Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins.

Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.     

Reconstitution of Bacillus stearothermophilus 50 S ribosomal subunits from purified molecular components.

Bacillus stearothermophilus 50 S ribosomal subunits have been reconstituted from a mixture of purified RNA and protein components. The protein fraction of 50 S subunits was separated into 27 components by a combination of various methods including ion exchange and gel filtration chromatography. The individual proteins showed single bands in a variety of polyacrylamide gel electrophoresis systems, and nearly all showed single spots on two-dimensional polyacrylamide gels. The molecular weights of the proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An equimolar mixture of the purified proteins was combined with 23 S RNA and 5 S RNA to reconstitute active 50 S subunits by the procedure of Nomura and Erdmann (Nomura, M., and Erdmann, V. A. (1970) Nature 226, 1214-1218). Reconstituted 52 S subunits containing purified proteins were slightly more active than subunits reconstituted with an unfractionated total protein extract in poly(U)-dependent polyphenylalanine synthesis and showed comparable activity in various assays for ribosomal function. The reconstitution proceeded more rapidly with the mixture of purified proteins than with the total protein extract. Reconstituted 50 S subunits containing purified proteins co-sedimented with native 50 S subunits on sucrose gradients and had a similar protein compsoition. Initial experiments on the roles of the individual proteins in ribosomal structure and function were performed. B. stearothermophilus protein 13 was extracted from 50 S subunits under the same conditions as escherichia coli L7/L12, and the extraction had a similar effect on ribosomal function. When single proteins were omitted from reconstitution mixtures, in most cases the reconstituted 50 S subunits showed decreased activity in polypheylalanine synthesis.     

Early assembly proteins of the large ribosomal subunit of the thermophilic archaebacterium Sulfolobus. Identification and binding to heterologous rRNA species

Studies of ribosome structure in thermophilic archaebacteria may provide valuable information on (i) the mechanisms involved in the stabilization of nucleic acid-protein complexes at high temperatures and (ii) the degree of evolutionary conservation of the ribosomal components in the primary kingdoms of cell descent. In this work we investigate certain aspects of RNA/protein interaction within the large ribosomal subunits of the extremely thermophilic archaebacterium Sulfolobus solfataricus. The ribosomal proteins involved in the early reactions leading to in vitro particle assembly have been identified; it is shown that they can interact with the RNA in a temperature-independent fashion, forming a thermally stable "core" particle that can subsequently be converted into complete 50 S ribosomes. Among the protein components of the core particle, those capable of independently binding to 23 and 5 S RNA species have also been identified. Finally, we show that the early assembly proteins of Sulfolobus large ribosomal subunits are able to interact cooperatively with 23 S RNAs from other archaebacteria or from eubacteria, thereby suggesting that RNA/protein recognition sites are largely conserved within prokaryotic ribosomes. By contrast, no specific binding of the archaebacterial proteins to eukaryotic RNA could be demonstrated.     

Targeting exposed RNA regions in crystals of the small ribosomal subunits at medium resolution.

Within the framework of ribosomal crystallography, the small subunits are being analyzed, using crystals diffracting to 3 A resolution. The medium resolution electron density map of this subunit, obtained by multiple isomorphous replacement, show recognizable morphologies, strikingly similar to the functional active conformer of the small ribosomal subunit. It contains elongated dense features, traceable as RNA chains as well as globular regions into which the structures determined for isolated ribosomal proteins, or other known structural motifs were fitted. To facilitate unbiased map interpretation, metal clusters are being covalently attached either to the surface of the subunits or to DNA oligomers complementary to exposed ribosomal RNA. Two surface cysteines and the 3' end of the 16S ribosomal RNA have been localized. Targeting several additional RNA regions shed light on their relative exposure and confirmed previous studies concerning their functional relevance.     

A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome

BACKGROUND: This study addresses the general problem of dividing a density map of a nucleic-acid-protein complex obtained by cryo-electron microscopy (cryo-EM) or X-ray crystallography into its two components. When the resolution of the density map approaches approximately 3 A it is generally possible to interpret its shape (i. e., the envelope obtained for a standard choice of threshold) in terms of molecular structure, and assign protein and nucleic acid elements on the basis of their known sequences. The interpretation of low-resolution maps in terms of proteins and nucleic acid elements of known structure is of increasing importance in the study of large macromolecular complexes, but such analyses are difficult. RESULTS: Here we show that it is possible to separate proteins from nucleic acids in a cryo-EM density map, even at 11.5 A resolution. This is achieved by analysing the (continuous-valued) densities using the difference in scattering density between protein and nucleic acids, the contiguity constraints that the image of any nucleic acid molecule must obey, and the knowledge of the molecular volumes of all proteins. CONCLUSIONS: The new method, when applied to an 11.5 A cryo-EM map of the Escherichia coli 70S ribosome, reproduces boundary assignments between rRNA and proteins made from higher-resolution X-ray maps of the ribosomal subunits with a high degree of accuracy. Plausible predictions for the positions of as yet unassigned proteins and RNA components are also possible. One of the conclusions derived from this separation is that 23S rRNA is solely responsible for the catalysis of peptide bond formation. Application of the separation method to any nucleoprotein complex appears feasible.     

Interaction of tetracycline with RNA: photoincorporation into ribosomal RNA of Escherichia coli.

Photolysis of [3H]tetracycline in the presence of Escherichia coli ribosomes results in an approximately 1:1 ratio of labelling ribosomal proteins and RNAs. In this work we characterize crosslinks to both 16S and 23S RNAs. Previously, the main target of photoincorporation of [3H]tetracycline into ribosomal proteins was shown to be S7, which is also part of the one strong binding site of tetracycline on the 30S subunit. The crosslinks on 23S RNA map exclusively to the central loop of domain V (G2505, G2576 and G2608) which is part of the peptidyl transferase region. However, experiments performed with chimeric ribosomal subunits demonstrate that peptidyltransferase activity is not affected by tetracycline crosslinked solely to the 50S subunits. Three different positions are labelled on the 16S RNA, G693, G1300 and G1338. The positions of these crosslinked nucleotides correlate well with footprints on the 16S RNA produced either by tRNA or the protein S7. This suggests that the nucleotides are labelled by tetracycline bound to the strong binding site on the 30S subunit. In addition, our results demonstrate that the well known inhibition of tRNA binding to the A-site is solely due to tetracycline crosslinked to 30S subunits and furthermore suggest that interactions of the antibiotic with 16S RNA might be involved in its mode of action.     

Assembly of the 30S ribosomal subunit

Ribosomes are large macromolecular complexes responsible for cellular protein synthesis. The smallest known cytoplasmic ribosome is found in prokaryotic cells; these ribosomes are about 2.5 MDa and contain more than 4000 nucleotides of RNA and greater than 50 proteins. These components are distributed into two asymmetric subunits. Recent advances in structural studies of ribosomes and ribosomal subunits have revealed intimate details of the interactions within fully assembled particles. In contrast, many details of how these massive ribonucleoprotein complexes assemble remain elusive. The goal of this review is to discuss some crucial aspects of 30S ribosomal subunit assembly.     

The ribosomes of Drosophila

Summary The assembly of proteins and RNA into mature ribosomal subunits has been studied in Drosophila cell cultures by pulse-chase experiments. Pulse labeled rRNA has a transit time of 3 h, while the transfer of ribosomal protein occurs completely within 30 min. Inhibition of protein synthesis by cycloheximide results in an almost immediate cessation of ribosome assembly, a result which indicates that no large pool of free ribosomal proteins exists in the cell. Substituting pre-ribosomal RNA with the analogue 5-fluorouridine (5-FU) results in a cessation of ribosome maturation. Under these conditions at least three large subunit proteins continue to accumulate on pre-existing cytoplasmic subunits, indicating an exchange. A portion of ribosomal subunit proteins synthesized in the presence of 5-FU can be recovered in cytoplasmic subunits once the effect of 5-FU has been reversed. This is most easily interpreted in terms of their stabilization on substituted pre-rRNA within the nucleolus, and subsequent utilization on unsubstituted RNA.Work supported by a grant from the NIH (GM 22866)     

Resistance of bacterial protein synthesis to double-stranded RNA

Double-stranded RNA fails to inhibit the formation of translation initiation complexes on R17 bacteriophage RNA, overall synthesis of R17 proteins, or the ability of bacterial initiation factor IF-3 to prevent the association of 30S and 50S ribosomal subunits into single ribosomes. Yet, IF-3 can form complexes with double-stranded RNA. However, IF-3 binds to double-stranded RNA with lower apparent affinity than to either R17 RNA or 30S ribosomal subunits; this may explain the resistance of bacterial protein synthesis to double-stranded RNA.     

Identification of several proteins involved in the messenger RNA binding site of the 30 S ribosome by inactivation with 2-methoxy-5-nitrotropone

Chemical modification of unwashed 30 S ribosomal subunits with 2-methoxy-5-nitrotropone causes a rapid loss of their capacity to bind bacteriophage Qβ RNA. Reconstitution experiments show that ribosomal protein is the functionally inactivated species. When purified unmodified ribosomal proteins were included in a mixture of 16 S ribosomal RNA and total protein derived from 2-methoxy-5-nitrotropone-treated subunits, four proteins (S1, S12, S13 and S21) were found to promote the reconstitution of particles capable of binding natural messenger RNA.     

Structure and function in the herpes simplex virus 1 RNA-binding protein U(s)11: mapping of the domain required for ribosomal and nucleolar association and RNA binding in vitro.

The herpes simplex virus 1 US11 protein is an RNA-binding regulatory protein that specifically and stably associates with 60S ribosomal subunits and nucleoli and is incorporated into virions. We report that US11/ beta-galactosidase fusion protein expressed in bacteria bound to rRNA from the 60S subunit and not the 40S subunit. This binding reflects the specificity of ribosomal subunit association. Analyses of deletion mutants of the US11 gene showed that specific RNA binding activity, nucleolar localization, and association with 60S ribosomal subunits were found to map to the amino acid sequences of the carboxyl terminus of US11 protein, suggesting that these activities all reflect specific binding of US11 to large subunit rRNA. The carboxyl-terminal half of the protein consists of a regular tripeptide repeat of the sequence RXP and constitutes a completely novel RNA-binding domain. All of the mutant US11 proteins could be incorporated into virus particles, suggesting that the signal for virion incorporation either is at the amino-terminal four amino acids or is redundant in the protein.     

Minimal set of ribosomal components for reconstitution of the peptidyltransferase activity.

A new approach is described to gain further information concerning the ribosomal components involved in the peptidyltransferase (PTF) activity exerted by Escherichia coli 50S subunits. A particle is reconstituted from highly purified proteins and RNA under modified incubation conditions. This particle contains only 16 out of the 34 distinct components constituting the native subunit, and yet still exhibits significant PTF activity. Single omission tests at the level of this "minimal ribosomal particle" indicate the limits set on a further reduction of the components, and in particular reveal that protein L18 can be excluded from the set of proteins which are essential for PTF activity, thus leaving L2, L3, L4, L15, and L16 as primary candidates for this function. 5S RNA is not needed for PTF activity of the "minimal ribosomal particle". Furthermore, a buffer condition is described which drastically improves the stability of total protein preparations and facilitates the isolation of individual proteins.     

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification.

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both LiCl-split and core proteins. The proteins L2 and L16 from the split fraction and L4 from the core fraction of unmodified ribosomes were together nearly as effective as total unmodified proteins in restoring peptidyl transferase activity to reconstituted ribosomes when added with proteins from modified ribosomes. These three proteins are therefore the most important targets identified as responsible for loss of peptidyl transferase activity on photo-oxidation of 50 S ribosomal subunits.     

Analysis of ribosomal proteins and ribosomal subunits of pea seeds by electrophoresis in polyacrylamide gel]

The amino acid composition of overall protein of ribosomes and ribosomal subunits of pea seeds has been found typical of ribosomal protein. Electrophoresis in polyacrylamide gel demonstrates that proteins extracted by the solution of 3 M LiCl-4 M urea from purified ribosomes of pea seeds move towards the cathode at pH 2.2 and separate into 41 components. Electrophoresis in a tris-glycine buffer at pH 9.2 does not reveal any substance corresponding to acid proteins. Similar distribution patterns are observed when ribosomal particles are isolated with or without triton (0,5%). The treatment of ribosomes by deoxycholate results in some changes, depending on the detergent concentration. All the protein components detected in ribosomes, except one, are present in the subunits. Proteins of large and small ribosome subunits produced 26 and 21 components respectively in polyacrylamide gel electrophoresis. The distribution patterns of proteins of the two subunits appear to be different. The majority of the components of the large and small subunits differ in mobility. The data obtained suggest considerable specificity of the protein composition of 60S and 40S subunits of 80S ribosomes in higher plants.     

Dynamics of the biosynthesis of components of the protein synthesizing apparatus of the rat liver at the stage of restoration of translation, inhibited by cycloheximide

The biosynthesis of proteins, ribosomal RNA and other components of the rat liver protein-synthesizing system during the reparation and subsequent activation of translation inhibited by a sublethal dose cycloheximide (CHI, 3 mg/kg) was studied. It was found that the incorporation of labeled precursors into proteins and ribosomal rRNA isolated from free and membrane-bound polysomes is repaired already 3 hours after CHI injection. 6-9 hours thereafter, the level of component labeling reaches control values, whereas the total protein biosynthesis is retarded. After 12-24 hours, marked stimulation of ribosome biosynthesis and the integration of ribosomes into polysomes are observed together with an asymmetric accumulation of excessive amounts of newly synthesized 40S subunits into polysomes 12 hours after CHI infection. The putative mechanisms of the activation of expression of the part of the genome responsible for protein and ribosomal rRNA synthesis as well as for the synthesis of other components of the protein-synthesizing system are discussed.     

Charge Segregation and Low Hydrophobicity Are Key Features of Ribosomal Proteins from Different Organisms

Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge.     

Factors causing release of ribosomal subunits from isolated nuclei of regenerating rat liver in vitro.

Incubation medium II causes release of ribosomal subunits from isolated prelabeled nuclei of regenerating rat liver in vitro (Sato, T., Ishikawa, K. and Ogato, K. (1976) Biochim. Biophys. Acta 000, 000-000). The effects of individual components of this medium on release of subunits were studied and the following results were obtained. 1. Dialyzed cytosol was effective in causing release of total labeled RNA, but its effect on release of labeled ribosomal subunits was rather lower than that of low molecular yeast RNA. Spermidine inhibited the release of total labeled RNA as well as that of labeled ribosomal subunits. 2. Low molecular yeast RNA was the most effective component for inducing release of labeled ribosomal subunits. Homologous ribosomal RNA was as effective as yeast RNA. Cytoplasmic ribosomes, prepared by washing with solution of high salt concentration, and their subunits were also effective. 3. Transfer RNA was not so effective as yeast RNA and ribosomal RNA and even after heat treatment it had little effect. 4. Among the homopolyribonucleotides tested, polyuridylic acid had a strong effect but polyadenylic acid, polycytidylic acid and polyinosinic acid had no effect. 5. The effects of yeast RNA and polyuridylic acid in causing release of labeled ribosomal subunits were dependent upon their concentrations in the reaction mixture. The characteristics of the factors which cause release of labeled ribosomal subunits in vitro are discussed on the basis of the results.