NEWS & VIEWS RESEARCH grooves. Naganuma et al. report that recognition of the acceptor stem of tRNAAla/GU is mediated by the tips of two α-helices (α11 and α14, located in the tRNA-recognition domain of AlaRS) that hold the acceptor stem tightly and position it for aminoacylation by clamping onto both the minor and major grooves. The strong clamp formed by α11 and α14 widens the major groove in the acceptor stem, preventing structural clashes with other AlaRS domains. The researchers also find that the G3∙U70 base pair is in the wobble geometry by which G3 orients to the minor groove. The amino group of G3 seems to form two hydrogen bonds with an aspartate amino-acid residue (Asp 450) in α14, which supports the idea9 that this group has a crucial role in aminoacylation by AlaRS. In contrast to other ARS∙tRNA complexes solved so far13, the L-shaped structure of tRNAAla in the complex is only mildly distorted compared with that of the free tRNA. Overall, Naganuma and colleagues’ structures show that AlaRS accommodates tRNA Ala/AU and tRNA Ala/GU in similar ways, but they reveal a previously unknown mechanism by which the local geometry of the G3∙U70 wobble pair is propagated through the acceptor-stem duplex towards the singlestranded 3ʹ-CCA region. In the GU-containing complex, the terminus of the 3ʹ-CCA end — the site of amino-acid attachment — reaches the active site of AlaRS (Fig. 1). But in the AU variant, the 3ʹ-CCA end bends away from the active site. This bent conformation might be stabilized by interactions between nearby nucleotides. Even though tRNAAla/AU can, in principle, switch between extended and bent conformations, such a conformational change is restricted by a ‘route separator’ in AlaRS, a structure formed by three consecutive glycine amino-acid residues and a glutamate residue. The authors’ kinetic analyses confirmed that the kcat value of the A. fulgidus AlaRS dimer for aminoacylation of tRNAAla/GU is about 100fold higher than for tRNAAla/AU, whereas the rates of overall product formation and binding affinities of the tRNA variants differed by only about twofold. Thus, tRNA discrimination by AlaRS seems to rely on a catalytic mechanism involving ‘reactive’ (straight acceptor stem) and ‘non-reactive’ (bent acceptor stem) states of the bound tRNA. Although Naganuma and colleagues’ findings provide exciting information about the mechanism by which AlaRS discriminates between tRNAAla/GU and tRNAAla/AU, higher-resolution structures are needed to assign specific hydrogen-bonding interactions with greater confidence. Moreover, we still do not know how AlaRSs recognize mitochondrial tRNAAla molecules, which do not usually contain a G3∙U70 base pair. And it is not clear how the 3ʹ-CCA end of tRNAAla/GU interacts with the second catalytic site (the editing domain) of AlaRS, which hydrolyses tRNAs to which the wrong
amino acids have been attached and which also has to recognize the G3∙U70 base pair14. Crystal structures of other ARSs bound to mutant tRNAs will be needed to establish whether those enzymes also use reactive and unreactive states, as well as route separators, to decode their distinct identity sets. Finally, it remains to be seen whether similar mechanisms are employed by other RNAs that use the G∙U wobble pair as a specific recognition signal. ■ Oscar Vargas-Rodriguez and Karin Musier-Forsyth are in the Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, Ohio 43210, USA. e-mail: [email protected]
2. Hou, Y.-M. & Schimmel, P. Nature 333, 140–145 (1988). 3. McClain, W. H. & Foss, K. Science 240, 793–796 (1988). 4. Giegé, R., Sissler, M. & Florentz, C. Nucleic Acids Res. 26, 5017–5035 (1998). 5. Beuning, P. J. & Musier-Forsyth, K. Biopolymers 52, 1–28 (1999). 6. Imura, N., Weiss, G. B. & Chambers, R. W. Nature 222, 1147–1148 (1969). 7. de Duve, C. Nature 333, 117–118 (1988). 8. Park, S. J., Hou, Y.-M. & Schimmel, P. Biochemistry 28, 2740–2746 (1989). 9. Musier-Forsyth, K. et al. Science 253, 784–786 (1991). 10. Musier-Forsyth, K. & Schimmel, P. Nature 357, 513–515 (1992). 11. McClain, W. H., Chen, Y.-M., Foss, K. & Schneider, J. Science 242, 1681–1684 (1988). 12. Gabriel, K., Schneider, J. & McClain, W. H. Science 271, 195–197 (1996). 13. Perona, J. J. & Hadd, A. Biochemistry 51, 8705–8729 (2012). 14. Beebe, K., Mock, M., Merriman, E. & Schimmel, P. Nature 451, 90–93 (2008).
1. Naganuma, M. et al. Nature 510, 507–511 (2014).
This article was published online on 11 June 2014.
A PPL I ED P H YS I CS
Trawling for complements A method has been invented for determining nanoscale variations in the distribution of electric charge on surfaces. It has so far been used to examine specific inorganic materials, but could find widespread applications in imaging. J. MARTY GREGG & AMIT KUMAR
T
hunderstorms are among nature’s most awe-inspiring spectacles. Lightning flashes pierce the gloom, causing vast amounts of electric charge — built up at the base of clouds — to find their way to Earth. Even without flashes, static electricity in the clouds affects the charge distribution on Earth’s surface. Localized regions of land, and all the objects contained in them, become either enriched in or denuded of electrons, producing a localized net charge that is opposite in sign to that of the clouds directly above. Although most of the cloud base is negative, charge densities vary. As the storm passes overhead, these variations are reflected by complementary charge variations on Earth. To facilitate charge redistribution, electric currents must flow. So, by monitoring the currents at fixed locations on Earth, variations in the charge densities of the passing storm clouds can be mapped. Writing in Proceedings of the National Academy of Sciences, Hong et al.1 report how they have done almost exactly this, except that, rather than mapping large-scale electrical storms, they have invented an analogous technique to determine nanoscale charge variations on the surfaces of inorganic materials. To do their experiments, Hong and
colleagues made relatively minor adaptations to an atomic force microscope (AFM), if anything, simplifying its mode of operation over that conventionally used. They brought a sharp conducting tip, positioned on the end of a flexible cantilever, into contact with the surface of a lithium niobate crystal. They then trawled this metallic tip across the crystal surface and, using a sensitive detection circuit, monitored minute currents associated with charge redistribution between the tip and its grounded electrode. Lithium niobate is an insulating oxide, and is important in telecommunications applications. Within each of the unit cells of this crystalline material, ions are naturally arranged such that the centres of positive and negative charge are slightly separated spatially. This results in what is termed an electric dipole, a vector entity that points from negative to positive within the crystal. In lithium niobate, the orientation of this dipole can be reversed by an electric field, defining the material as ferroelectric. For practical applications, reversals in dipole orientation induced by an electric field are engineered to make spatially periodic stripe patterns, in which positive and negative charges are alternately closer to the crystal surface, in distinct regions called domains. When electrodes are brought into 2 6 J U N E 2 0 1 4 | VO L 5 1 0 | NAT U R E | 4 8 1
© 2014 Macmillan Publishers Limited. All rights reserved
RESEARCH NEWS & VIEWS Grounded electrode A
A
Tip
–
+
Charge on tip
Ground
–
–
–
–
–
+
+
+
+
+
+
+
+
+
+
–
–
–
–
–
Current left to right Current right to left 0
Figure 1 | Charge gradient mapping. Hong and colleagues1 have developed a technique for determining charge variations on the surface of ferroelectric materials. A metallic tip connected to the ground by means of an electrode scans rapidly across the surface of the material, which is also connected to the ground. The tip records a current, in a device equivalent to an ammeter (A), only when it encounters a charge gradient at a junction between two distinct domains, in which positive and negative charges are alternately closer to the surface; white arrows denote the electric-dipole vectors in each domain. The magnitude of the current (blue and brown curves), which is associated with charge redistribution in the tip (grey curve), is proportional to the charge gradient as well as to the tip velocity; its sign is governed by the direction of the charge gradient, as well as by the scan direction.
contact, the proximity of the near-surface charges in the ferroelectric material induces charge redistribution in the electrode: charges of opposite sign to the near-surface charges in the material are attracted to the interface between the electrode and the ferroelectric material, and like charges are repelled. The same thing happens when an AFM tip is used as an electrode (Fig. 1). If the tip is moved within a single domain, because dipoles are uniformly oriented, the initial charge redistribution that occurs in the tip is unchanged. However, whenever the tip passes over the junction between one domain and the next (over a domain wall), dipole reversal causes complementary charges to flow between the tip and its grounded electrode. Because the current associated with this charge redistribution between the tip and the ground is proportional to the rate of change of charge at the tip, it increases with the scanning speed of the AFM tip (complete charge redistribution is forced to occur in a shorter time). Moreover, because the nature of the charge redistribution in the tip depends on the direction in which a specific domain wall is traversed, the sign of the current changes whenever the scan direction is reversed. Hong et al. point out that the total current that is generated as the tip passes over each domain wall equates to a charge-density change at the ferroelectric material’s surface that is around twice that of the dipole density in lithium niobate, exactly as would be expected. The finding confirms that this method can quantitatively, as well as qualitatively, map nanoscale charge-density variations. The authors highlight the possibly confounding influence of ions and dipolar molecules in the air that nullify the electric fields generated by the near-surface charges in the ferroelectric material. However, they note
that their imaging technique, which they call charge gradient microscopy, is largely immune to such airborne layers, because it seems that the tip scrapes them away. Although this method for imaging charge variation is conceptually quite simple, its potential importance cannot be overstated. It has become evident that the domain walls of ferroelectric materials can have diverse functional characteristics that are completely different from the domains that they delineate: they can be conducting2, or even superconducting3, when the domains themselves are insulating. Moreover, they can be moved, injected and annihilated by external electric fields4, and so could be used in new
two-dimensional electronic devices. Hong and colleagues’ imaging technique rapidly locates ferroelectric-material domain walls and could therefore be invaluable in supporting the quest for “domain wall nanoelectronics”5. Furthermore, its discovery comes at a time when shortcomings in the most common method for domain imaging in ferroelectric materials (piezoresponse force microscopy) are starting to become uncomfortably apparent: for example, high-quality images of ‘domains’ have been obtained using piezoresponse force micro scopy in materials in which ferroelectricity is extremely unlikely6,7. Restricting the authors’ charge gradient microscopy to imaging ferroelectric domain walls, exciting as this is, would probably be a mistake. Variation in nanoscale charge distribution must be crucial in many aspects of science, from understanding communications in cell biology to teasing out the details of battery electrochemistry. Perhaps the adoption of this imaging technique by other areas of science will allow its full potential to be realized. ■ J. Marty Gregg and Amit Kumar are in the Centre for Nanostructured Media, School of Mathematics and Physics, Queen’s University Belfast, Belfast BT7 1NN, UK. e-mails: [email protected]; [email protected] 1. Hong, S. et al. Proc. Natl Acad. Sci. USA 111, 6566–6569 (2014). 2. Sluka, T., Tagantsev, A. K., Bednyakov, P. & Setter, N. Nature Commun. 4, 1808 (2013). 3. Aird, A. & Salje, E. K. H. J. Phys. Condens. Matter 10, L377–L380 (1998). 4. Whyte, J. R. et al. Adv. Mater. 26, 293–298 (2014). 5. Catalan, G., Seidel, J., Ramesh, R. & Scott, J. F. Rev. Mod. Phys. 84, 119–156 (2012). 6. Bark, C. W. et al. Nano Lett. 12, 1765–1771 (2012). 7. Kim, Y. et al. ACS Nano 6, 7026–7033 (2012).
STR UC TUR A L B I OLOGY
Enzyme assembly line pictured Many enzymes form ‘assembly lines’ containing a series of catalytic modules. Visualization of how the structure of a module shifts during catalysis provides a clearer idea of how such enzymes work. See Article p.512 & Letter p.560 P E T E R F. L E A D L AY
L
iving cells contain many molecular machines that integrate and orchestrate essential processes. One of the most familiar is the ribosome, which polymerizes amino acids to form proteins. In the early 1990s, a radically different type of polymerizing assembly line was discovered1,2: poly ketide synthase (PKS) multi-enzymes, which
4 8 2 | NAT U R E | VO L 5 1 0 | 2 6 J U N E 2 0 1 4
© 2014 Macmillan Publishers Limited. All rights reserved
make polyketide compounds, including many important antibiotics. Remarkably, these complexes use a different module of catalytic sites for each cycle of a chemical-chain extension. Efforts to understand these sophisticated catalysts have received a great boost from two studies in this issue from the same research group: Dutta et al.3 (page 512) report the three-dimensional structure of a PKS protein that houses an intact catalytic module, and
amino acids have been attached and which also has to recognize the G3∙U70 base pair14. Crystal structures of other ARSs bound to mutant tRNAs will be needed to establish whether those enzymes also use reactive and unreactive states, as well as route separators, to decode their distinct identity sets. Finally, it remains to be seen whether similar mechanisms are employed by other RNAs that use the G∙U wobble pair as a specific recognition signal. ■ Oscar Vargas-Rodriguez and Karin Musier-Forsyth are in the Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, Ohio 43210, USA. e-mail: [email protected]
2. Hou, Y.-M. & Schimmel, P. Nature 333, 140–145 (1988). 3. McClain, W. H. & Foss, K. Science 240, 793–796 (1988). 4. Giegé, R., Sissler, M. & Florentz, C. Nucleic Acids Res. 26, 5017–5035 (1998). 5. Beuning, P. J. & Musier-Forsyth, K. Biopolymers 52, 1–28 (1999). 6. Imura, N., Weiss, G. B. & Chambers, R. W. Nature 222, 1147–1148 (1969). 7. de Duve, C. Nature 333, 117–118 (1988). 8. Park, S. J., Hou, Y.-M. & Schimmel, P. Biochemistry 28, 2740–2746 (1989). 9. Musier-Forsyth, K. et al. Science 253, 784–786 (1991). 10. Musier-Forsyth, K. & Schimmel, P. Nature 357, 513–515 (1992). 11. McClain, W. H., Chen, Y.-M., Foss, K. & Schneider, J. Science 242, 1681–1684 (1988). 12. Gabriel, K., Schneider, J. & McClain, W. H. Science 271, 195–197 (1996). 13. Perona, J. J. & Hadd, A. Biochemistry 51, 8705–8729 (2012). 14. Beebe, K., Mock, M., Merriman, E. & Schimmel, P. Nature 451, 90–93 (2008).
1. Naganuma, M. et al. Nature 510, 507–511 (2014).
This article was published online on 11 June 2014.
A PPL I ED P H YS I CS
Trawling for complements A method has been invented for determining nanoscale variations in the distribution of electric charge on surfaces. It has so far been used to examine specific inorganic materials, but could find widespread applications in imaging. J. MARTY GREGG & AMIT KUMAR
T
hunderstorms are among nature’s most awe-inspiring spectacles. Lightning flashes pierce the gloom, causing vast amounts of electric charge — built up at the base of clouds — to find their way to Earth. Even without flashes, static electricity in the clouds affects the charge distribution on Earth’s surface. Localized regions of land, and all the objects contained in them, become either enriched in or denuded of electrons, producing a localized net charge that is opposite in sign to that of the clouds directly above. Although most of the cloud base is negative, charge densities vary. As the storm passes overhead, these variations are reflected by complementary charge variations on Earth. To facilitate charge redistribution, electric currents must flow. So, by monitoring the currents at fixed locations on Earth, variations in the charge densities of the passing storm clouds can be mapped. Writing in Proceedings of the National Academy of Sciences, Hong et al.1 report how they have done almost exactly this, except that, rather than mapping large-scale electrical storms, they have invented an analogous technique to determine nanoscale charge variations on the surfaces of inorganic materials. To do their experiments, Hong and
colleagues made relatively minor adaptations to an atomic force microscope (AFM), if anything, simplifying its mode of operation over that conventionally used. They brought a sharp conducting tip, positioned on the end of a flexible cantilever, into contact with the surface of a lithium niobate crystal. They then trawled this metallic tip across the crystal surface and, using a sensitive detection circuit, monitored minute currents associated with charge redistribution between the tip and its grounded electrode. Lithium niobate is an insulating oxide, and is important in telecommunications applications. Within each of the unit cells of this crystalline material, ions are naturally arranged such that the centres of positive and negative charge are slightly separated spatially. This results in what is termed an electric dipole, a vector entity that points from negative to positive within the crystal. In lithium niobate, the orientation of this dipole can be reversed by an electric field, defining the material as ferroelectric. For practical applications, reversals in dipole orientation induced by an electric field are engineered to make spatially periodic stripe patterns, in which positive and negative charges are alternately closer to the crystal surface, in distinct regions called domains. When electrodes are brought into 2 6 J U N E 2 0 1 4 | VO L 5 1 0 | NAT U R E | 4 8 1
© 2014 Macmillan Publishers Limited. All rights reserved
RESEARCH NEWS & VIEWS Grounded electrode A
A
Tip
–
+
Charge on tip
Ground
–
–
–
–
–
+
+
+
+
+
+
+
+
+
+
–
–
–
–
–
Current left to right Current right to left 0
Figure 1 | Charge gradient mapping. Hong and colleagues1 have developed a technique for determining charge variations on the surface of ferroelectric materials. A metallic tip connected to the ground by means of an electrode scans rapidly across the surface of the material, which is also connected to the ground. The tip records a current, in a device equivalent to an ammeter (A), only when it encounters a charge gradient at a junction between two distinct domains, in which positive and negative charges are alternately closer to the surface; white arrows denote the electric-dipole vectors in each domain. The magnitude of the current (blue and brown curves), which is associated with charge redistribution in the tip (grey curve), is proportional to the charge gradient as well as to the tip velocity; its sign is governed by the direction of the charge gradient, as well as by the scan direction.
contact, the proximity of the near-surface charges in the ferroelectric material induces charge redistribution in the electrode: charges of opposite sign to the near-surface charges in the material are attracted to the interface between the electrode and the ferroelectric material, and like charges are repelled. The same thing happens when an AFM tip is used as an electrode (Fig. 1). If the tip is moved within a single domain, because dipoles are uniformly oriented, the initial charge redistribution that occurs in the tip is unchanged. However, whenever the tip passes over the junction between one domain and the next (over a domain wall), dipole reversal causes complementary charges to flow between the tip and its grounded electrode. Because the current associated with this charge redistribution between the tip and the ground is proportional to the rate of change of charge at the tip, it increases with the scanning speed of the AFM tip (complete charge redistribution is forced to occur in a shorter time). Moreover, because the nature of the charge redistribution in the tip depends on the direction in which a specific domain wall is traversed, the sign of the current changes whenever the scan direction is reversed. Hong et al. point out that the total current that is generated as the tip passes over each domain wall equates to a charge-density change at the ferroelectric material’s surface that is around twice that of the dipole density in lithium niobate, exactly as would be expected. The finding confirms that this method can quantitatively, as well as qualitatively, map nanoscale charge-density variations. The authors highlight the possibly confounding influence of ions and dipolar molecules in the air that nullify the electric fields generated by the near-surface charges in the ferroelectric material. However, they note
that their imaging technique, which they call charge gradient microscopy, is largely immune to such airborne layers, because it seems that the tip scrapes them away. Although this method for imaging charge variation is conceptually quite simple, its potential importance cannot be overstated. It has become evident that the domain walls of ferroelectric materials can have diverse functional characteristics that are completely different from the domains that they delineate: they can be conducting2, or even superconducting3, when the domains themselves are insulating. Moreover, they can be moved, injected and annihilated by external electric fields4, and so could be used in new
two-dimensional electronic devices. Hong and colleagues’ imaging technique rapidly locates ferroelectric-material domain walls and could therefore be invaluable in supporting the quest for “domain wall nanoelectronics”5. Furthermore, its discovery comes at a time when shortcomings in the most common method for domain imaging in ferroelectric materials (piezoresponse force microscopy) are starting to become uncomfortably apparent: for example, high-quality images of ‘domains’ have been obtained using piezoresponse force micro scopy in materials in which ferroelectricity is extremely unlikely6,7. Restricting the authors’ charge gradient microscopy to imaging ferroelectric domain walls, exciting as this is, would probably be a mistake. Variation in nanoscale charge distribution must be crucial in many aspects of science, from understanding communications in cell biology to teasing out the details of battery electrochemistry. Perhaps the adoption of this imaging technique by other areas of science will allow its full potential to be realized. ■ J. Marty Gregg and Amit Kumar are in the Centre for Nanostructured Media, School of Mathematics and Physics, Queen’s University Belfast, Belfast BT7 1NN, UK. e-mails: [email protected]; [email protected] 1. Hong, S. et al. Proc. Natl Acad. Sci. USA 111, 6566–6569 (2014). 2. Sluka, T., Tagantsev, A. K., Bednyakov, P. & Setter, N. Nature Commun. 4, 1808 (2013). 3. Aird, A. & Salje, E. K. H. J. Phys. Condens. Matter 10, L377–L380 (1998). 4. Whyte, J. R. et al. Adv. Mater. 26, 293–298 (2014). 5. Catalan, G., Seidel, J., Ramesh, R. & Scott, J. F. Rev. Mod. Phys. 84, 119–156 (2012). 6. Bark, C. W. et al. Nano Lett. 12, 1765–1771 (2012). 7. Kim, Y. et al. ACS Nano 6, 7026–7033 (2012).
STR UC TUR A L B I OLOGY
Enzyme assembly line pictured Many enzymes form ‘assembly lines’ containing a series of catalytic modules. Visualization of how the structure of a module shifts during catalysis provides a clearer idea of how such enzymes work. See Article p.512 & Letter p.560 P E T E R F. L E A D L AY
L
iving cells contain many molecular machines that integrate and orchestrate essential processes. One of the most familiar is the ribosome, which polymerizes amino acids to form proteins. In the early 1990s, a radically different type of polymerizing assembly line was discovered1,2: poly ketide synthase (PKS) multi-enzymes, which
4 8 2 | NAT U R E | VO L 5 1 0 | 2 6 J U N E 2 0 1 4
© 2014 Macmillan Publishers Limited. All rights reserved
make polyketide compounds, including many important antibiotics. Remarkably, these complexes use a different module of catalytic sites for each cycle of a chemical-chain extension. Efforts to understand these sophisticated catalysts have received a great boost from two studies in this issue from the same research group: Dutta et al.3 (page 512) report the three-dimensional structure of a PKS protein that houses an intact catalytic module, and
