Formation of Pseudo- and Hybrid-11S Globulins from Subunits of Soybean and Broad Bean 11S Globulins

Pseudo- and hybrid-11S globulins were reconstituted from native acidic and basic subunits of soybean and broad bean 11S globulins. The subunit structures of these two globulins are known to be similar to each other. Pseudo-11S globulins were formed in combinations between glycinin acidic subunit (G-AS_{1 + 2}) and glycinin basic subunit (G-BS) and between legumin acidic subunit (L-ASII) and legumin basic subunit (L-BS). Hybrid-11S globulins were formed in combinations between G-AS_{1 + 2} and L-BS and between L-ASII and G-BS. The yields of the reconstituted 11S components of G-AS_{1 +2} + G-BS and G-AS_{1 + 2} + L-BS were lower than those of L-ASII + G-BS and L-ASII + L-BS. These pseudo- and hybrid-11S globulins were similar to native 11S globulins; they all consisted of reconstituted intermediary subunits which were composed of acidic and basic subunits linked by disufide bridges and had molecular weights similar to those of native 11S globulins. However, the dissociation-association behaviors of pseudo-glycinin and hybrid-11S globulins were different from those of native 11S globulins.     

Interaction Involving Disulfide Bridges between Subunits of Soybean Seed Globulin and between Subunits of Soybean and Sesame Seed Globulins

Native subunit proteins of glycinin, the acidic and the basic subunits designated as AS₁₊₂, AS₂₊₃, AS₄, AS₅, and AS₆ and BS, respectively, were isolated by DEAE-Sephadex A-50 column chromatography in the presence of 6 m urea and 0.2 m 2-mercaptoethanol.

Reconstitution of intermediary subunits involving a disulfide bridge from native acidic and basic subunits was investigated. Formation of the intermediary subunit was observed in combinations between BS and each acidic subunit except AS₆. The yields of the reconstituted intermediary subunits differed from one another.

Further, formation of the intermediary complexes was observed when native acidic and basic subunits of soybean glycinin and sesame 13 S globulin, respectively (or reverse combinations), were mixed under reductively denatured condition and subjected to the reconstitution procedure. Considerring the overall evidence, we may conclude that the complexes are probably a hybrid intermediary subunit.     

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.     

Effect of removal of 160 nucleotides from the 3′ end of Escherichia coli 16S rRNA on the reconstitution and activity of 30S ribosomes

$\text{E. coli}$ 16S rRNA deprived of 160 nucleotides from its 3′ end was obtained by digestion with polynucleotide phosphorylase. Such rRNA was used for the reconstitution of 30S subunits and the resulting particles contained all proteins present in native 30S ribosomes. Their sedimentation coefficient was estimated as 26.5S. Poly AUG-dependent binding of fMet-tRNA to subunits reconstituted with shortened rRNA was the same as to 30S particles reconstituted with the native 16S rRNA. Subunits reconstituted with shortened rRNA were also active in poly U-dependent phenylalanine incorporation; however, their activity reached only 50% of that obtained with 30S subunits reconstituted with native 16S rRNA.     

Partial reassembly of yeast 60 S ribosomal subunits in vitro following controlled dissociation under nondenaturing conditions

Previously it has been shown that 12 of the yeast ribosomal proteins were extractable from 60 S subunits under a specific nondenaturing condition [J. C. Lee, R. Anderson, Y. C. Yeh, and P. Horowitz (1985) Arch. Biochem. Biophys. 237, 292-299]. In the present paper, we showed that these proteins could be reassembled with the corresponding protein-deficient core particles to form biologically active ribosomal subunits. Effects of time, temperature, and varying concentrations of monovalent cations, divalent cations, cores, and ribosomal proteins on reconstitution were examined. Reconstitution was determined by binding of radiolabeled proteins to the nonradiolabeled cores as well as activity for polypeptide synthesis in a cell-free protein-synthesizing system. The optimal conditions for reconstitution were established. Whereas the core particles were about 10-20% as active as native 60 S subunits in an in vitro yeast cell-free protein-synthesizing system, the reconstituted particles were 80% as active. The activity of the reconstituted particles was proportional to the amount of extracted proteins added to the reconstitution mixture. About 55 +/- 7% of the core particles recombined with the extracted proteins to form reconstituted particles. These reconstituted particles cosedimented with native 60 S subunits in glycerol gradients and contained all of the 12 extractable proteins.     

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 active 50 S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis.

Active 50 S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis can be reconstituted in vitro from dissociated RNA and proteins. The reconstituted 50 S sub-units are indistinguishable from native 50 S subunits in sedimentation on sucrose gradients and in protein composition. The procedure used is similar to that developed for reconstitution of Bacillus stearothermophilus 50 S subunits, though the optimal conditions are somewhat different. Hybrid ribosomes can be reconstituted with 23 S RNA and proteins from different sources (B. stearothermophilus and B. licheniformis or B. subtilis). The thermal stability of these ribosomes depends on the source of the proteins, and not on the source of 23 S RNA.     

Polymerization of Soybean 11S Globulin Due to Reactions with Peroxidizing Linoleic Acid

Soybean 11S globulin was polymerized by incubating with peroxidizing linoleic acid. The molar ratio of the acidic subunits to the basic subunits of 11S globulin decreased with the elapse of the incubation time. The acidic subunits were lost faster and formed polymers more easily than the basic subunits. The acidic and basic subunits in 11S globulin were fractionated by DEAE-Sephadex gel chromatography. Each of the acidic and basic subunits was allowed to react with peroxidizing linoleic acid individually. The results also showed that the acidic subunits formed polymers faster than the basic subunits. Both succinylated and acetylated 11S globulins were also submitted to the incubation with peroxidizing linoleic acid. The polymerization of the modified protein was suppressed by masking ε-amino groups.     

Purification and subunit structure of legumin of Vicia faba L. (broad bean)

Zonal isoelectric precipitation was shown to be an effective method for the preparation of legumin which was homogeneous as judged by ultracentrifugation and polyacrylamide-gel electrophoresis. The subunit structure of legumin was investigated by preparative sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and ion-exchange chromatography in urea. Five distinct subunits, of which two were acidic (alpha) and had a molecular weight of 37000, and three were basic (beta) with molecular weights of 20100, 20900 and 23800, were identified. The alpha and beta subunits were present in equimolar amounts in the legumin molecule and, in view of this and molecular-weight considerations, an alpha(6)beta(6) subunit model was proposed for legumin.     

Processing and assembly in vitro of engineered soybean beta-conglycinin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins

A short interdomain sequence between the N- and C-terminal domains of beta-conglycinin, the major 7S seed storage protein of soybean, was selected as a target for insertion of amino acid residues specifically cleaved by an asparaginyl endopeptidase that processes globulins into acidic and basic chains. Modified beta-conglycinin subunits containing the proteolytic cleavage site self-assembled into trimers in vitro at an efficiency similar to that of the unmodified subunit. In contrast to the absence of cleavage of the unmodified subunits, however, the modified beta-conglycinin trimers were processed by purified soybean asparaginyl endopeptidase into two polypeptides, each the size expected for the beta-conglycinin N- and C-terminal domains, respectively. The cleavage did not alter the assembly of mutant beta-conglycinins and the cleaved mutant trimers remained stable to further proteolytic attack. To examine the possibility of coassembly between the cleaved 11S and 7S subunits, in vitro processed mutant beta-conglycinin subunits were mixed with native dissociated 11S globulin preparations. Reassembly at a high ionic condition did not induce the 7S subunits to interact with 11S subunits to form hexameric complexes. Thus, cleavage of 7S globulin subunits into acidic and basic domains may not be sufficient for hexamer assembly to occur. Biotechnological implications of the engineered proteins are discussed.     

Ribosomes with unique breaks in 16S RNA: isolation and biological activity

A set of Escherichia coli 16S rRNA having unique breaks were prepared using the method of oligodeoxyribonucleotide-directed fragmentation with RNAse H. 16S RNA remained compact or dissociated to separate fragments, depending on the cleavage site location in the RNA structure. 16S rRNAs which have been split at different sites or their isolated fragments were used for a reconstitution of the 30S ribosomal subunits. These reconstituted 30S subunits carrying unique breaks at positions 301, 772, 1047 have the same sedimentation coefficients and electron microscopy images as the native subunit. They were active in the poly(U)-directed cell-free system of synthesis of polyphenylalanine.     

Total reconstitution of active large ribosomal subunits of the thermoacidophilic archaebacterium Sulfolobus solfataricus.

The large ribosomal subunit of the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus has been reconstituted from the completely dissociated RNA and proteins by a two-step incubation procedure at high temperatures. Successful reconstitution requires a preliminary incubation of the ribosomal components for 45 min at 65 degrees C, followed by a second heat-treatment at 80 degrees C for 60 min. Structural reassembly depends upon high concentrations of K+ (300-400 mM) and Mg2+ (20-40 mM) ions. In addition, complete recovery of subunit function stringently requires the presence of a polyamine, thermine (or spermine). The reconstituted archaebacterial subunits are essentially indistinguishable from the native ones by a number of structural and functional criteria.     

Specific subunit pairs of legumin from Vicia faba

Four pairs of disulphide-linked acidic (α) and basic (β) subunits were isolated from legumin of Vicia faba. Pairing between α- and β-subunits is nonrandom, supporting the view that each subunit pair arises from a common precursor polypeptide, already containing intramolecular disulphide bonds, when cleavage to the subunit pair takes place. The subunit pairs belong to two structural types: type A contains Met, whereas type B lacks Met. In addition to these four subunit pairs, at least two more pairs are present in legumin in minor amounts.     

Immunochemical accessibility of ribosomal protein S4 in the 30 S ribosome. The interaction of S4 with S5 and S12

The reactivity of protein S4-specific antibody preparations with 30 S ribosomal subunits and intermediates of in vitro subunit reconstitution has been characterized using a quantitative antibody binding assay. Anti-S4 antibody preparations did not react with native 30 S ribosomal subunits; however, they did react with various subunit assembly intermediates that lacked proteins S5 and S12. The inclusion of proteins S5 and S12 in reconstituted particles resulted in a large decrease in anti-S4 reactivity, and it was concluded that proteins S5 and S12 are primarily responsible for the masking of S4 antigenic determinants in the 30 S subunit. The effect of S5 and S12 on S4 accessibility is consistent with data from a variety of other approaches, suggesting that these proteins form a structural and functional domain in the small ribosomal subunit.     

Osmolytes stimulate the reconstitution of functional 50S ribosomes from in vitro transcripts of Escherichia coli 23S rRNA

Functional Escherichia coli 50S ribosomal subunits can be reconstituted from their natural rRNA and protein components. However, when the assembly is performed with in vitro-transcribed 23S rRNA, the reconstitution efficiency is diminished by four orders of magnitude. We tested a variety of chemical chaperones (compounds that are typically used for protein folding), putative RNA chaperones (proteins) and ribosome-targeted antibiotics (small-molecule ligands) that might be reasoned to aid in folding and assembly. Addition of the osmolyte trimethylamine-oxide (TMAO) and the ketolide antibiotic telithromycin (HMR3647) to the reconstitution stimulates its efficiency up to 100-fold yielding a substantially improved system for the in vitro analysis of mutant ribosomes.     

Isolation and Partial Characterization of Heat-denatured Products of Soybean 11S Globulin and Their Analysis by Electrophoresis

Heat denaturation of soybean 11S globulin was examined at 70° and 100°C in phosphate buffer (pH 7.6), at 0.01 and 0.5 ionic strength. Gel electrophoresis (Davis system) indicated that heat-denatured soybean 11S globulin contained two major components (buffer-soluble form). But they were not identified at 70°C-0.5 ionic strength. Gel filtration followed by SDS-gel electrophoresis showed that the major components were composed of a monomer and at least three of kinds of oligomers containing only an acidic subunit. Gel filtration of the precipitate formed at 100°C at 0.5 ionic strength gave two peaks. SDS-gel electrophoresis indicated that the first peak contained aggregates of highly polymerized subunits, and the second peak contained a monomer of basic subunit and seven kinds of oligomers with various proportions of basic subunits to an acidic subunit.     

Effect of antibiotics on large ribosomal subunit assembly reveals possible function of 5 S rRNA.

Functional large ribosomal subunits of Thermus aquaticus can be reconstituted from ribosomal proteins and either natural or in vitro transcribed 23 S and 5 S rRNA. Omission of 5 S rRNA during subunit reconstitution results in dramatic decrease of the peptidyl transferase activity of the assembled subunits. However, the presence of some ribosome-targeted antibiotics of the macrolide, ketolide or streptogramin B groups during 50 S subunit reconstitution can partly restore the activity of ribosomal subunits assembled without 5 S rRNA. Among tested antibiotics, macrolide RU69874 was the most active: activity of the subunits assembled in the absence of 5 S rRNA was increased more than 30-fold if antibiotic was present during reconstitution procedure. Activity of the subunits assembled with 5 S rRNA was also slightly stimulated by RU69874, but to a much lesser extent, approximately 1.5-fold. Activity of the native T. aquaticus 50 S subunits incubated in the reconstitution conditions in the presence of RU69874 was, in contrast, slightly decreased. The presence of antibiotics was essential during the last incubation step of the in vitro assembly, indicating that drugs affect one of the last assembly steps. The 5 S rRNA was previously shown to form contacts with segments of domains II and V of 23 S rRNA. All the antibiotics which can functionally compensate for the lack of 5 S rRNA during subunit reconstitution interact simultaneously with the central loop in domain V (which is known to be a component of peptidyl transferase center) and a loop of the helix 35 in domain II of 23 S rRNA. It is proposed that simultaneous interaction of 5 S rRNA or of antibiotics with the two domains of 23 S rRNA is essential for the successful assembly of ribosomal peptidyl transferase center. Consequently, one of the functions of 5 S rRNA in the ribosome can be that of assisting the assembly of ribosomal peptidyl transferase by correctly positioning functionally important segments of domains II and V of 23 S rRNA.     

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.