Three-dimensional structure of 50 S Escherichia coli ribosomal subunits depleted of proteins L7/L12

A structural study of Escherichia coli 50 S ribosomal subunits depleted selectively of proteins L7/L12 and visualized by low-dose electron microscopy has been carried out by multivariate statistical analysis, classification schemes and the new reconstruction technique from single-exposure, random-conical tilt series. This approach has allowed us to solve the three-dimensional structure of the depleted 50 S subunits at a resolution of 3 nm-1. In addition, two distinct morphological populations of subunits (cores) have been identified in the electron micrographs analyzed and have been separately studied in three dimensions. Depleted subunits in the two morphological states present as main features common to these two structures but different from those of the non-depleted subunit (1) the absence of the stalk, (2) a rearrangement of the stalk-base that changes the overall structure of this region. This morphological change is quite noticeable and important, since this region is mapped as a part of the GTPase center. The two conformations differ mainly in the orientation of the area between the L1 region and the head (the probable localization of the peptidyl transferase center) and in the accessibility of the region located below the head. A possible relationship of these structural changes to the functional dynamics of the ribosome is suggested.     

Chemical reactivity of E. coli 5S RNA in situ in the 50S ribosomal subunit.

E. coli 50S ribosomal subunits were reacted with monoperphthalic acid under conditions in which non-base paired adenines are modified to their 1-N-oxides. 5S RNA was isolated from such chemically reacted subunits and the two modified adenines were identified as A73 and A99. The modified 5S RNA, when used in reconstitution of 50S subunits, yielded particles with reduced biological activity (50%). The results are discussed with respect to a recently proposed three-dimensional structure for 5S RNA, the interaction of the RNA with proteins E-L5, E-L18 and E-L25 and previously proposed interactions of 5S RNA with tRNA, 16S and 23S ribosomal RNAs.     

Ribosomal protein L7/L12 cross-links to proteins in separate regions of the 50 S ribosomal subunit of Escherichia coli

The 50 S ribosomal subunits from Escherichia coli were modified by reaction with 2-iminothiolane under conditions in which 65 sulfhydryl groups, about 2/protein, were added per subunit. Earlier work showed that protein L7/L12 was modified more extensively than the average but that nearly all 50 S proteins contained sulfhydryl groups. Mild oxidation led to the formation of disulfide protein-protein cross-links. These were fractionated by urea gel electrophoresis and then analyzed by diagonal gel electrophoresis. Cross-linked complexes containing two, three, and possibly four copies of L7/L12 were evident. Cross-links between L7/L12 and other ribosomal proteins were also formed. These proteins were identified as L5, L6, L10, L11, and, in lower yield, L9, L14, and L17. The yields of cross-links to L5, L6, L10, and L11 were comparable to the most abundant cross-links formed. Similar experiments were performed with 70 S ribosomes. Protein L7/L12 in 70 S ribosomes was cross-linked to proteins L6, L10, and L11. The strong L7/L12-L5 cross-link found in 50 S subunits was absent in 70 S ribosomes. No cross-links between 30 S proteins and L7/L12 were observed.     

Gel electrophoretic studies on ribosomal proteins L7L12 and the Escherichia coli 50 s subunit

Three forms of the 50 S ribosomal subunit of Escherichia coli have been separated by agarose/acrylamide gel electrophoresis. The slowest migrating form, S-50 S, corresponded to native 50 S subunits and contained four copies of proteins $\text{L}\text{7}\text{L}\text{12}$. Removal of the four copies of this protein produced a more rapidly migrating form, M-50 S. The M-50 S form was then converted to the fastest migrating form, F-50 S, by removal of additional proteins, including L10 and L11. A one-step removal of a pentameric complex of four copies of $\text{L}\text{7}\text{L}\text{12}$ plus L10 converted the S-50 S subunit directly to the F-50 S subunit. These proteins recombined specifically with the appropriate protein-deficient 50 S subunit at 3 °C to reform the S-50 S subunit, i.e. the M-50 S subunit was converted back to the S-50 S form by the addition of purified proteins $\text{L}\text{7}\text{L}\text{12}$; and the F-50 S subunit bound the pentameric complex of $\text{L}\text{7}\text{L}\text{12}$ and L10 to form S-50 S. The binding of the pentameric complex, isolated by glycerol gradient centrifugation, supports the model that all four copies of proteins $\text{L}\text{7}\text{L}\text{12}$ are together in one part of the ribosome called the “$\text{L}\text{7}\text{L}\text{12}$ stalk”. Only the four copies of $\text{L}\text{7}\text{L}\text{12}$ were removed from the 50 S subunit in low salt (0.125 m-NH₄Cl) plus 50% ethanol at 0 °C. These ribosomes (in the M-50 S form) had less than 5% of the peptide-synthesizing activity of untreated control ribosomes as measured by a poly(U) translation system in vitro. Peptide-synthesizing activity was restored, upon addition of $\text{L}\text{7}\text{L}\text{12}$, back to the treated ribosomes to give 50 S subunits (S-50 S) with a full complement of four copies of $\text{L}\text{7}\text{L}\text{12}$. Antibody to proteins $\text{L}\text{7}\text{L}\text{12}$ bound only to the S-50 S subunits, producing four new bands separated by gel electrophoresis. The bands represented complexes of one, two, three and four antibodies bound to a 50 S subunit. This result was obtained using either 50 S subunits or 70 S tight couples and indicated that all four copies of $\text{L}\text{7}\text{L}\text{12}$ are either located at a single site in the $\text{L}\text{7}\text{L}\text{12}$ stalk or, much less likely, are divided between two symmetrical sites. Proteins $\text{L}\text{7}\text{L}\text{12}$ were not only accessible to their specific antibody but could also be removed from 70 S ribosomes and polyribosomes without causing their dissociation into subunits. The ribosomes and polyribosomes had an increased gel electrophoretic mobility which was reversed by addition of proteins $\text{L}\text{7}\text{L}\text{12}$.     

Investigation of structure of the ribosomal L12/P stalk

This review contains recent data on the structure of the functionally important ribosomal domain, L12/P stalk, of the large ribosomal subunit. It is the most mobile site of the ribosome; it has been found in ribosomes of all living cells, and it is involved in the interaction between ribosomes and translation factors. The difference between the structures of the ribosomal proteins forming this protuberance (despite their general resemblance) determines the specificity of interaction between eukaryotic and prokaryotic ribosomes and the respective protein factors of translation. In this review, works on the structures of ribosomal proteins forming the L12/P-stalk in bacteria, archaea, and eukaryotes and data on structural aspects of interactions between these proteins and rRNA are described in detail.     

Structure of protein-deficient 50 S ribosomal subunits. Particles without 5 S RNA-protein complex retain the L7/L12 stalk and associate with 30 S subunits

50 S ribosomal subunit derivatives without the 5 S RNA-protein complex obtained either by splitting with EDTA or by reconstitution from the 23 S RNA and proteins have been studied by electron microscopy. Removal of the 5 S RNA-protein complex is shown to affect neither the overall morphology of the larger ribosomal subunit nor the mode of its association with the small subunit.     

Reaction of celite-bound fluorescein isothiocyanate with the 50S E. coli ribosomal subunit

The reaction of Celite-bound fluorescein isothiocyanate with E. coli 50S ribosomes and the 50S moiety of the intact 70S particle has been studied. Approximately five dyes react per 50S particle at pH 8.6 or 9.0. Substantial biological activity is retained. No significant difference between the pattern of reactivity of free and complexed 50S particle can be detected. This suggests the absence of major shielding or conformational changes induced in the 50S by combination with the 30S subunit. The most reactive proteins are L1, L2, L3, and L21. Protein L3 is found in 1.5 and 3 m LiCl core particles where it is still very reactive toward fluorescein. Some other core particle proteins are more reactive than they are in the intact ribosome. In general this work supports previous findings that the proteins of the intact 50S subunit are much less exposed than those of the 30S particle.     

Characterization of Mg2+-induced conformational change in the 50S ribosomal subunit by differential hydrogen exchange.

The technique of differential hydrogen exchange allows detection of a conformational change in the 50S subunit of Escherichia coli ribosome when the magnesium concentration is lowered in a range where ribosomal activity is fully preserved. This change is characterized by a seventy-fold acceleration of about thirty labile hydrogens in the case of a Mg2+ jump from 10 mM to 2 mM. The small number of hydrogens involved can explain the difficulty in detecting this change by other methods.     

The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

Ribosomal protein L2 from Escherichia coli binds to and protects from nuclease digestion a substantial portion of 'domain IV' of 23S rRNA. In particular, oligonucleotides derived from the sequence 1757-1935 were isolated and shown to rebind specifically to protein L2 in vitro. Other L2-protected oligonucleotides, also derived from domain IV (i.e. from residues 1955-2010) did not rebind to protein L2 in vitro nor did others derived from domain I. Given that protein L2 is widely believed to be located in the peptidyl transferase centre of the 50S ribosomal subunit, these data suggest that domain IV of 23S rRNA is also present in that active site of the ribosomal enzyme.     

Localization of the protein L2 in the 50 S subunit and the 70 S E. coli ribosome

The protein L2 is found in all ribosomes and is one of the best conserved proteins of this mega-dalton complex. The protein was localized within both the isolated 50 S subunit and the 70 S ribosome of the Escherichia coli bacteria with the neutron-scattering technique of spin-contrast variation. L2 is elongated, exposing one end of the protein to the surface of the intersubunit interface of the 50 S subunit. The protein changes its conformation slightly when the 50 S subunit reassociates with the 30 S subunit to form a 70 S ribosome, becoming more elongated and moving approximately 30 A into the 50 S matrix. The results support a recent observation that L2 is essential for the association of the ribosomal subunits and might participate in the binding and translocation of the tRNAs.     

Shape of protein L11 from the 50S ribosomal subunit of Escherichia coli.

Protein L11 from the 50S ribosomal subunit of Escherichia coli A19 was purified by a method using nondenaturing conditions. Its shape in solution was studied by hydrodynamic and low-angle x-ray scattering experiments. The results from both methods are in good agreement. In buffers similar to the ribosomal reconstitution buffer, the protein is monomeric at concentrations up to 3 mg/mL and has a molecular weight of 16 000-17 000. The protein molecule resembles a prolate ellipsoid with an axial ratio of 5-6:1 a radius of gyration of 34 A, and a maximal length of 150 A. From the low-angle x-ray diffraction data, a more refined model of the protein molecule has been constructed consisting of two ellipsoids joined by their long axes.     

Studies on the binding of the ribosomal protein complex L7/12-L10 and protein L11 to the 5''-one third of 23S RNA: a functional centre of the 50S subunit.

The RNA binding sites of the protein complex of L7/12 dimers and L10, and of protein L11, occur within the 5'-one third of 23S RNA. Binding of the L7/12-L10 protein complex to the 23S RNA is stimulated by protein L11 and vice-versa. This is the second example to be established of mutual stimulation of RNA binding by two ribosomal proteins or protein complexes, and suggests that this may be an important principle governing ribosomal protein-RNA assembly. When the L7/12-L10 complex is bound to the RNA, L10 becomes strongly resistant to trypsin. Since the L7/12 dimer does not bind specifically to the 23S RNA, this suggests that L10 constitutes a major RNA binding site of the protein complex. Only one of the L7/12 dimers is bound strongly in the (L7/12-L10)-23S RNA complex; the other can dissociate with no concurrent loss of L10.     

Preparation and characterization of fluorescent 50S ribosomes. Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli

So that the topographic and dynamic properties of the L7/L12--L10 complex in the 50S ribosome of Escherichia coli could be studied, methods and reagents were developed in order to introduce fluorescent groups at specific positions of these proteins. In the case of L7/L12, this was done by attaching an aldehyde group to Lys-51 of the protein by using 4-(4-formylphenoxy)butyrimidate or by converting the amino terminus of L12 into an aldehyde group by periodate oxidation. Subsequent reaction of the aldehyde groups with newly developed hydrazine derivatives of fluorescein and coumarin resulted in specifically labeled L7/L12 derivatives. L10 was modified at the single cysteine residue with N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide. The fluorescent proteins L10 and L7/L12 could be reconstituted into 50S ribosomes. The resulting specifically labeled 50S ribosomes show 25--100% activity in elongation factor G dependent GTPase as well as in polyphenylalanine synthesis. The fluorescent properties of the labeled 50S ribosomes show that these fluorescent derivatives are suitable for energy transfer studies.     

Functional homology between the 30S ribosomal protein S7 from E. coli K12 and S7 from E. coli MRE600

Summary It is known that the 30S protein S7 from E. coli strain MRE600 is chemically different from the S7 from E. coli strain K12 (Q13). We have reconstituted 30S subunits using S7 from MRE600 and all other molecular components from K12 and compared the functional activity of the reconstituted particles with those of the particles reconstituted using the S7 from K12. Both reconstituted particles showed the same activity in several functions tested. Since the presence of S7 is essential for the reconstitution of active 30S subunits, we conclude that the S7 from strain K12 is functionally equivalent to the S7 from strain MRE600.This is paper No. 1612 of the Laboratory of Genetics and paper XVIII in the series, Structure and Function of Bacterial Ribosomes. Papers XVI and XVII in this series are Fahnestock, Held, and Nomura (1972) and Fahnestock, Erdmann, and Nomura (1973), respectively.     

Cooperative interactions among protein and RNA components of the 50S ribosomal subunit of Escherichia coli.

Copperative interactions among constituents of the 50S ribosomal subunit of Escherichia coli have been analyzed in order to elucidate its assembly and structural organization. Proteins L5 and L18 were shown to be necessary and sufficient to effect the association of the 5S and 23S RNAs into a quaternary complex that contains equimolar amounts of all four components. Measurement of diffusion constants by laser light scattering revealed that integration of the 5S RNA induced the 23S RNA to adopt a somewhat more open conformation. An investigation of relationships among proteins associated with the central and 3' portions of the 23S RNA demonstrated that attachment of L5, L10 + L11, and L28 depends upon the RNA-binding proteins L16, L2, and L1 + L3 + L6, respectively, and that L2 interacts with the central segment of the 23S RNA. These data, as well as the results of others, have been used to construct a scheme that depicts both direct and indirect associations of the 5S RNA, the 23S RNA, and over two-thirds of the subunit proteins. The 5' third of the 23S RNA apparently organizes the proteins required to nucleate essential reactions, whereas a region within 500 to 1500 bases of its 3' terminus is associated primarily with proteins involved in the major functional activities of the 50S ribosomal particle.     

Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation

The L7/12 stalk of the large subunit of bacterial ribosomes encompasses protein L10 and multiple copies of L7/12. We present crystal structures of Thermotoga maritima L10 in complex with three L7/12 N-terminal-domain dimers, refine the structure of an archaeal L10E N-terminal domain on the 50S subunit, and identify these elements in cryo-electron-microscopic reconstructions of Escherichia coli ribosomes. The mobile C-terminal helix alpha8 of L10 carries three L7/12 dimers in T. maritima and two in E. coli, in concordance with the different length of helix alpha8 of L10 in these organisms. The stalk is organized into three elements (stalk base, L10 helix alpha8-L7/12 N-terminal-domain complex, and L7/12 C-terminal domains) linked by flexible connections. Highly mobile L7/12 C-terminal domains promote recruitment of translation factors to the ribosome and stimulate GTP hydrolysis by the ribosome bound factors through stabilization of their active GTPase conformation.     

Binding of Escherichia coli ribosomal proteins to 23S RNA under reconstitution conditions for the 50S subunit.

The RNA binding capacity of 50S proteins from E. coli ribosomes has been tested under improved conditions; purified proteins active in reconstitution assays were used, and the binding was studied under the conditions of the total reconstitution procedure for the 50S subunit. The results are: 1) Interaction of 23S RNA was found with 17 proteins, namely L1, L2, L3, L4, L7/L12, L9, L10, L11, L15, L16, L17, L18, L20, L22, L23, L24 and L29. 2) The proteins L1, L2, L3, L4, L9, L23 and L24 bound to 23S RNA at a level of about one copy per RNA molecule, whereas L20 could bind more than one copy (no saturation was observed at 1.8 copies per 23S RNA), and the other proteins bound 0.2--0.6 copies per RNA. 3) L1, L3, L7/L12 showed a slight binding to 16S RNA, L26 (identical with S20) strong binding to 16S RNA. 4) The binding of L2, L7/L12, L10, L11, L15, L16 and L18 was preparation sensitive, i.e. the binding ability changed notably from preparation to preparation. 5) All proteins bound equally well to 23S RNA in presence of 4 and 20 mM Mg2+, respectively, except L2, L3, L4, L7/L12, L9, L10, L15, L16 and L18, which bound less strongly at 20 mM than at 4 mM Mg2+.