, at atomic resolution. The proposed structural and biochemical experiments will provide unique contributions to our understanding of protein biosynthesis as it occurs in bacteria, and how antibiotics interfere with bacterial translation.

Summary. Mechanical signals from a cell's microenvironment are known to alter cell behavior and gene expression profiles, but the role of mRNA processing is unclear. The Cell Mechanics Core provides as set of resources that, together with the computational (Core 4) and RNA profiling resources (Cores 1 and 2) in the Center for RNA Systems Biology, will provide a new perspective on the intersection of cell mechanics and gene regulation.

SUMMARY Our overall goal is to systematically define c/s-regulatory elements in mRNAs that control translation initiation under defined physiological conditions. While the sequence requirements for translation start codon selection have been studied for individual genes, a comprehensive understanding of the rules for start site selection is not known for the human transcriptome.

Summary Our overall goal is to systematically define c/s-regulatory elements in mRNAs that control miRNA-mediated silencing under defined physiological conditions. While many miRNA targets have been predicted, and some individual targets have been investigated experimentally, the ways in which mRNA structures enhance or inhibit miRNA targeting are not known for the human transcriptome.

DESCRIPTION (provided by applicant): Human biology depends on the way the human genome is expressed. Protein levels in cells rely on the fate of messenger RNA-how pre-mRNAs are spliced, how and when mRNAs are translated, and finally when mRNAs are degraded. Defects in these steps can lead to diseases ranging from inherited disorders to cancer. By their nature as RNA polymers, pre-mRNAs and mRNAs may contain secondary and tertiary structural elements that serve as regulators of mRNA abundance and protein synthesis. Despite the central importance of mRNA regulation in biology, there has not been a systems-level study of how pre-mRNA and mRNA structure controls mRNA fate in living cells. The Center for RNA Systems Biology will use new methods to establish a fundamental basis for understanding and predicting the control of mRNA fate due to RNA structure embedded in pre-mRNA and mRNA sequences. The Center will combine new in vivo chemical probing methods with control of the physical environment of cells to address the following Specific Aims: 1) Determine the roles of RNA structure in pre-mRNAs in controlling alternative splicing and their Relationship to human genetic variation. 2) Define mRNA structures that control translation initiation and protein synthesis in response to a cell's physical environment. 3) Map RNA structural regulation of miRNA-mediated turnover. Ultimately the goal of the Center is to develop maps of relationships between the placement of RNA structure in pre-mRNA or mRNA sequences and mRNA fate. These maps will provide many new insights into human biology and the mechanisms underlying genotypic variation and human disease.

RNA; Systems Biology;

DESCRIPTION (provided by applicant): Protein synthesis by the ribosome directly couples genotype to phenotype in the cell, and its regulation is central to cellular physiology. Although many steps of translation have diverged since the last common ancestor, translation initiation and ribosome recycling remain intimately coupled in both bacteria and humans. These coupled events in the translation cycle are the focus of the present application. Our understanding of the molecular mechanism of protein synthesis has undergone a revolution in the last decade, built on rapid advances in the structural biology of the ribosome. High-resolution structures are now available for the entire ribosome in both bacteria and eukaryotes, and of the large ribosomal subunit in archaea. However, important questions relating to dynamic events in translation remain unanswered due to the challenge of isolating structural intermediates. In this application, we propose to probe the molecular mechanism of ribosome recycling in bacteria, a process catalyzed by the GTPase elongation factor G and a validated target of antibiotics. We will build on our prior groundbreaking results in this area by combining x-ray crystallography, cryo-electron microscopy (cryo-EM) and single-molecule biophysics. By using x-ray crystallography, cryo-EM, and new methods and tools we have developed to study human translation initiation factor eIF3, we will also probe human translation initiation, one of the most dynamic steps in protein synthesis. We will use our unique system to functionally reconstitute human translation initiation factor eIF3, cryo-EM, genetics in Neurospora crassa, and revolutionary new methods in genome engineering to dissect the contribution of eIF3 to start codon selection on human cellular mRNAs. The combination of eIF3 reconstitution in E. coli, genetics in N. crassa, mutagenesis in human cells, and cutting-edge cryo-EM provides a powerful and unique means to unravel the molecular contributions of eIF3 to translation initiation in humans. Taken together, the three aims of this application build on the fundamental insights into ribosome structure and function obtained in the prior funding period, and address key mechanisms in translational control that could be exploited for the development of new antibiotics and therapeutics.

Core - Improved High-resolution cryo-EM Methodology Jamie Cate and Kenneth Downing PROJECT SUMMARY/ABSTRACT The capabilities of cryo-EM have begun to match or surpass those of X-ray crystallography in many areas of structural biology. However, there remain crucial bottlenecks in cryo-EM that hamper reconstructions of fragile complexes. The goal of this Core Project is to develop methods that will further enhance the capabilities of cryo-EM, in order to support the work of the Projects and Associated Projects of this Program. We address two key areas where single-particle cryo-EM requires further improvement. We will first address the cryo-preservation of delicate samples on cryo-EM grids. Many samples are subject to denaturation when exposed to the extensive air-water interface during blotting, or to a continuous carbon grid. To prepare specimens in a more structure-friendly way by avoiding specimen contact with the air-water interface, streptavidin monolayer crystals will be used as a specimen-support film for affinity-binding interactions. We will test a series of affinity adaptor and affinity tagging methods, to ensure uniform particle orientations on the streptavidin monolayer crystal lattice. These experiments will demonstrate the general utility of using streptavidin monolayer crystals as a support material. Second, we will develop means to correct beam-induced sample movement. When using direct electron detectors in ?movie? mode, the initial frames of exposures are presently discarded, although they potentially contain the highest resolution information. In subsequent frames, the high-resolution information is no longer available due to electron beam induced damage. We propose to use the image of the streptavidin support film as a fiducial, to better characterize the frame-to-frame motions that occur. We will use human 48S translation preinitiation complexes and Escherichia coli 70S ribosomes as test systems to validate the methods in these two aims. Once we have established these methods, they will be of wide use in the cryo-EM community, and should greatly expand the scope of cryo-EM into areas of structural biology that were previously inaccessible, or accessible only to x-ray crystallography.

Multiexon skipping to restore dystrophin protein expression using tethered RNPs Project Summary Mutations in the dystrophin (DMD) gene often lead to frameshift mutations in the mature messenger RNA, causing nonsense mediated decay of the mRNA and loss of dystrophin protein in muscle cells. The most severe of these mutations cause Duchenne muscular dystrophy (DMD), an X-linked disease in males that leads to progressive loss of muscle function and early death. Given the large variety of mutations within the dystrophin gene and its very large size, it has not been possible to derive a general treatment for DMD. One promising strategy to treat a subset of patients is to trigger exon skipping during pre-mRNA splicing, thereby restoring the reading frame in the mature mRNA. Although the resulting dystrophin protein may be shorter than the wild-type version, some of these shorter versions are reasonably functional and can lessen the disease phenotype. Many labs and companies are exploring exon skipping induced by antisense oligonucleotides, exemplified by phosphorodiamidate morpholino oligomers (PMOs), some of which are now in clinical trials. Although the FDA has approved one PMO that leads to skipping of exon 51, this approval is provisional on demonstrating a clear clinical benefit. Further, each single antisense oligonucleotide can only treat a small percentage of patients. To overcome these limitations, one promising strategy would be to induce skipping of exons 45-55 in the DMD mRNA. If successful, such a treatment would generate a truncated dystrophin protein seen in Becker muscular dystrophy (BMD) patients that leads to a much milder phenotype, and could be used to treat ~45% of DMD patients. Although it is possible to induce skipping of exons 45-55 using a cocktail of 10 modified PMOs, these treatments could cause toxicity and induce variably spliced mRNAs due to inefficient exon skipping. We propose to take a fundamentally new approach to induce multiexon skipping. We will program bacterial Argonautes with guide RNAs targeting exons 45 and 55 to form RNA-protein complexes (RNPs) to induce exon skipping. To achieve multiexon skipping, we will tether the RNPs to each other. Tethering the RNPs should bring exons 44 and 56 into closer proximity, which we propose should increase skipping of exons 45-55. In the longer term, the use of tethered guide-RNA/Argonaute RNPs could be a general means to induce multiexon skipping to treat DMD.

ABSTRACT Protein targets for many human diseases remain ?undruggable? due to their underlying biochemical behavior. These limits to discovery of small molecule drugs hold back the promise of developing affordable therapeutics. Here we propose to develop an entirely new mechanism of action that could enable targeting previously ?undruggable? proteins, by selectively blocking their translation by the human ribosome. Most drugs and drug candidates known to modulate human translation target translation initiation factor complexes or upstream signaling pathways such as mammalian target of Rapamycin (mTOR). These generally modulate translation of a large number of mRNAs. To date, only one type of compound that selectively targets the ribosome?to induce premature stop codon readthrough?is being evaluated in the clinic. We recently demonstrated that small molecules can selectively stall the translation of human proteins, with very little off-target activity. The compound we analyzed, PF-06446846 (PF846), directly and selectively inhibits the translation of PCSK9 during translation elongation, by stalling the ribosome on the nascent polypeptide residing in the ribosome exit tunnel. However, it remains unclear how this and related compounds selectively stall translation. The few examples of off-target proteins we identified as stalled by PF846 (less than 0.4 percent of the human proteome) exhibit substantial primary structure variability, making it difficult to predict target sequences for future development of selective ribosome-targeting drugs. We have preliminary evidence that PF846 interacts with these diverse nascent chain sequences to induce ribosome stalling by subtly different mechanisms. We propose to elucidate the full molecular mechanism of PF846 stalling of translation, so that this family of compounds can be further optimized to target proteins whose expression or overexpression causes diverse human diseases for which no treatments are now available. This family of compounds could also be optimized to target viruses, which use human translation to synthesize their proteome. These efforts have the potential to open up an entirely new mechanism of action for human therapeutic development.

PROJECT SUMMARY Protein biosynthesis directly couples genotype to phenotype in the cell, and its regulation is central to cellular physiology. Our understanding of the molecular mechanism of protein synthesis has undergone a revolution in the last decade, built on rapid advances in structural biology and systems biology. However, important questions relating to dynamic events in translation remain unanswered, as the transient nature of these events makes them difficult to isolate. In this application, we propose to decipher the molecular mechanisms of how eukaryotic translation initiation factor 3 (eIF3) in humans regulates translation initiation. We recently discovered that human eIF3 serves multiple roles in translation. Human eIF3 generally helps assemble translation preinitiation complexes at the start codon, but also directly controls the translation of specific mRNAs. We found that eIF3 can either activate or repress the translation of an mRNA, depending on how eIF3 binds to the mRNA's 5' untranslated region (5' UTR). Furthermore, we discovered that eIF3 harbors a subunit?EIF3D?that binds the 5'-m7G cap on certain mRNAs in an RNA structure-dependent manner. These discoveries indicate that eIF3 may integrate multiple signals to control the translational output of individual mRNAs, much like the Mediator complex in transcription. The aims in this application build on these groundbreaking results to address fundamental questions of how eIF3 functions. We propose to determine the structural basis for eIF3-mediated control of specialized translation, using cryo-electron microscopy (cryo-EM) and in vitro biochemistry. We will also probe how mRNAs regulated by eIF3 are structured in living cells, and dissect the functional importance of these structures to eIF3 regulation and the translational capacity of these mRNAs. Finally, we will use systems biological approaches to explore how eIF3 contributes to the regulation of translation mediated by N-6- methyladenosine (m6A) modifications in mRNAs. By combining advances in cryo-EM to our expertise in human cell engineering, we are in a unique position to unravel the molecular contributions of eIF3 to translation initiation in humans. Taken together, the three aims of this application build on the fundamental insights into eIF3 structure and function obtained in the prior funding period, and address key mechanisms in translational control that could be widespread in human biology. In the long run, our insights into the molecular mechanisms used in human cells to direct eIF3-mediated activation and repression of specific mRNAs could pave the way for the development of new small-molecule and cell-based therapeutics.