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Chapter 6 : Studies of Elongation Factor G-Dependent tRNA Translocation by Three-Dimensional Cryo-Electron Microscopy

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Studies of Elongation Factor G-Dependent tRNA Translocation by Three-Dimensional Cryo-Electron Microscopy, Page 1 of 2

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Abstract:

This chapter presents a detailed analysis of cryo-EM results, which reveal conformational changes of both elongation factors (EFs)-G and the ribosome. Fusidic acid was used to stabilize the binding of EF-G in the GDP state; its presence, following GTP hydrolysis, prevents the dissociation of EF-G from the ribosome. The binding position of the anticodon stem of the A-site tRNA and the tip portion of domain IV of EF-G show substantial overlap. The chapter makes use of information from hydroxyl radical probing of EF-G on the ribosome to locate positions of various ribosomal RNA fragments on the ribosome. Burma and coworkers and Mesters and coworkers showed that EF-dependent GTPase activity changes the conformation of the naked ribosome. However, in the absence of a 3-D map, the nature of these conformational changes was unknown. The results conclusively show that a number of ribosomal regions undergo a change of conformation upon EF-G binding. A very recent cryo-EM localization study of Tet(O) protein reveals that Tet(O) indeed has a structure similar to that of EF-G and binds in the same position on the 70S ribosome. From the comparison of the binding of all these factors to the ribosome, it appears that they use the same anchoring points on the ribosome and make contact with the same GTPase-associated center while differing substantially in their functions.

Citation: Agrawal R, Heagle A, Frank J. 2000. Studies of Elongation Factor G-Dependent tRNA Translocation by Three-Dimensional Cryo-Electron Microscopy, p 53-62. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch6

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Figures

Image of Figure 1.
Figure 1.

Stereo-view presentation of 3-D cryo-EM maps (transparent blue) of the 70S•EF-G•GMPP(CH)P complex ( Table 1 , complex 2) (top) and the 70S•(tRNA)•EF-G•GDP•fusidic acid complex ( Table 1 , complex 3) (bottom). The 30S subunit is below the 50S subunit. In the top panel, the stalk is bifurcated and no connection is formed between the stalk base and EF-G (red and transparent dark blue). In this panel, the difference masses corresponding to A-site (pink) and P-site (green) tRNAs were obtained from the 70S•(tRNA)•EF-G•GMPP(CH)P complex ( Table 1 , complex 4), prepared with a lower concentration of EF-G (0.8 M), and the fragmented mass corresponding to domains I, II, and III of EF-G (red) was obtained from the 70S•(tRNA)•EF-G•GMPP(CH2)P complex Table 1 , complex 5), prepared with a higher concentration of EF-G (1.6 ) ( ). The mass shown in transparent dark blue corresponds to EF-G in the 70S•EF-G•GMPP(CH)P complex ( Table 1 , complex 2). Landmarks of the 30S subunit are as follows: h, head; pt, platform; sp, spur. Landmarks of the 50S subunit are as follows: CP, central protuberance; L1, L1 protein; St, stalk. In the bottom panel, the L12 stalk is in an extended conformation and an arc-like connection (arc) is present between the stalk base and EF-G. In addition to EFG (red), a distinct mass corresponding to the P-site tRNA (green) and a smeared mass corresponding to the E-site tRNAs (yellow) are seen in the intersubunit space.

Citation: Agrawal R, Heagle A, Frank J. 2000. Studies of Elongation Factor G-Dependent tRNA Translocation by Three-Dimensional Cryo-Electron Microscopy, p 53-62. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch6
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Image of Figure 2
Figure 2

(Top) Stereo-view presentation showing the positions of various domains of EF-G on the ribosome (white wire mesh) obtained by fitting the X-ray crystal structure of EF-G into the mass corresponding to EF-G in the 70S•(tRNA)•EF-G •GDP•fusidic acid complex. A detailed description of the fitting procedure is provided elsewhere ( ). Various domains of the crystal structure of EF-G are shown in different colors: magenta and pink, domain I (magenta, G domain, and pink, G′domain); blue, domain II; green, domain III; yellow, domain IV; and red, domain V. Landmarks of the 30S subunit are as follows: b, body; h, head; sp, spur. Landmarks of the 50S subunit are as follows: CP, central protuberance; St, stalk. The arrow points to the mRNA channel passing through the neck of the 30S subunit (see chapter 5). (Middle) Stereo-view presentation showing the proximity of various amino acids of EF-G that are expected to be close to the specific nucleotide residues of rRNAs ( ). Amino acid residues are shown as beads (5-Å radius) of different colors. Magenta represents amino acid 196 of domain I (G domain) of EF-G, which is proximal to nucleotides 2650 to 2653 and 2668 to 2669 of 23S RNA. Blue 1 and 2 represent amino acids 301 and 314, respectively, of domain II of EF-G, which are proximal to nucleotides 37 to 39, 441 to 445, 496 to 497, and 537 to 539 and to nucleotides 368 to 370, 384 to 385, and 493 to 497 of 16S RNA, respectively. Yellow represents amino acid 541 of domain IV of EF-G, which is proximal to nucleotides 1213 to 1214 of 16S RNA. Orange 1 and 2 represent amino acids 506 and 585, respectively, of domain IV of EF-G. These two amino acids are proximal to specific residues of both 16S and 23S RNAs, indicating that 23S RNA reaches the decoding region of the 30S subunit, thus strongly supporting Brimacombe's cross-linking data ( ). Amino acid residue 506 is proximal to nucleotides 1228 to 1230 of the 16S RNA and nucleotides 1920 to 1925 of the 23S RNA, whereas amino acid residue 585 is proximal to nucleotides 790, 1229 to 1230, 1339 to 1340, and 1397 to 1400 (decoding region) of the 16S RNA and nucleotides 1921 to 1923 of the 23S RNA. Red 1 and 2 represent amino acids 650 and 655, respectively, of domain V of EF-G, which are proximal to nucleotides 1100 and 2659 and 1065 to 1068 of 23S RNA, respectively. (Bottom) Fitting of the crystal structures of the L11-23S RNA fragment (nucleotides 1051 to 1108) complex ( ) and the -sarcin-ricin stem-loop ( ) into the 15-Å-resolution map ( ) of the 70S ribosome. the placement of crystal structures is based on proximities of 23S RNA nucleotides to amino acids of EF-G (see the middle panel), and is strongly supported by the similarity between the structural features of the crystal structure and the cryo-EM map. The L11-23S RNA fragment was refitted in line with the placement of this structure into the 5Å-resolution X-ray map of . Green, L11 protein portion of the complex; magenta, 58-nucleotide portion of the same complex; yellow, -sarcin-ricin stem-loop structure.

Citation: Agrawal R, Heagle A, Frank J. 2000. Studies of Elongation Factor G-Dependent tRNA Translocation by Three-Dimensional Cryo-Electron Microscopy, p 53-62. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch6
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Figure 3

Relative movement of the 30S subunit with respect to the 50S subunit upon EF-G•GMPP(CH)P binding to the 70S ribosome. Two volumes, the 70S•fMet-tRNAMet complex ( ) (shown as solid blue) and the 70S•EF-G•GMPP(CH)P complex (transparent pink), were superimposed so that the 50S subunits of the two volumes were perfectly aligned. The 50S subunits of both volumes have been removed to avoid visual confusion. (Reproduced from ) The landmarks are the same as in Fig. 2 , top panel.

Citation: Agrawal R, Heagle A, Frank J. 2000. Studies of Elongation Factor G-Dependent tRNA Translocation by Three-Dimensional Cryo-Electron Microscopy, p 53-62. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch6
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Image of Figure 4
Figure 4

Side by side comparison of binding positions of EF-G (red; adapted from ) (left) and ternary complex (orange) of Phe-tRNA, EF-Tu, and GDP ( ) on the 15-Å-resolution map ( ) of the 70S ribosome. The binding of the ternary complex was arrested by using a kirromycin-stalled ribosome. The landmarks are the same as those introduced in Fig. 1 (top panel) and 2 (top panel). ch, mRNA channel.

Citation: Agrawal R, Heagle A, Frank J. 2000. Studies of Elongation Factor G-Dependent tRNA Translocation by Three-Dimensional Cryo-Electron Microscopy, p 53-62. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch6
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Image of Figure 5
Figure 5

Stereo-view presentation to show the proximity of the tip of domain IV of EF-G (yellow) to the anticodon (green) end of the A-site tRNA (red).

Citation: Agrawal R, Heagle A, Frank J. 2000. Studies of Elongation Factor G-Dependent tRNA Translocation by Three-Dimensional Cryo-Electron Microscopy, p 53-62. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch6
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Tables

Generic image for table
Table 1

70S ribosome•EF-G complexes used in this study

Citation: Agrawal R, Heagle A, Frank J. 2000. Studies of Elongation Factor G-Dependent tRNA Translocation by Three-Dimensional Cryo-Electron Microscopy, p 53-62. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch6

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