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Chapter 13 : Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography

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Abstract:

The study of the process of protein synthesis, which encompasses translation of the genetic code, has been intensively under way for about 4 decades. This chapter summarizes recent studies of the structure and function of ribosomes by a combination of biochemical and X-ray-crystallographic approaches, focusing primarily on work from the laboratory. Ribosomologists have traditionally used a wide variety of experimental strategies to study the many molecular interactions of the translational machinery. EF-G was bound in the presence of fusidic acid and GTP to ribosomes containing mRNA and tRNA bound to the P site, and hydroxyl radicals were initiated. Three-dimensional crystals of ribosomes and ribosomal subunits were obtained more than a decade ago, but only recently has X-ray crystallography begun to provide detailed information about the large-scale structure of the ribosome. In the X-ray map, bridges from elements originating in the 50S subunit can be seen to contact the minor groove of this helix at positions corresponding closely to those predicted from the foot printing studies. The arrangement of the S8-16S rRNA interaction has been investigated extensively by directed hydroxyl radical probing. Modification interference studies with kethoxal showed that modification of a set of guanines in 16S rRNA interferes with subunit association. The RNA chemical-probing studies left unaddressed the possibility that protein-RNA (or protein-protein) interactions might play a role in subunit association.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13

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Image of Figure 1
Figure 1

Hybrid-states model for the translocation cycle ( ). The ribosomal binding sites (A, P, and E) for tRNA on the 30S and 50S subunits are schematized as rectangles. tRNAs are shown as sticks, with a squiggle and "aa" representing the peptidyl and aminoacyl moieties, respectively. The elongation factors EF-Tu and EF-G are represented as circles. The different binding states for tRNA (P /P, A/T, A/A, P/E, A/P, and E) are indicated at the bottom.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Figure 2

Extended hybrid-states model ( ). Additional steps in the translocational cycle have been added, based on more recent studies described in the text.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 3
Figure 3

Crystal structure of EF-G•GDP ( ), showing several of the positions of tethering Fe(II) used for directed hydroxyl radical probing of rRNA in the ribosome ( ). The tethering positions shown in yellow target 16S rRNA, those in red target 23S rRNA, and those in blue target both 16S and 23S rRNAs. Roman numerals in red indicate targeted domains of 23S rRNA; black roman numerals indicate domains of EF-G.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 4
Figure 4

Predicted position of EF-G in the ribosome, based on directed hydroxyl radical probing results ( ). The locations of different ribosomal features are as indicated.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 5
Figure 5

(top). Fourier difference map (yellow) of ribosome cocrystals containing A-site tRNA versus crystals with a vacant A site, at 25-Å resolution ( ), superimposed on electron density of the complete 70S ribosome (blue), viewed from the L12 side of the ribosome. The 30S subunit is on the left, and the 50S subunit is on the right. The absence of visible calculated negative density (red) is indicative of the low noise level of the difference map at this resolution.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 6
Figure 6

(bottom). (A) Stereo view of an 8.8-Å Fourier difference map of a ribosome cocrystal containing a full-length P-site tRNA versus a cocrystal containing only a P-site-bound ASL. Positive density is shown in blue, and negative density is shown in red. (B) Calculated electron density for tRNA at 8.8Å, showing the ASL region in gray and the rest of the tRNA in blue.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 7
Figure 7

(left). Electron density map of the complete 70S ribosome from at 7.8-Å resolution ( ). The 30S subunit (blue) is in the foreground, with the head at the top and the platform to the left. The 50S subunit (gray) is behind, with its L1 ridge protruding at the left and the L12 stalk region to the right.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 8
Figure 8

(right). View of the 30S subunit excised from the electron density map of the 70S ribosome, viewed from the 50S subunit. The head is at the top, the platform is at the right, and body is to the left. The penultimate stem can be seen as an ~100-Å-long helix extending from just below the middle of the head, angling slightly to the left, to the bottom of the subunit.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 9
Figure 9

Chemical footprints of ribosomal protein S8 on 16S rRNA, using hydroxyl radicals generated by free Fe(II)-EDTA ( ) and base-specific probes ( ). The relative strengths of protection are indicated by the sizes of the solid circles.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 10
Figure 10

The S8 binding region of the 30S ribosomal subunit. A pseudoatom model for 16S rRNA (yellow) is superimposed on the 7.8-Å electron density map. Nucleotides protected from hydroxyl radicals by S8 ( ) are shown in red. Density corresponding to protein S8 is indicated. The 620 stem runs from left to right at the bottom, and the 820 and 840 helices run from right to left at the upper left.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 11
Figure 11

Directed hydroxyl radical probing of 16S rRNA from six different positions on the surface of protein S8 ( ). The relative strengths of cleavage are indicated by the sizes of the solid circles.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 12
Figure 12

Protection of 16S (A) and 23S (B) rRNAs from hydroxyl radical and base-specific probes ( ). The relative strengths of protection are indicated by the sizes of the solid circles.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 13
Figure 13

Protection of 16S rRNA in 30S ribosomal subunits from hydroxyl radical probing by initiation factor IF-3 ( ). The relative strengths of protection are indicated by the sizes of the solid circles.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 14
Figure 14

(A) View of the subunit interface region of the 7.8-Å electron density map of the 70S ribosome, showing an RNA helical bridge extending from the 50S subunit to the bottom of the platform of the 30S subunit. (B) Fitting the solution structure of the U2 snRNA loop IIA ( ) to the electron density of the intersubunit bridge. The loop region is at the right, in contact with the 30S subunit.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 15
Figure 15

(Left and center) Cleavage targets in 23S rRNA after directed hydroxyl radical probing from positions 12 and 46 of protein S15 (Culver and Noller, unpublished). (Right) Protection of 23S rRNA from free hydroxyl radicals by association of 30S and 50S subunits ( ). The relative strengths of cleavage and protection are indicated by the sizes of the solid circles.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Figure 16

Similarity between sequences of the 715 loop of 23S rRNA and the U2 snRNA loop IIA. Nucleotides in bold are conserved in all three loop structures.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 17
Figure 17

Nucleotides in 16S rRNA protected from base-specific probes by P-site tRNA ( ) (A) and those whose modification interferes with P-site tRNA binding ( ) (B). Solid circles and triangle are (A) protections or (B) selected against for tRNA binding. The triangle indicates N7 position.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Image of Figure 18
Figure 18

Calculated allowed positions of targets of directed hydroxyl radical probing from Fe(II) tethered to ribosome-bound ASLs ranging in length from 4 to 33 bp ( ). Colored clouds are shown for nucleotide targets in 16S rRNA (A and B), 23S rRNA (C and D), and 5S rRNA (E), relative to P-site tRNA (left) and A-site tRNA (right).

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Figure 19

(A) View of the P-site ASL bound to the ribosome in the 7.8-Å electron density map, from the right-hand side (subunit interface at the right). The ASL is shown in yellow, with its anticodon in green; the P-site codon is red. (B) Axial view of the P-site ASL, viewed from the 50S subunit. a, b, and c, bridges to the ASL stem from the 30S subunit; d and e, bridges to the anticodon and codon, respectively; f, an apparent stacking interaction between the 30S subunit and the wobble base pair.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Figure 20

Elements of 23S rRNA associated with the PT function of the ribosome. Closed and open circles indicate bases protected by the acceptor ends of A- and P-site tRNAs ( ). Several sites cross-linked by the acceptor ends of A- and P-site tRNAs are indicated by small arrows ( ). Features that base-pair with the C74 of P-tRNA ( ) and C75 of A-tRNA ( ) are indicated as the P loop and A loop, respectively.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Figure 21

Fourier difference map of P-tRNA (yellow) superimposed on 7.8-Å electron density map of the 70S ribosome in the PT region of the 50S subunit (green), showing the pinching of the CCA tail of the tRNA by features of the 50S subunit.

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
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Tables

Generic image for table
Table 1

MAD phasing of 70S ribosome structure

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13
Generic image for table
Table 2

Directed hydroxyl radical probing of rRNA from specific positions on ribosomal proteins and translation factors

Citation: Noller H, Cate J, Dallas A, Culver G, Green R, Holmberg L, Joseph S, Lancaster L, Lieberman K, Merryman C, Newcomb L, Samaha R, Von Ahsen U, Yusupov M, Yusupova G, Wilson K, Earnest T. 2000. Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, p 127-150. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch13

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