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Expanding the Ribosomal Universe
Structure
Previews Expanding the Ribosomal Universe Jonathan D. Dinman1 and Terri Goss Kinzy2,* 1Department
of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA of Molecular Genetics, Microbiology, & Immunology, UMDNJ Robert Wood Johnson Medical School, Piscataway, NJ, USA *Correspondence: [email protected] DOI 10.1016/j.str.2009.11.003 2Department
In this issue of Structure, Taylor et al. (2009) present the most complete model of an eukaryotic ribosome to date. This achievement represents a critical milestone along the path to structurally defining the unique aspects of the eukaryotic protein synthetic machinery. Since Palade and coworkers used transmission electron microscopy (EM) methods to visualize eukaryotic ribosomes (Figure 1) (Kirsch et al., 1960), our modern understanding of the relationship between molecular structure and function have time and again returned to the analysis of this ancient molecule. Ribosomes serve a fundamental role in all kingdoms of life as the platform for the synthesis of proteins. While peptide-bond formation occurs as an intrinsic activity of the ribosome, the efficiency and accuracy of the initiation, elongation, and termination steps of translation require the interaction of the ribosome with soluble protein factors and aminoacylated transfer RNAs. Thus, understanding the structures of the factors and the ribosome, as well as the structural and functional interactions between these essential components of the translational apparatus, is essential for understanding this key step in gene regulation. Enhanced computational resources and technical prowess have dramatically expanded our ability to solve extremely complex molecular structures. Currently, X-ray crystallography and cryo-EM provide complementary approaches for meshing atomic resolution structure with conformational dynamics. As evidenced by the Nobel Prizes recently awarded for the atomic-scale resolution of archael and eubacterial ribosome structures (Wittmann et al., 1982; Ban et al., 2000; Brodersen et al., 2001), the ribosome has re-emerged as the ‘‘molecule of the century.’’ The past thirty years have seen tremendous advances in single-particle reconstruction applied elegantly to the ribosome (Frank, 2009). Looking forward, the determination of the structure of the eukaryotic ribosome represents the next major achievement. The work of Taylor et al. (2009) represents a critical milestone toward this goal,
identifying structural elements specific to eukaryotic ribosomes. In addition, it highlights the key advances that occur when the structure is considered in the presence of trans-acting factors. This includes eukaryotic elongation factors for which all the X-ray structures are known and for which a battery of mutants alleles in yeast are available (Taylor et al., 2006), using a method that can shed light on the dynamics of the steps in protein synthesis. The current study employed a combination of cryo-EM and structural homology modeling to create the most comprehensive model of the eukaryotic ribosome to date. Based on a structure initially determined by Taylor et al. (2007), this work partnered several teams with key tools and insights into modeling the eukaryotic ribosome and continuing the task of fitting eukaryote-specific ribosomal proteins (rps) into the overall scheme. It provides a critical update of the eukaryotic ribosome structure and pays careful attention to those parts that have remained mysterious even in the context of recent structural studies. A critical breakthrough in this work was the fitting of Saccharomyces cerevisiae rRNA and rp sequence into the Thermomyces lanuginosus structure by capitalizing on both being over 85% identical. As such, it is a terrific boon to the eukaryotic ribosome community in general, and provides a well-defined roadmap for yeast geneticists to test the hypotheses developed from these models. One of the major differences between archael/eubacterial ribosomes and their eukaryotic counterparts is size: eukaryotic ribosomes are significantly larger, primarily due to the presence of an additional 12 small and 41 large ribosomal subunit rRNA-based structures known as expansion segments (ES). The structural elucidation of these is of tremendous
value, as they give form to elements that are thought to play important roles in both the enormously complex eukaryotic ribosome biogenesis program, and as receptors for eukaryote-specific ribosomal protein and trans-acting regulatory factors. The new resolution allows not only for enhanced visualization of major ES, such as the 18S ES6, but also for ES-ES interactions to be proposed such as between 18S ES3 and ES6. The location of 18S ES7 supports a role in recruiting the more complicated eukaryotic initiation factors. Previously merged S. cerevisiae 25S ES7 and ES39 regions are now found to be not only distinct from but in different conformations, as compared to the thermophilic and more stable T. lanuginosus ribosomes. The identification of additional (but not all) rps is tremendous useful for researchers employing the power of yeast molecular genetics. The clustering of the ES structures with eukaryotic specific rps indicates their likely link in ribosome function and assembly. In addition, Table 1 in Taylor et al. (2009) represents a ‘‘Rosetta Stone’’ for translating eubacterial, archael, and eukaryotic ribosomal protein equivalents, and already adorns numerous surfaces of our laboratories as a quick reference guide. The docking of available atomic-resolution apo-structures of unique eukaryotic rps onto the ribosome greatly enhances our understanding of these elements. For example, this analysis clearly reveals details of the interaction between the rp components of the rpL1 stalk base, rpP0, and eEF2. Similarly, docking of the RACK complex onto the small subunit reveals unique functional interactions between these two elements that could not be discerned in previous structures. The localization of rpS19, utilizing the Pyrococcus abyssi
Structure 17, December 9, 2009 ª2009 Elsevier Ltd All rights reserved 1547
Structure
Previews RPS19 structure (Gregory establish the unanswered et al., 2007), allows for localiquestions in the eukaryotic zation of a protein linked ribosome structure research, to Diamond-Blackfan anemia and demonstrate a pathway (DBA; see Taylor et al., 2006). to their solution. There are This allows for a structural even more challenges ahead, interpretation and genetic including how the eukaryotic dissection of the interface specific rps and rRNA ES between this clearly important may affect other ribosomerp and the 18S rRNA. This associated processes such analysis also demonstrates as IRES-mediated translation the value of individual strucand miRNA-directed regulatures of the ‘‘missing’’ rps tion of gene expression. This and the importance of ribowork represents both a rich some structures determined source of new information in the context of associated and a challenge in the quest factors such as eEF2. Hypothto understand the eukaryotic eses are developed when ribosome. modeling allows for two potential locations (e.g., for rpS24e, integrating biochemREFERENCES ical, and immunolocalization data). While some limitations Ban, N., Nissen, P., Hansen, J., Moore, P.B., and Steitz, T.A. still exist to mapping the (2000). Science 289, 905–920. apo-rp structures onto the Brodersen, D.E., Carter, A.P., model, this represents an Clemons, W.M., Jr., Morganimportant advancement in Figure 1. The Eukaryotic Ribosome over 40 Years Warren, R.J., Murphy, F.V., Ogle, The pioneering work of Palade and coworkers demonstrated ribonucleic partithe field and highlights the J.M., Tarry, M.J., Wimberly, B.T., cles from eukaryotes (rats) (Kirsch et al., 1960), which has evolved (insert) into and Ramakrishnan, V. (2001). Cold need for more structures to molecular level understanding of the ribosome (Taylor et al., 2009), showing the Spring Harb. Symp. Quant. Biol. augment our understanding. 80S density map (mesh) and fitted atomic structure, reproduced from Frank 66, 17–32. (2009). Structure in front, in red, is eEF2. Courtesy of Lila Rubenstein and JoaThe supplemental informachim Frank. Dinman, J.D. (2009). J. Biol. Chem. tion is of great value and 284, 11761–11765. should not be dismissed. The discussion of the rps19e mutations associ- function, while the continuing evolution Frank, J. (2009). Q. Rev. Biophys., in press. ated with DBA is highly insightful and is the of the cryo-EM structures and modeling Gregory, L.A., Aguissa-Toure, A.H., Pinaud, N., first example of human mutations whose techniques reveal the structurally dy- Legrand, P., Gleizes, P.E., and Fribourg, S. functional consequences may benefit namic nature of this complex molecular (2007). Nucleic Acids Res. 35, 5913–5921. from this analysis. The extended discus- machine. The ability to identify and clarify Kirsch, J.F., Siekevitz, P., and Palade, G.E. (1960). sions of yeast large subunit-ribosomal the expansion of both the rRNA and the J. Biol. Chem. 235, 1419–1424. proteins, the ES structures, and the protein repertoire in the eukaryotic riboTaylor, D.J., Frank, J., and Kinzy, T.G. (2006). changes in the B1b/B1c bridge during in- some promises to illuminate critical new Eukaryotic Ribosomes and the Elongation tersubunit ratcheting serve as a timely information, such as locations of muta- Pathway. In Translational Control of Gene Exprescomplement to the functional, biochem- tions linked to human diseases and sion, N. Sonenberg, J.W.B. Hershey, and M.B. Mathews, eds. (Cold Spring Harbor, NY: Cold ical, and structural analyses of rpl11p structural elements critical for eukaryotic Spring Harbor Laboratory Press), pp. 59–85. mutants in this region currently being regulatory processes. The use of fungal systems also establishes a linkage to the Taylor, D.J., Nilsson, J., Merrill, A.R., Andersen, analyzed in the Dinman laboratory. G.R., Nissen, P., and Frank, J. (2007). EMBO J. It is clear that the eukaryotic ribosome wealth of genetic and biochemical infor- 26, 2421–2431. represents the next great challenge in mation generated from many laboratories D.J., Devkota, B., Huang, A.D., Topf, M., the structural analysis of translation. The exploring rRNA processing and ribosome Taylor, Narayanan, E., Sali, A., Harvey, S.C., and Frank, foundation for this effort is strong; the assembly to the structural biology and the J. (2009). Structure 17, this issue, 1591–1604. high-resolution structures of bacterial functional biochemistry of these unique Wittmann, H.G., Mussig, J., Piefke, J., Gewitz, ribosomes illuminate many of the con- rRNA and rp expansions (Dinman, 2009). H.S., Rheinberger, H.J., and Yonath, A. (1982). served elements of the structure and In summary, Taylor et al. (2009) clearly FEBS Lett. 146, 217–220.
1548 Structure 17, December 9, 2009 ª2009 Elsevier Ltd All rights reserved
Previews Expanding the Ribosomal Universe Jonathan D. Dinman1 and Terri Goss Kinzy2,* 1Department
of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA of Molecular Genetics, Microbiology, & Immunology, UMDNJ Robert Wood Johnson Medical School, Piscataway, NJ, USA *Correspondence: [email protected] DOI 10.1016/j.str.2009.11.003 2Department
In this issue of Structure, Taylor et al. (2009) present the most complete model of an eukaryotic ribosome to date. This achievement represents a critical milestone along the path to structurally defining the unique aspects of the eukaryotic protein synthetic machinery. Since Palade and coworkers used transmission electron microscopy (EM) methods to visualize eukaryotic ribosomes (Figure 1) (Kirsch et al., 1960), our modern understanding of the relationship between molecular structure and function have time and again returned to the analysis of this ancient molecule. Ribosomes serve a fundamental role in all kingdoms of life as the platform for the synthesis of proteins. While peptide-bond formation occurs as an intrinsic activity of the ribosome, the efficiency and accuracy of the initiation, elongation, and termination steps of translation require the interaction of the ribosome with soluble protein factors and aminoacylated transfer RNAs. Thus, understanding the structures of the factors and the ribosome, as well as the structural and functional interactions between these essential components of the translational apparatus, is essential for understanding this key step in gene regulation. Enhanced computational resources and technical prowess have dramatically expanded our ability to solve extremely complex molecular structures. Currently, X-ray crystallography and cryo-EM provide complementary approaches for meshing atomic resolution structure with conformational dynamics. As evidenced by the Nobel Prizes recently awarded for the atomic-scale resolution of archael and eubacterial ribosome structures (Wittmann et al., 1982; Ban et al., 2000; Brodersen et al., 2001), the ribosome has re-emerged as the ‘‘molecule of the century.’’ The past thirty years have seen tremendous advances in single-particle reconstruction applied elegantly to the ribosome (Frank, 2009). Looking forward, the determination of the structure of the eukaryotic ribosome represents the next major achievement. The work of Taylor et al. (2009) represents a critical milestone toward this goal,
identifying structural elements specific to eukaryotic ribosomes. In addition, it highlights the key advances that occur when the structure is considered in the presence of trans-acting factors. This includes eukaryotic elongation factors for which all the X-ray structures are known and for which a battery of mutants alleles in yeast are available (Taylor et al., 2006), using a method that can shed light on the dynamics of the steps in protein synthesis. The current study employed a combination of cryo-EM and structural homology modeling to create the most comprehensive model of the eukaryotic ribosome to date. Based on a structure initially determined by Taylor et al. (2007), this work partnered several teams with key tools and insights into modeling the eukaryotic ribosome and continuing the task of fitting eukaryote-specific ribosomal proteins (rps) into the overall scheme. It provides a critical update of the eukaryotic ribosome structure and pays careful attention to those parts that have remained mysterious even in the context of recent structural studies. A critical breakthrough in this work was the fitting of Saccharomyces cerevisiae rRNA and rp sequence into the Thermomyces lanuginosus structure by capitalizing on both being over 85% identical. As such, it is a terrific boon to the eukaryotic ribosome community in general, and provides a well-defined roadmap for yeast geneticists to test the hypotheses developed from these models. One of the major differences between archael/eubacterial ribosomes and their eukaryotic counterparts is size: eukaryotic ribosomes are significantly larger, primarily due to the presence of an additional 12 small and 41 large ribosomal subunit rRNA-based structures known as expansion segments (ES). The structural elucidation of these is of tremendous
value, as they give form to elements that are thought to play important roles in both the enormously complex eukaryotic ribosome biogenesis program, and as receptors for eukaryote-specific ribosomal protein and trans-acting regulatory factors. The new resolution allows not only for enhanced visualization of major ES, such as the 18S ES6, but also for ES-ES interactions to be proposed such as between 18S ES3 and ES6. The location of 18S ES7 supports a role in recruiting the more complicated eukaryotic initiation factors. Previously merged S. cerevisiae 25S ES7 and ES39 regions are now found to be not only distinct from but in different conformations, as compared to the thermophilic and more stable T. lanuginosus ribosomes. The identification of additional (but not all) rps is tremendous useful for researchers employing the power of yeast molecular genetics. The clustering of the ES structures with eukaryotic specific rps indicates their likely link in ribosome function and assembly. In addition, Table 1 in Taylor et al. (2009) represents a ‘‘Rosetta Stone’’ for translating eubacterial, archael, and eukaryotic ribosomal protein equivalents, and already adorns numerous surfaces of our laboratories as a quick reference guide. The docking of available atomic-resolution apo-structures of unique eukaryotic rps onto the ribosome greatly enhances our understanding of these elements. For example, this analysis clearly reveals details of the interaction between the rp components of the rpL1 stalk base, rpP0, and eEF2. Similarly, docking of the RACK complex onto the small subunit reveals unique functional interactions between these two elements that could not be discerned in previous structures. The localization of rpS19, utilizing the Pyrococcus abyssi
Structure 17, December 9, 2009 ª2009 Elsevier Ltd All rights reserved 1547
Structure
Previews RPS19 structure (Gregory establish the unanswered et al., 2007), allows for localiquestions in the eukaryotic zation of a protein linked ribosome structure research, to Diamond-Blackfan anemia and demonstrate a pathway (DBA; see Taylor et al., 2006). to their solution. There are This allows for a structural even more challenges ahead, interpretation and genetic including how the eukaryotic dissection of the interface specific rps and rRNA ES between this clearly important may affect other ribosomerp and the 18S rRNA. This associated processes such analysis also demonstrates as IRES-mediated translation the value of individual strucand miRNA-directed regulatures of the ‘‘missing’’ rps tion of gene expression. This and the importance of ribowork represents both a rich some structures determined source of new information in the context of associated and a challenge in the quest factors such as eEF2. Hypothto understand the eukaryotic eses are developed when ribosome. modeling allows for two potential locations (e.g., for rpS24e, integrating biochemREFERENCES ical, and immunolocalization data). While some limitations Ban, N., Nissen, P., Hansen, J., Moore, P.B., and Steitz, T.A. still exist to mapping the (2000). Science 289, 905–920. apo-rp structures onto the Brodersen, D.E., Carter, A.P., model, this represents an Clemons, W.M., Jr., Morganimportant advancement in Figure 1. The Eukaryotic Ribosome over 40 Years Warren, R.J., Murphy, F.V., Ogle, The pioneering work of Palade and coworkers demonstrated ribonucleic partithe field and highlights the J.M., Tarry, M.J., Wimberly, B.T., cles from eukaryotes (rats) (Kirsch et al., 1960), which has evolved (insert) into and Ramakrishnan, V. (2001). Cold need for more structures to molecular level understanding of the ribosome (Taylor et al., 2009), showing the Spring Harb. Symp. Quant. Biol. augment our understanding. 80S density map (mesh) and fitted atomic structure, reproduced from Frank 66, 17–32. (2009). Structure in front, in red, is eEF2. Courtesy of Lila Rubenstein and JoaThe supplemental informachim Frank. Dinman, J.D. (2009). J. Biol. Chem. tion is of great value and 284, 11761–11765. should not be dismissed. The discussion of the rps19e mutations associ- function, while the continuing evolution Frank, J. (2009). Q. Rev. Biophys., in press. ated with DBA is highly insightful and is the of the cryo-EM structures and modeling Gregory, L.A., Aguissa-Toure, A.H., Pinaud, N., first example of human mutations whose techniques reveal the structurally dy- Legrand, P., Gleizes, P.E., and Fribourg, S. functional consequences may benefit namic nature of this complex molecular (2007). Nucleic Acids Res. 35, 5913–5921. from this analysis. The extended discus- machine. The ability to identify and clarify Kirsch, J.F., Siekevitz, P., and Palade, G.E. (1960). sions of yeast large subunit-ribosomal the expansion of both the rRNA and the J. Biol. Chem. 235, 1419–1424. proteins, the ES structures, and the protein repertoire in the eukaryotic riboTaylor, D.J., Frank, J., and Kinzy, T.G. (2006). changes in the B1b/B1c bridge during in- some promises to illuminate critical new Eukaryotic Ribosomes and the Elongation tersubunit ratcheting serve as a timely information, such as locations of muta- Pathway. In Translational Control of Gene Exprescomplement to the functional, biochem- tions linked to human diseases and sion, N. Sonenberg, J.W.B. Hershey, and M.B. Mathews, eds. (Cold Spring Harbor, NY: Cold ical, and structural analyses of rpl11p structural elements critical for eukaryotic Spring Harbor Laboratory Press), pp. 59–85. mutants in this region currently being regulatory processes. The use of fungal systems also establishes a linkage to the Taylor, D.J., Nilsson, J., Merrill, A.R., Andersen, analyzed in the Dinman laboratory. G.R., Nissen, P., and Frank, J. (2007). EMBO J. It is clear that the eukaryotic ribosome wealth of genetic and biochemical infor- 26, 2421–2431. represents the next great challenge in mation generated from many laboratories D.J., Devkota, B., Huang, A.D., Topf, M., the structural analysis of translation. The exploring rRNA processing and ribosome Taylor, Narayanan, E., Sali, A., Harvey, S.C., and Frank, foundation for this effort is strong; the assembly to the structural biology and the J. (2009). Structure 17, this issue, 1591–1604. high-resolution structures of bacterial functional biochemistry of these unique Wittmann, H.G., Mussig, J., Piefke, J., Gewitz, ribosomes illuminate many of the con- rRNA and rp expansions (Dinman, 2009). H.S., Rheinberger, H.J., and Yonath, A. (1982). served elements of the structure and In summary, Taylor et al. (2009) clearly FEBS Lett. 146, 217–220.
1548 Structure 17, December 9, 2009 ª2009 Elsevier Ltd All rights reserved