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Category: Microbial Genetics and Molecular Biology
Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography, Page 1 of 2
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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.
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Hybrid-states model for the translocation cycle ( Moazed and Noller, 1989a ). 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.
Hybrid-states model for the translocation cycle ( Moazed and Noller, 1989a ). 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.
Extended hybrid-states model ( Wilson and Noller, 1998b ). Additional steps in the translocational cycle have been added, based on more recent studies described in the text.
Extended hybrid-states model ( Wilson and Noller, 1998b ). Additional steps in the translocational cycle have been added, based on more recent studies described in the text.
Crystal structure of EF-G•GDP ( Czworkowski et al., 1994 ), showing several of the positions of tethering Fe(II) used for directed hydroxyl radical probing of rRNA in the ribosome ( Wilson and Noller, 1998a ). 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.
Crystal structure of EF-G•GDP ( Czworkowski et al., 1994 ), showing several of the positions of tethering Fe(II) used for directed hydroxyl radical probing of rRNA in the ribosome ( Wilson and Noller, 1998a ). 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.
Predicted position of EF-G in the ribosome, based on directed hydroxyl radical probing results ( Wilson and Noller, 1998a ). The locations of different ribosomal features are as indicated.
Predicted position of EF-G in the ribosome, based on directed hydroxyl radical probing results ( Wilson and Noller, 1998a ). The locations of different ribosomal features are as indicated.
(top). Fourier difference map (yellow) of ribosome cocrystals containing A-site tRNA versus crystals with a vacant A site, at 25-Å resolution ( Cate et al., 1999 ), 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.
(top). Fourier difference map (yellow) of ribosome cocrystals containing A-site tRNA versus crystals with a vacant A site, at 25-Å resolution ( Cate et al., 1999 ), 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.
(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 tRNAPhe at 8.8Å, showing the ASL region in gray and the rest of the tRNA in blue.
(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 tRNAPhe at 8.8Å, showing the ASL region in gray and the rest of the tRNA in blue.
(left). Electron density map of the complete 70S ribosome from T. thermophilus at 7.8-Å resolution ( Cate et al., 1999 ). 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.
(left). Electron density map of the complete 70S ribosome from T. thermophilus at 7.8-Å resolution ( Cate et al., 1999 ). 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.
(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.
(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.
Chemical footprints of ribosomal protein S8 on 16S rRNA, using hydroxyl radicals generated by free Fe(II)-EDTA ( Powers and Noller, 1995 ) and base-specific probes ( Svensson et al., 1988 ). The relative strengths of protection are indicated by the sizes of the solid circles.
Chemical footprints of ribosomal protein S8 on 16S rRNA, using hydroxyl radicals generated by free Fe(II)-EDTA ( Powers and Noller, 1995 ) and base-specific probes ( Svensson et al., 1988 ). The relative strengths of protection are indicated by the sizes of the solid circles.
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 ( Powers and Noller, 1995 ) 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.
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 ( Powers and Noller, 1995 ) 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.
Directed hydroxyl radical probing of 16S rRNA from six different positions on the surface of protein S8 ( Lancaster et al., unpubished ). The relative strengths of cleavage are indicated by the sizes of the solid circles.
Directed hydroxyl radical probing of 16S rRNA from six different positions on the surface of protein S8 ( Lancaster et al., unpubished ). The relative strengths of cleavage are indicated by the sizes of the solid circles.
Protection of 16S (A) and 23S (B) rRNAs from hydroxyl radical and base-specific probes ( Merryman et al., 1999a , 1999b ). The relative strengths of protection are indicated by the sizes of the solid circles.
Protection of 16S (A) and 23S (B) rRNAs from hydroxyl radical and base-specific probes ( Merryman et al., 1999a , 1999b ). The relative strengths of protection are indicated by the sizes of the solid circles.
Protection of 16S rRNA in 30S ribosomal subunits from hydroxyl radical probing by initiation factor IF-3 ( Dallas and Noller, unpublished ). The relative strengths of protection are indicated by the sizes of the solid circles.
Protection of 16S rRNA in 30S ribosomal subunits from hydroxyl radical probing by initiation factor IF-3 ( Dallas and Noller, unpublished ). The relative strengths of protection are indicated by the sizes of the solid circles.
(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 ( Stallings and Moore, 1997 ) to the electron density of the intersubunit bridge. The loop region is at the right, in contact with the 30S subunit.
(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 ( Stallings and Moore, 1997 ) to the electron density of the intersubunit bridge. The loop region is at the right, in contact with the 30S subunit.
(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 ( Merryman et al., 1999b ). The relative strengths of cleavage and protection are indicated by the sizes of the solid circles.
(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 ( Merryman et al., 1999b ). The relative strengths of cleavage and protection are indicated by the sizes of the solid circles.
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.
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.
Nucleotides in 16S rRNA protected from base-specific probes by P-site tRNA ( Moazed and Noller, 1986 , 1990 ) (A) and those whose modification interferes with P-site tRNA binding ( von Ahsen and Noller, 1995 ) (B). Solid circles and triangle are (A) protections or (B) selected against for tRNA binding. The triangle indicates N7 position.
Nucleotides in 16S rRNA protected from base-specific probes by P-site tRNA ( Moazed and Noller, 1986 , 1990 ) (A) and those whose modification interferes with P-site tRNA binding ( von Ahsen and Noller, 1995 ) (B). Solid circles and triangle are (A) protections or (B) selected against for tRNA binding. The triangle indicates N7 position.
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 ( Joseph et al., 1997 ). 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).
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 ( Joseph et al., 1997 ). 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).
(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.
(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.
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 ( Moazed and Noller, 1989b , 1991 ). Several sites cross-linked by the acceptor ends of A- and P-site tRNAs are indicated by small arrows ( Wower et al., 1989 ; Mitchell et al., 1993 ; Hall et al., 1988 ; Steiner et al., 1988 ). Features that base-pair with the C74 of P-tRNA ( Samaha et al., 1995 ) and C75 of A-tRNA ( Green et al., 1998 ; Kim and Green, in press ) are indicated as the P loop and A loop, respectively.
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 ( Moazed and Noller, 1989b , 1991 ). Several sites cross-linked by the acceptor ends of A- and P-site tRNAs are indicated by small arrows ( Wower et al., 1989 ; Mitchell et al., 1993 ; Hall et al., 1988 ; Steiner et al., 1988 ). Features that base-pair with the C74 of P-tRNA ( Samaha et al., 1995 ) and C75 of A-tRNA ( Green et al., 1998 ; Kim and Green, in press ) are indicated as the P loop and A loop, respectively.
Fourier difference map of P-tRNA (yellow) superimposed on 7.8-Å electron density map of the T. thermophilus 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.
Fourier difference map of P-tRNA (yellow) superimposed on 7.8-Å electron density map of the T. thermophilus 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.
MAD phasing of 70S ribosome structure
MAD phasing of 70S ribosome structure
Directed hydroxyl radical probing of rRNA from specific positions on ribosomal proteins and translation factors
Directed hydroxyl radical probing of rRNA from specific positions on ribosomal proteins and translation factors