1887

Chapter 4 : Visualization of the Translational Elongation Cycle by Cryo-Electron Microscopy

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $15.00

Preview this chapter:
Zoom in
Zoomout

Visualization of the Translational Elongation Cycle by Cryo-Electron Microscopy, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555818142/9781555811846_Chap04-1.gif /docserver/preview/fulltext/10.1128/9781555818142/9781555811846_Chap04-2.gif

Abstract:

A detailed structural knowledge of the complex and dynamic interplay of the ribosome with mRNA, tRNA, and other protein factors is necessary to understand translation in molecular terms. Cryo-electron microscopy together with single-particle analysis has enabled direct visualization of the ribosome in different functional states. The complexes are either biochemically stable or trapped by antibiotics which arrest the elongation factors Tu and G in their respective binding sites on the ribosome. This chapter reviews the functional implications of these findings and proposes a comprehensive structural model for the elongation cycle. Cryo-electron microscopy of individual noncrystallized molecules (single particles) together with angular reconstitution is a standard approach for probing the three-dimensional (3-D) structure of biological macromolecules. After the localization of tRNA molecules in the pre- and post-translocation state of the ribosome, the binding sites of EF-Tu and EF-G in two different states on the ribosome were determined. The antibiotic does not affect the ribosome binding of EF-G·GTP or GTP hydrolysis, whereas it strongly inhibits translocation and blocks EF-G turnover after GTP hydrolysis and translocation. The body of EF-G is in a position slightly different from that of EF-Tu. This may result from the fact that EF-Tu has not yet undergone its conformational change from the GTP- to the GDP-bound form, which leads to its dissociation from the ribosome.

Citation: Pape T, Matadeen R, Orlova E, Van Heel M, Stark H. 2000. Visualization of the Translational Elongation Cycle by Cryo-Electron Microscopy, p 37-44. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch4

Key Concept Ranking

Elongation Factor Tu
0.5364644
Cryo-Electron Microscopy
0.41294977
0.5364644
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

Figures

Image of Figure 1
Figure 1

Synopsis of the 3-D reconstruction of the 70S ribosome. Electron-microscopic images of individual ribosomes are aligned by multireference alignment techniques (a), and similar images are grouped into classes by multivariate statistical classification procedures. All images belonging to the same class are averaged to produce noise-reduced class averages or characteristic views (b). An Euler angle orientation is assigned to each class average by the angular reconstitution technique. A 3-D reconstruction is computed (c) and used to produce reprojected images (d), which in turn are used as reference images for realignment of the original raw image data set and for improving the accuracy of the Euler angle determinations.

Citation: Pape T, Matadeen R, Orlova E, Van Heel M, Stark H. 2000. Visualization of the Translational Elongation Cycle by Cryo-Electron Microscopy, p 37-44. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 2
Figure 2

Visualization of the elongation cycle by 3-D reconstructions of different ribosome complexes at ˜20-Å resolution. The ternary complex (red) binds to the posttranslocation state (A) with the peptidyl-tRNA in the P site (green) and forms a “GTPase state” on the ribosome (B). After dissociation of EF-Tu•GDP and accommodation of the aa-tRNA (blue) in the A site, peptidyl transfer takes place, leading to the pretranslocation state (C). EF-G•GTP (purple) binding and instantaneous GTP hydrolysis leads to a pre*-translocation state (D), which rearranges after translocation into a post*-translocation state (E). Finally, EF-G•GDP and deacylated tRNA dissociate to give the posttranslocation state again. The numbers indicate the domains of the elongation factors as revealed by fitting the X-ray structures into the respective densities. The views are from the L7-L12 side along the intersubunit space, with the 30S subunit on the left and the 50S subunit on the right. For morphological details, refer to Fig. 3 5 . As the EF-G•GTP X-ray structure is unknown, the EF-G•GDP coordinates were used and are therefore shown in quotation marks.

Citation: Pape T, Matadeen R, Orlova E, Van Heel M, Stark H. 2000. Visualization of the Translational Elongation Cycle by Cryo-Electron Microscopy, p 37-44. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 3
Figure 3

Comparison of the positions of EF-Tu in the ternary complex and EF-G on the ribosome. In the pre* complex the position of the ternary complex (red) is almost orthogonal to the position of EF-G (purple). In the post* complex both factors are pointing into the decoding site, showing similar overall arrangements. The views are the same as in Fig. 2 .

Citation: Pape T, Matadeen R, Orlova E, Van Heel M, Stark H. 2000. Visualization of the Translational Elongation Cycle by Cryo-Electron Microscopy, p 37-44. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 4
Figure 4

Models of the 16S and 23S rRNAs. rRNA elements possibly involved in elongation are highlighted. In the 30S subunit the decoding site (A site) is depicted in blue, the 530 stem-loop is in cyan, helix 34 is in green, and the 912 region (helix 27) is in red. In the 50S subunit the 1070 loop region (the GTPase-associated center) is shown in magenta and the 2660 stem-loop (-sarcin stem-loop) is in purple. Shown in gold are the 790 loop of 16S rRNA and the 1920 loop of 23S rRNA, which probably form the major intersubunit bridge. The morphological details are indicated CP, central protuberance; PTC, peptidyltransferase center. (Coordinates of the models courtesy of R. Brimacombe.)

Citation: Pape T, Matadeen R, Orlova E, Van Heel M, Stark H. 2000. Visualization of the Translational Elongation Cycle by Cryo-Electron Microscopy, p 37-44. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 5
Figure 5

Structural changes of the 30S subunit during translocation viewed from the solvent side. Characteristic morphological features are indicated; those which are not always visible are in parentheses.

Citation: Pape T, Matadeen R, Orlova E, Van Heel M, Stark H. 2000. Visualization of the Translational Elongation Cycle by Cryo-Electron Microscopy, p 37-44. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555818142.chap4
1. Abel, K.,, M. D. Yoder,, R. Hilgenfeld,, and F. Jurnak. 1996. An α to β conformational switch in EF-Tu. Structure 4:11531159.
2. Ævarsson, A.,, E. Brazhnikov,, M. Garber,, J. Zheltonosova,, Y. Chirgadze,, S. al-Karadaghi,, L. A. Svensson,, and A. Liljas. 1994. Three-dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus. EMBO J. 13:36693677.
3. Agrawal, R. K.,, P. Penczek,, R.A. Grassucci,, and J. Frank. 1998. Visualisation of elongation factor G on the Escherichia coli 70S ribosome: the mechanism of translocation. Proc. Natl. Acad. Sci. USA 95:61346138.
4. Allen, P. N.,, and H. F. Noller. 1989. Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA. J. Mol. Biol. 208:457468.
5. Czworkowski, J.,, J. Wang,, T. A. Steitz,, and P. B. Moore. 1994. The crystal structure of elongation factor G complexed with GDP, at 2.7 Å resolution. EMBO J. 13:36613668.
6. Döring, T.,, P. Mitchell,, M. Osswald,, D. Bochkariov,, and R. Brimacombe. 1994. The decoding region of 16S RNA: a crosslinking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop. EMBO J. 13: 26772685.
7. Dubochet, J.,, M. Adrian,, J. J. Chang,, J. C. Homo,, J. Lepault,, A. W. McDowall,, and P. Schultz. 1988. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21:129228.
8. Gabashvili, I. S.,, R. K. Agrawal,, R. Grassucci,, and J. Frank. 1999. Structure and structural variations of the Escherichia coli 30 S ribosomal subunit as revealed by three-dimensional cryoelectron microscopy. J. Mol. Biol. 286:12851291.
9. Harauz, G.,, and M. van Heel. 1986. Exact filters for general geometry three-dimensional reconstruction. Optik 73:146156.
10. Lodmell, J. S.,, and A. E. Dahlberg. 1997. A conformational switch in Escherichia coli 16S ribosomal RNA during decoding of messenger RNA. Science 277:12621267.
11. Moazed, D.,, and H. F. Noller. 1989. Intermediate states in the movement of transfer RNA in the ribosome. Nature 342:142148.
12. Moazed, D.,, J. M. Robertson,, and H. F. Noller. 1988. Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA. Nature 334:362364.
13. Mueller, F.,, and R. Brimacombe. 1997. A new model for the threedimensional folding of Escherichia coli 16S ribosomal RNA. I. Fitting the RNA to a 3D electron microscopic map at 20 Å. J. Mol. Biol. 271:524544.
14. Mueller, F.,, I. Sommer,, P. Baranov,, R. Matadeen,, M. van Heel,, and R. Brimacombe. 1999. The 3D arrangement of the RNA in the E. coli 50S ribosomal subunit. I. Fitting the 23S and 5S rRNA to a cryo-electron microscopic map of the 70S ribosome at 13 Å resolution. Submitted for publication.
15. Nissen, P.,, M. Kjeldgaard,, S. Thirup,, G. Polekhina,, L. Reshetnikova,, B. F. Clark,, and J. Nyborg. 1995. Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. Science 270:14641472.
16. Noller, H. F.,, T. Powers,, P. N. Allen,, D. Moazed,, and S. Stern,. 1996. rRNA and translation: tRNA selection and movement in the ribosome, p. 239258. In R. A. Zimmermann, and A. E. Dahlberg, (ed.), Ribosomal RNA Structure, Evolution, Processing, and Function in Protein Biosynthesis. CRC Press, Boca Raton, Fla.
17. Pape, T. 1998. Induced fit in aminoacyl-tRNA selection on the ribosome. Ph.D. thesis. Witten/Herdecke University, Witten, Germany.
18. Pape, T.,, W. Wintermeyer,, and M. V. Rodnina. 1998. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome. EMBO J. 17:74907497.
19. Pape, T.,, W. Wintermeyer,, and M. V. Rodnina. 1999. Induced fit in initial selection and proofreading of aminoacyl-tRNA on the ribosome. EMBO J. 18:38003807.
20. Paulsen, H.,, J. M. Robertson,, and W. Wintermeyer. 1983. Topological arrangement of two transfer RNAs on the ribosome. Fluorescence energy transfer measurements between A and P site-bound tRNAPhe. Nucleic Acids Res. 10:26512663.
21. Polekhina, G.,, S. Thirup,, M. Kjeldgaard,, P. Nissen,, C. Lippmann,, and J. Nyborg. 1996. Helix unwinding in the effector region of elongation factor EF-Tu-GDP. Structure 4:11411151.
22. Porse, B. T.,, I. Leviev,, A. S. Mankin,, and R. A. Garrett. 1998. The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase center. J. Mol. Biol. 276: 391404.
23. Powers, T.,, and H. F. Noller. 1994. The 530 loop of 16S rRNA: a signal to EF-Tu? Trends Genet. 10:2731.
24. Radermacher, M. 1988. Three-dimensional reconstruction of single particles from random and non-random tilt series. J. Electron Microsc. Tech. 9:359394.
25. Rodnina, M. V.,, R. Fricke,, and W. Wintermeyer. 1994. Transient conformational states of aminoacyl-tRNA during ribosome binding catalysed by elongation factor Tu. Biochemistry 33:1226712275.
26. Rodnina, M. V.,, R. Fricke,, L. Kuhn,, and W. Wintermeyer. 1995. Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome. EMBO J. 14: 26132619.
27. Rodnina, M. V.,, T. Pape,, R. Fricke,, L. Kuhn,, and W. Wintermeyer. 1996. Initial binding of the elongation factor Tu•GTP•aminoacyl-tRNA complex preceding codon recognition on the ribosome. J. Biol. Chem. 271:646652.
28. Rodnina, M. V.,, A. Savelsbergh,, V. I. Katunin,, and W. Wintermeyer. 1997. Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome. Nature 385:3741.
29. Rodnina, M. V.,, A. Savelsbergh,, N. B. Matassova,, V. I. Katunin,, Y. P. Semenkov,, and W. Wintermeyer. 1999. Thiostrepton inhibits the turnover but not the GTPase of elongation factor G on the ribosome. Proc. Natl. Acad. Sci. USA 96:95869590.
30. Stark, H.,, E. V. Orlova,, J. Rinke-Appel,, N. Junke,, F. Mueller,, M. Rodnina,, W. Wintermeyer,, R. Brimacombe,, and M. van Heel. 1997a. Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy. Cell 88:1928.
31. Stark, H.,, M. V. Rodnina,, J. Rinke-Appel,, R. Brimacombe,, W. Wintermeyer,, and M. van Heel. 1997b. Visualisation of elongation factor Tu on the Escherichia coli ribosome. Nature 389: 403406.
32. Stark, H.,, M. V. Rodnina,, F. Zemlin,, W. Wintermeyer,, and M. van Heel. The 13Å structure of the E. coli ribosome: conformational changes of elongation factor Tu upon ribosome binding. Unpublished data.
33. Stark, H.,, M. V. Rodnina,, M. van Heel,, and W. Wintermeyer. Large-scale movement of elongation factor G and extensive conformational changes of the ribosome during translocation as visualised by electron cryomicroscopy. Submitted for publication.
34. Stern, S.,, T. Powers,, L. M. Changchien,, and H. F. Noller. 1989. RNA-protein interactions in 30S ribosomal subunits: folding and function of 16S rRNA. Science 244:783790.
35. Tapprich, W. E.,, and A. E. Dahlberg. 1990. A single base mutation at position 2661 in E. coli 23S ribosomal RNA affects the binding of ternary complex to the ribosome. EMBO J. 9:26492655.
36. Traut, R. R.,, D. Dey,, D. E. Bochkariov,, A. V. Oleinikov,, G. G. Jokhadze,, B. Hamman,, and D. Jameson. 1995. Location and domain structure of Escherichia coli ribosomal protein L7/L12: site specific cysteine cross-linking and attachment of fluorescent probes. Biochem. Cell Biol. 73:949958.
37. van Heel, M. 1987. Angular reconstitution: a posteriori assignment of projection directions for 3D reconstruction. Ultramicroscopy 21:111124.
38. van Heel, M. 1989. Classification of very large electron microscopical image data sets. Optik 82:114126.
39. van Heel, M.,, and J. Frank. 1981. Use of multivariate statistics in analysing the images of biological macromolecules. Ultramicroscopy 6:187194.
40. van Heel, M.,, G. Harauz,, E. Orlova,, R. Schmidt,, and M. Schatz. 1996. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116:1724.
41. Willie, G. R.,, N. Richman,, W. P. Godtfredsen,, and J. W. Bodley. 1975. Some characteristics of and structural requirements for the interaction of 24,25-dihydrofusidic acid with ribosome-elongation factor G complexes. Biochemistry 14:17131718.
42. Wilson, K. S.,, and H. F. Noller. 1998. Mapping the position of translational elongation factor EF-G in the ribosome by directed hydroxyl radical probing. Cell 92:131139.
43. Wimberly, B. T.,, R. Guymon,, J. P. McCutcheon,, S. W. White,, and V. Ramakrishnan. 1999. A detailed view of a ribosomal active site: the structure of the L11-RNA complex. Cell 97:491502.
44. Zimmermann, R. A., 1996. The decoding domain, p. 277309. In R. A. Zimmermann, and A. E. Dahlberg (ed.), Ribosomal RNA Structure, Evolution, Processing, and Function in Protein Biosynthesis. CRC Press, Boca Raton, Fla.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error