Approaching the molecular structure of ribosomes

Fifteen forms of three-dimensional crystals and three forms of two-dimensional sheets from ribosomal particles have been grown. In all cases only biologically active particles could be crystallized, the crystalline material retaining its integrity and biological activity for months. Cryastallographic data have been collected from crystals of 50 S ribosomal subunits, using synchrotron radiation, at temperatures between 19 and -180 degree C. Although at around 0 degrees C in the synchrotron X-ray beam the crystals rapidly lose their high-resolution reflections, at cryo-temperatures hardly any radiation damage occurs over long periods, and a complete set of diffraction data to about 6 A resolution could be collected from a single crystal. Heavy-atom clusters were used for soaking as well as for specific binding to the surface of the ribosomal subunits prior to crystallization. The 50 S ribosomal subunits from a mutant of Bacillus stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with the native form. Models of the entire 70 S ribosome and of the 50 S subunit have been reconstructed from two-dimensional sheets at 47 and 30 A, respectively. These models demonstrate the overall shape of the particles, the contact areas between large and small subunits, the space where protein biosynthesis may take place and a tunnel through the 50 S subunit which could provide a path for the nascent polypeptide chain.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

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.     

Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme

Succinate dehydrogenase consists of two protein subunits and contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated S-1, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as +80, -240, and -25 mV, respectively. Magnetic spin interactions between clusters S-1 and S-2 and between S-1 and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin in the enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the histidine that binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehydrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residues downstream from the histidyl in the Fp subunit can also effect flavinylation.     

Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase

Succinate dehydrogenase is a conserved membrane-bound enzyme consisting of two nonidentical subunits: a flavo iron-sulfur protein (Fp) subunit, containing a covalently bound flavin, and an iron-sulfur protein (Ip) subunit. Bacillus subtilis succinate dehydrogenase in wild type bacteria and 12 well characterized succinate dehydrogenase-defective mutants were examined by low temperature EPR spectroscopy to characterize the enzyme and study subunit location and biosynthesis of its iron-sulfur clusters. The wild type B. subtilis enzyme contains iron-sulfur clusters which are analogous to clusters S-1 and S-3 of bovine heart succinate dehydrogenase but with slightly different EPR characteristics. Spins from cluster S-2 were not detectable as in the case of the intact form of bovine heart succinate dehydrogenase. However, dithionite reduction of the B. subtilis enzyme greatly enhanced spin relaxation of the ferredoxin-type cluster S-1, indicating the presence of the cluster S-2. Iron-sulfur cluster S-1 was found to be assembled in soluble succinate dehydrogenase subunits in the cytoplasm, but only if full-length Fp polypeptides and relatively large fragments of Ip polypeptides were present. Cluster S-1 was not detected in mutants with soluble mutated Fp polypeptides or in a mutant totally lacking Ip subunit polypeptide. Iron-sulfur clusters S-1, S-2, and S-3 were assembled also when the covalently bound flavin in the Fp subunit was absent. Clusters S-1 and S-3 in the membrane-bound flavin-deficient succinate dehydrogenase were not reduced by succinate but could be reduced by electron transfer from NADH dehydrogenase via the menaquinone pool.     

Crystals of wild-type, mutated, derivatized and complexed 50 S ribosomal subunits from Bacillus stearothermophilus suitable for X-ray analysis

Three-dimensional single crystals of wild-type and mutated 50 S ribosomal subunits from Bacillus stearothermophilus, as well as crystals of reconstituted subunits containing heavy-atom clusters and complexes of these subunits with tRNA and a short nascent polypeptide chain, were grown from polyethylene glycol in the presence of salts at low concentrations. Within experimental error, all these crystals are isomorphous, packed with monoclinic symmetry (C2) in unit cells of a = 300 A, b = 546 A, c = 377 (+/- 1%) A and beta = 112 degrees. Using synchrotron radiation at 85 to 100 K they diffract to 11 A resolution and can be irradiated for hours without disintegrating, so that a complete data set could be collected from a single crystal.     

Reconstitution and crystallisation experiments with isolated split proteins from Bacillus stearothermophilus ribosomes

Six proteins (B-L1, B-L6, B-L10, B-L11, B-L12 and B-L16) were removed from 50S ribosomal subunits of Bacillus stearothermophilus by treatment with ethanol and ammonium chloride. The proteins were isolated in a pure form, and one of them (B-L6) was crystallized. Five of the six proteins (in various combinations) were added back to the core particles, resulting in 50S subunits lacking one protein. The biological activities of these ribosomal particles as determined in the poly(U)-system varied over a wide range, depending on the protein which was omitted. The particles lacking one protein provide useful tools for heavy-atom derivation necessary for our crystallographic studies on the 50S subunits of Bacillus stearothermophilus.     

The suitability of a monofunctional reagent of an undecagold cluster for phasing data collected from the large ribosomal subunits from Bacillus stearothermophilus

An electron density map of the large ribosomal subunit from Bacillus stearothermophilus was obtained at 26 Å resolution by single isomorphous replacement (SIR) from a derivative formed by specific quantitative labeling with a dense undecagold cluster. For derivalization, a mono-functional reagent of this cluster was bound to a sulfhydryl group of a purified ribosomal protein. which was in turn reconstituted with core particles of a mutant lacking this protein. The native, mutated, and derivatzed 50S ribosomal subunits crystallize under the same conditions in the same space group. Under favorable conditions, crystals of the derivatized subunit proved to be isomorphous with the native ones, whereas the crystals of the mutant may have somewhat different packing. After resolving the SIR phase ambiguity by solvent flattening, the electron density shows a packing that is consistent with the noncrystallographic symmetry found by Patterson searches as well as with the motif observed in electron micrographs of thin sections of the crystals. These studies established that phase information can be obtained from heavy metal clusters, even when the crystals under investigation are unstable and weakly diffracting. These results encouraged further effort at the construction of specifically derivatized crystals from other ribosomal particles that diffract to higher resolution. © 1994 John Wiley & Sons, Inc.     

Binding of S21 to the 50S subunit and the effect of the 50S subunit on nonradiative energy transfer between the 3' end of 16S RNA and S21

Escherichia coli ribosomal protein S21 was labeled at its single cysteine group with a fluorescent probe. Labeled S21 showed full activity in supporting MS2 RNA-dependent binding of formylmethionyl-tRNAf to 30S ribosomal subunits. Fluorescence anisotropy measurements and direct analysis on glycerol gradients demonstrate conclusively that labeled S21 binds to 50S ribosomal subunits as well as to 30S and 70S particles. The relative binding affinities are in the order 70S greater than 30S greater than 50S. Other results presented appear to indicate that S21 is bound in the same position on either 50S subunits or 30S subunits as in 70S ribosomes, suggesting that the protein is bound simultaneously to both subunits in the latter. Addition of 50S subunits to 30S particles containing probes on S21 and at the 3' end of 16S RNA caused a decrease in the energy transfer between these points. The results correspond to an apparent change in distance from 51 to 61 A.     

Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients

In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins.     

The fate of membrane-bound ribosomes following the termination of protein synthesis

Contemporary models for protein translocation in the mammalian endoplasmic reticulum (ER) identify the termination of protein synthesis as the signal for ribosome release from the ER membrane. We have utilized morphometric and biochemical methods to assess directly the fate of membrane-bound ribosomes following the termination of protein synthesis. In these studies, tissue culture cells were treated with cycloheximide to inhibit elongation, with pactamycin to inhibit initiation, or with puromycin to induce premature chain termination, and ribosome-membrane interactions were subsequently analyzed. It was found that following the termination of protein synthesis, the majority of ribosomal particles remained membrane-associated. Analysis of the subunit structure of the membrane-bound ribosomal particles remaining after termination was conducted by negative stain electron microscopy and sucrose gradient sedimentation. By both methods of analysis, the termination of protein synthesis on membrane-bound ribosomes was accompanied by the release of small ribosomal subunits from the ER membrane; the majority of the large subunits remained membrane-bound. On the basis of these results, we propose that large ribosomal subunit release from the ER membrane is regulated independently of protein translocation.     

Cross-linking of mRNA to initiation factor eIF-3, 24 kDa cap binding protein and ribosomal proteins S1, S3/3a, S6 and S11 within the 48S pre-initiation complex

Native small ribosomal subunits from rabbit reticulocytes contain all initiation factors necessary for the formation of the mRNA-containing 48S pre-initiation complex. The complex formed in the presence of Met-tRNAf and 125I-labelled globin mRNA was cross-linked with diepoxybutane, and the covalent mRNA-protein complexes were isolated under denaturating conditions. The proteins of the covalent complex were identified as the 110, 95 and 66/64 kDa subunits of eIF-3. In addition, the 24 kDa cap binding protein and the ribosomal proteins S1, S3/3a, S6 and S11 were found covalently linked to the mRNA. Ribosomal proteins S3/3a and S6 were also involved in the ribosomal mRNA-binding domain of reticulocyte polysomes.     

ATTACHMENT OF RIBOSOMES TO MEMBRANES DURING POLYSOME FORMATION IN MOUSE SARCOMA 180 CELLS

Addition of nutrients to starved mouse S-180 cells leads to rapid conversion of ribosomal monomers to polysomes. During this process, a portion of the ribosomes originally found in the 17,000 g (10 min centrifugation) supernatant of cell lysates becomes firmly attached to structures sedimenting at 500 g (5 min centrifugation). Electron microscopy of sections of the intact cells showed the change from randomly distributed ribosomal particles to clusters. Association with membranes also became evident. The material sedimenting at 500 g comprised nuclei enclosed in an extensive endoplasmic reticulum (ER) network. This fraction prepared from recovering cells showed numerous ribosome clusters associated with the ER network. The appearance of many of these clusters indicated that the ribosomal particles were not directly bound to the membranes. RNase treatment released about 40% of the attached ribosomes as monomers, and ethylenediaminetetraacetic acid released 60% as subunits. It is suggested that during polysome formation a portion of the ribosomes becomes attached to the membranes through the intermediary of messenger RNA.     

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.     

Mobile domains in ribosomes revealed by proton nuclear magnetic resonance

Ribosomes and subunits from eukaryotic and prokaryotic sources were studied by high-resolution proton magnetic-resonance spectroscopy. If all ribosomal components are firmly bound within the particle, then only broad spectra would be expected. However, relatively sharp resonances were found both in ribosomal subunits and in 70 or 80 S ribosomes. The regions of these mobile protein domains have been partially assigned in Escherichia coli ribosomes. Large and small ribosomal subunits were treated to remove selectively proteins L7/12 and S1, respectively. Sharp proton magnetic resonance spectra were not observed for the stripped large subunit showing that proteins L7/12 comprise the flexible protein region and that there is little other flexibility in the stripped subunit. Complete removal of S1 from the small subunit greatly reduced but did not abolish the sharp protein resonance peaks, indicating that protein S1 contains a substantial flexible component but that other flexible components remain in the stripped small subunit. Evidence for generality of these features of ribosome organization is provided by similar studies on ribosomes from eukaryotic sources.     

The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro.

Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.     

Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose

Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20° C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.     

Reactivity of proteins in ribosomes from Saccharomyces cerevisiae with trypsin

The susceptibility of 40S and 60S ribosomal subunits from Saccharomyces cerevisiae to digestion with varying concentrations of trypsin was studied by two-dimensional electrophoresis and quantitative measurements of the protein remaining with the ribosomal particles after trypsin treatment. Proteins from both subunits can be classified into three groups according to their rate of digestion by trypsin. These results are in good agreement with those obtained on the order of ribosomal assembly in vivo, i.e., proteins which are most susceptible to trypsin digestion have been shown to associate with the ribosomal particles at a relatively late stage of ribosome assembly.     

Partial reconstitution of 60S ribosomal subunits from yeast

Summary Ribosomal 60S subunits active in polyphenylalanine synthesis can be reconstituted from core particles lacking 20–40% of the total protein. These core particles were obtained by treatment of yeast 60S subunits with dimethylmaleic anhydride, a reagent for protein amino groups. Upon reconstitution a complementary amount of split proteins is incorporated into the ribosomal particles, which have the sedimentation coefficient of the original subunits. Ribosomal protein fractions obtained by extraction with 1.25 M NH₄Cl, 4 M LiCl, 7 M LiCl, or 67% acetic acid, are much less efficient in the reconstitution of active subunits from these core particles than the corresponding released fraction prepared with dimethylmaleic anhydride. Attempts to reconstitute active subunits from protein-deficient particles obtained with 1.25 M NH₄Cl plus different preparations of ribosomal proteins, including the fraction released with dimethylmaleic anhydride, were unsuccessful. Therefore, under our conditions, of the disassembly procedures assayed only dimethylmaleic anhydride allows partial reconstitution of active 60S subunits.Abbreviation DMMA dimethylmaleic anhydride