Reconstitution of active 30-S ribosomal subunits in vitro using heat-denatured 16-S rRNA.

30-S ribosomal subunits which have been reconstituted using heat-denatured 16-S rRNA can participate in the synthesis of lysosyme in vitro. Therefore all the information contributed by 16-S rRNA to the reconstitution process is carried in the primary sequence of this RNA. The specific protein-synthesizing activity of 30-S subunits reconstituted from 30-S subunit proteins and heat-denatured 16-S rRNA is about one third of that observed if unheated 16-S rRNA is used and is comparable to the activity of 30-S particles isolated after dissociation of 70-S ribosomes in the presence of 0.1 mM Mg2+.     

In vitro reassembly of active large ribosomal subunits of the halophilic archaebacterium Haloferax mediterranei.

The large ribosomal subunits of the halophilic archaebacterium Haloferax mediterranei have been reconstituted in vitro from the dissociated RNA and protein components. Efficient reassembly of particles fully active in poly(U)-directed polyphenylalanine synthesis requires a 2-h incubation at 42 degrees C in the presence of no less than 2.5 M concentrations of monovalent cations and of 60 mM magnesium. K+ and NH4+ ions are equally effective in promoting subunit reconstitution; however, maximal efficiency is attained when they are combined in a 1:2 molar ratio. The reassembly process requires no heat activation step, as under the appropriate ionic conditions it takes place spontaneously within the temperature range optimal for growth of H. mediterranei cells (40-45 degrees C).     

Characterisation of RNA fragments obtained by mild nuclease digestion of 30-S ribosomal subunits from Escherichia coli.

When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5'-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9 S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3' end of the 16-S RNA, but lacking the 3'-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3'-terminal 50 nucleotides) was not found. This result suggests that the 3' region of 16-S RNA is not involved in stable interactions with protein.     

Specific ribonucleoprotein fragments from 40-S ribosomal subunits.

Well-defined ribonucleoprotein fragments, resulting from the action of endogenous nuclease on 40-S subunits, were able to be separated when using high concentrations of LiCl. The ribonucleoproteins obtained sedimented at 12, 17 S, 23 S and 30 S and contained 8 S, 12 S and 17 S RNA, respectively, associated with a few proteins. The proteins extracted from the fragments were [3H] labeled by reductive methylation and their molar proportion was determined. The smallest fragment (12, 17 S) contained only three proteins, S8, S9 and S24. The 23-S and 30-S materials contained some proteins in common, S15, S19, S22, S25; S16 was found mainly in 30 S. Two proteins, S26 and "protein y" were found mainly in 23 S material. Thus, these results can give information on the relative location of certain proteins in the 40-S subunits.     

Photo-induced protein-RNA cross-linking in mammalian 60-S ribosomal subunits.

Rat liver 60-S ribosomal subunits were submitted to increasing doses of radiation (253.7 nm), at 4 degrees C and 25 degrees C, as previously reported fro 40-S subunits. The existence of protein-RNA cross-linking was demonstrated by two methods. The first consisted in the separation of protein-RNA complex; the second was indirect, and took into account alteration either in the electrophoretic mobility of cross-linked proteins or the separability of 28-S RNA in a 4 M urea/3 M LiCl buffer. The peptide synthetase activity and the sedimentation characteristics of the particles irradiated at 4 degrees C were well preserved, but at 25 degrees C the large subunits were progressively inactivated and unfolded for doses higher than 2 x 10(18) quanta. The dose-dependent variations of protein cross-linkage determined by two-dimensional gel electrophoresis allowed us to distinguish those proteins which reacted at the lowest doses with a first-order reaction from those which cross-linked to RNA after a subtle modification of the subunit structure. At 25 degrees C, all proteins became low-dose reactive. The curve obtained for 28-S RNA cross-linkage was similar to that of the total protein moiety, while those obtained fro the 5-S and 5.8-S RNA (which were parallel) suggest a lower reactivity of these RNAs. As a general rule, proteins from the large subunits were more reactive to RNA than those from the small subunits. This could indicate differences in the organisation of the two subunits.     

Specific association of two homologous DNA-binding proteins to the native 30-S ribosomal subunits of Escherichia coli.

The native 30-S ribosomal subunits from Escherichia coli are shown to be associated with two proteins which are different from the known ribosome-associated and ribosomal proteins. Neither protein is foune on native 50-S subunits or on intact ribosomes in the cell extract. The purified proteins re-bind in vitro to free 30-S subunits, but do not bind to either free 50-S subunits or intact ribosomes. The proteins, denoted NS1 and NS2, have been purified and characterized. Both proteins showed the same molecular weight of 9500 by sodium dodecyl sulfate gel electrophoresis but 34 000 by gel filtration. Upon treatment with cross-linking reagents the purified proteins gave higher molecular weight species up to the tetrameric ones showing that they exist in solution as tetramers. The amino acid compositions, tryptic fingerprint patterns and N-terminal sequences of the two proteins have been determined. These data show that NS1 and NS2 possess distinct primary structures but with extensive sequence homology. Antibodies raised against the purified proteins cross-reacted in double immuno-diffusion tests confirming further the homology. Because of the similarity in properties a sample of the DNA-binding protein HD (Berthold, V. and Geider, K. (1976) Eur. J. Biochem. 71, 443--449) was compared to NS1 and NS2. In terms of several criteria, the protein HD is found to be a mixture of two proteins, namely NS1 and NS2. The present report is the first instance of an association of DNA-binding proteins to the ribosome.     

Partial reassembly of active 60S ribosomal subunits from rat liver following treatment with dimethylmaleic anhydride

Rat liver 60S ribosomal subunits were treated with dimethylmaleic anhydride, a reagent for protein amino groups, at a 1/15,000 mol/mol ratio. This caused the dissociation of specific proteins, which were separated from the 56S residual core particles by centrifugation and identified by two-dimensional gel electrophoresis. The core particles lacking 30% of the total proteins retained most of the initial activity measured by the puromycin reaction but only small percentages of activities measured by polyphenylalanine synthesis, elongation-factor-2(EF-2)-dependent GTP hydrolysis and EF-2-mediated GDP binding. Upon reconstitution, the complementary amount of split proteins was incorporated into ribosomal particles, which had almost the same catalytic activities and biophysical properties (density, sedimentation coefficient and capability to reassociate to 40S subunits) as the original subunits.     

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.     

Defect in the split proteins of 30-S ribosomal subunits and under-methylation of 16-S ribosomal RNA in a polyamine-requiring mutant of Escherichia coli grown in the absence of polyamines.

Polyphenylalanine synthesis was carried out with Escherichia coli Q13 50-S ribosomal subunits and reconstituted 30-S particles containing different combinations of 23-S core particles and 30-S subunit split proteins obtained from a polyamine-requiring mutant of E. coli during its growth in the presence or absence of putrescine. It was concluded that the defect in the amount of some kinds of 30-S subunit split proteins was responsible for the decrease of polypeptide synthesis in a polyamine-requiring mutant of E. coli grown in the absence of polyamines. The methylation of 16-S RNA during growth in the absence of putrescine was decreased, while the degree of methylation of 23-S RNA did not change significantly. The decrease in methylation of 16-S RNA in the absence of putrescine was due mainly to a decrease of methylation of adenine. The relationship between the decrease of polypeptide synthetic activity of 30-S ribosomal subunits obtained from a polyamine-requiring mutant of E. coli grown in the absence of polyamines and the decrease of methylation of 16-S RNA is discussed.     

30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15

Assembly of 30S ribosomal subunits from Escherichia coli has been dissected in detail using an in vitro system. Such studies have allowed characterization of the role for ribosomal protein S15 in the hierarchical assembly of 30S subunits; S15 is a primary binding protein that orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit. In vitro S15 is the sole primary binding protein in this cascade, performing a critical role during assembly of these four proteins. To investigate the role of S15 in vivo, the essential nature of rpsO, the gene encoding S15, was examined. Surprisingly, E. coli with an in-frame deletion of rpsO are viable, although at 37 degrees C this DeltarpsO strain has an exaggerated doubling time compared to its parental strain. In the absence of S15, the remaining four platform proteins are assembled into ribosomes in vivo, and the overall architecture of the 30S subunits formed in the DeltarpsO strain at 37 degrees C is not altered. Nonetheless, 30S subunits lacking S15 appear to be somewhat defective in subunit association in vivo and in vitro. In addition, this strain is cold sensitive, displaying a marked ribosome biogenesis defect at low temperature, suggesting that under nonideal conditions S15 is critical for assembly. The viability of this strain indicates that in vivo functional populations of 70S ribosomes must form in the absence of S15 and that 30S subunit assembly has a plasicity that has not previously been revealed or characterized.     

Intra-RNA cross-linking in Escherichia coli 30S ribosomal subunits: selective isolation of cross-linked products by hybridization to specific cDNA fragments.

M13 clones were constructed with cDNA inserts corresponding to specific regions of E. coli ribosomal RNA. The DNA from the clones was immobilized by coupling to diazobenzyloxymethyl cellulose, and was used for the selective isolation by hybridization of cross-linked RNA complexes containing the complementary sequences. Immobilized DNA samples with inserts complementary to four different regions covering bases 735-1384 of the 16S RNA were hybridized with a mixture of 16S RNA fragments generated by partial digestion of 30S subunits that had been cross-linked by ultraviolet irradiation in vivo. After dehybridization, the individual RNA fragments and cross-linked complexes were separated by gel electrophoresis and analysed by our usual procedures. Nine cross-links are described; four of these are hitherto unobserved "secondary structural" cross-links, and one is a new "tertiary structural" cross-link between positions 243-247 and 891-894 of the 16S RNA.