A method of preparing Escherichia coli 16 S RNA possessing previously unobserved 30 S ribosomal protein binding sites

A method of preparing 16 S RNA has been developed which yields RNA capable of binding specifically at least 12, and possibly 13, 30 S ribosomal proteins. This RNA, prepared by precipitation from 30 S subunits using a mixture of acetic acid and urea, is able to form stable complexes with proteins S3, S5, S9, S12, S13, S18 and possibly S11. In addition, this RNA has not been impaired in its capacity to interact with proteins S4, S7, S8, S15, S17 and S20, which are proteins that most other workers have shown to bind RNA prepared by the traditional phenol extraction procedure (Held et al., 1974; Garrett et al., 1971; Schaup et al., 1970,1971).We have applied several criteria of specificity to the binding of proteins to 16 S RNA prepared by the acetic acid-urea method. First, the new set of proteins interacts only with acetic acid-urea 16 S RNA and not with 16 S RNA prepared by the phenol method or with 23 S RNA prepared by the acetic acid-urea procedure. Second, 50 S ribosomal proteins do not interact with acetic acidurea 16 S RNA but do bind to 23 S RNA. Third, in the case of protein S9, we have shown that the bound protein co-sediments with acetic acid-urea 16 S RNA in a sucrose gradient. Additionally, a saturation binding experiment showed that approximately one mole of protein S9 binds acetic acid-urea 16 S RNA at saturation. Thus, we conclude that the method employed for the preparation of 16 S RNA greatly influences the ability of the RNA to form specific protein complexes. The significance of these results is discussed with regard to the in vitro assembly sequence.     

Evidence that 16S RNA from E. coli can assume two different biologically active conformations.

We have recently shown that 16S RNA can be extracted from 30S ribosomes by an acetic acid-urea precipitation procedure which yields RNA capable of binding 13 individual ribosomal proteins. This is in contrast to phenol extracted 16S RNA which can specifically associate with only 7 proteins2-7. In the experiments reported here, we demonstrate that the difference in protein binding capacities is due to a relatiely more "open" configuration possessed by the acetic acid-urea 16S RNA. Under identical conditions, acetic acid-urea 16S RNA is more susceptible to limited T1-RNase digestion than is phenol-16S RNA. In addition, acetic acid-urea RNA shows a relatively slower electrophoretic mobility. The observable difference in conformation between the two types of RNA is lost by storage at-70 degrees C. This loss is accompanied by a reduction in protein binding capacity of the acetic acid-urea 16S RNA.     

Stoichiometric measurements of 30S and 50S ribosomal proteins from Escherichia coli

Summary Stoichiometric investigations were made for all 21 proteins of the 30S subunit and for 31 proteins of the 50S subunit. While the results for the 30S proteins strongly suggest the existence of two different stoichiometric classes, the so-called fractional (F) and unit (U) proteins with average molar amounts of 0.1–0.5 and 0.8–1.2 per mole 30S respectively, at least one further group is present in isolated 50S particles. Therefore 50S proteins may be subdivided in the following way: a) 9–10 proteins with average molar amounts of 0.2–0.6 (F-proteins), b) 15–16 proteins with 0.8–1.2 copies per 50S particle (U-proteins), c) at least one protein with an average value of 1.8–2.2 copies, the repeated (R) proteins, d) a group of 7 proteins with 1.4–1.7 copies per particle; they can be named fractional repeated (FR) proteins. These findings indicate that 50S ribosomal particles show a more differentiated degree of structural heterogeneity at least in vitro than has been shown for 30S subunits.Paper No. 35 on Ribosomal Proteins. Preceding paper is by E. Deusser and H. G. Wittmann, Nature 238, 269 (1972).     

Properties of the interaction of ribosomal protein S4 and 16S RNA in Escherichia coli revertants from Streptomycin dependence to independence

Summary The binding properties of altered S4 proteins from E. coli revertants from streptomycin dependence to independence were investigated. Three of the proteins with the same length as the wild type protein, from mutants N424, N428 and N430, exhibited unchanged binding and conformational properties. However, three proteins with an altered length, from mutants N422, N425 and N433, bound more weakly to the 16S RNA, and their conformations were different from that of the wild type S4 protein. In the presence of the other 16S RNA binding proteins, no stimulation of the binding of the latter three proteins could be detected.Paper No. 40 on Ribosomal Proteins. Preceding paper is in press in Molec. gen. Genetics (1972).     

Location of proteins S5, S13 and S14 on the surface of the 3oS ribosomal subunit from Escherichia coli as determined by immune electron microscopy

Summary The structure of negatively stained 30 S ribosome-IgG-30 S ribosome complexes (dimers) was examined by electron microscopy to locate proteins S5, S13 and S14 on the surface of the 30S ribosomal subunit from Escherichia coli. The attachment points of non-crossreacting antibodies, specific to each of the three ribosomal proteins, were visualized and correlated to distinctive structural features of the 30S subunit. The 30S particle showed a bipartite structure of two globular sections unequal in size and connected by a narrow bridge (neck). Protein S5 was located at several sites on the surface of this neck region. Proteins S13 and S14 were localized in the smaller globular section and were found to be in close proximity to one another. A model of the 30 S subunit with the location of the three proteins is presented.     

30S ribosomal proteins cross-linked to 16S RNA by periodate oxidation followed by borohydride reduction

Gel electrophoretic techniques have been used to reexamine the RNA-protein cross-linking reaction induced by periodate oxidation and borohydride reduction of 30S ribosomal subunits. The results show that a number of 30S ribosomal proteins become attached to intact 16S RNA by this method, in addition to those already published. It follows that this cross-linking technique as it stands is of little value as a topographical probe of the environment of the 3-terminus of the 16S RNA.     

Complementary binding of oligonucleotides with 16S RNA and ribosomal ribonucleoproteins

The accessibility of single-stranded sequences of 16S RNA in free state and in ribonucleoprotein particles (RNP) to complementary binding with isoplith fractions of oligonucleotides was studied. RNP had different protein composition and corresponded to intermediate stages of E. coli 30S subunit assembly in vitro. Gel-filtration was used to detect the most strong binding. It was found that S4 essentially inhibited the hexamer binding to RNA. Core proteins bound to 16S RNA strongly increased the shielding of single-stranded regions while split proteins insignificantly changed the hexamer binding. Nevertheless evidence is presented that split proteins might also interact directly with 16S RNA in the 30S subunit.     

Variability of bacterial gene-directed enzyme production in human genetically deficient cells

Summary Human -galactosidase-deficient skin fibroblasts from a patient with generalized gangliosidosis (GM₁-gangliosidosis type I) were treated with phage plac DNA, coding for Escherichia coli -galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.23). New -galactosidase activity detected in cell extracts of phage DNA-treated GM₁-gangliosidosis fibroblasts continued to vary considerably from one experiment to another. It behaved like the E. coli z-gene product upon immunochemical and physicochemical investigation. In some experiments the antigenic behavior of resultant -galactosidase activity in plac DNA-treated cells resembled that of mutant E. coli -galactosidase. Among the factors and variables that may be responsible for the variation in the results obtained here and elsewhere, low physical binding between prokaryotic mRNA sequences and fibroblast ribosomal RNA could play a part connected with effective translation. This hypothesis is discussed under the aspect of a comparison of the ribosomal binding site of lac z mRNA with the 3-terminus of the eukaryotic 18s ribosomal RNA, which shows limited possibilities for base-pairing interactions.More extensive possibilities for forming Watson-Crick base pairs between their initiation site and the eukaryotic ribosomal binding site exist for other prokaryotic messengers, such as those of Q-replicase, f 1-coat protein, or UDPG-4-epimerase.     

Site of synthesis of spinach chloroplast ribosomal proteins and formation of incomplete ribosomal particles in isolated chloroplasts

Chloroplast ribosomal proteins from spinach have been prepared in the presence of a protease inhibitor and some modifications have been introduced to the previous characterization of the 50S subunits (Mache et al., MGG, 177, 333, 1980): 33 ribosomal proteins are detected instead of 34. No change has been observed for the 30S subunits.Using a light-driven system of protein synthesis it is shown that up to ten ribosomal proteins of the 30S and eight proteins of the 50S subunits are made in the chloroplast.Newly synthesized ribosomal subunits have been analysed on CsCl gradients after sedimentation at equilibrium, allowing the separation of fully assembled subunits from incomplete ribosomal particles. Most of the newly made 50S subunits are fully assembled (=1.634). A small amount of incomplete 50S particles (=1.686) is detectable. Newly made 30S subunits (=1.598) and incomplete 30S particles (=1.691) are also observed. The ribosomal proteins of the incomplete 30S have been determined. They contain eight or nine of the 30S-proteins, seven of which are synthesized within the chloroplast. It is suggested that incomplete ribosomal particles resulted from a step in the assembly of ribosomal subunits.     

Structural changes of tRNA and 5S rRNA induced with magnesium and visualized with synchrotron mediated hydroxyl radical cleavage

The structure of native yeast tRNA^Phe and wheat germ ribosomal 5S RNA induced by different magnesium ion concentrations was studied in solution with a synchrotron mediated hydroxyl radical RNA cleavage reaction. We showed that very small amounts of Mg⁺² can induce significant changes in the hydroxyl radical cleavage pattern of tRNA^Phe. It also turned out that a reactivity of tRNA^Phe towards OH coincides with the strong metal binding sites. Because of the Mg ions are heavily hydrated one can suggest the strong correlation of the observed nucleosides reactivity in vicinity of Mg²⁺ binding sites with availability of water molecules as a source of hydroxyl radical. On the other hand the structure of wheat germ 5S rRNA is less sensitive to the hydroxyl radical reaction than tRNA^Phe although some changes are visible at 4 mM Mg ions. It is probably due to the lack of strong Mg⁺² binding sites in that molecule. The reactivity of nucleotides in loops C and D of 5S rRNA is not effected, what suggests their flexibility or involvement in higher order structure formation. There is different effect of magnesium on tRNA and 5S rRNA folding. We found that nucleotides forming strong binding sites for magnesium are very sensitive to X-ray generated hydroxyl radical and can be mapped with OH. The results show, that guanine nucleotides are preferentially hydrated. X-ray footprinting mediated hydroxyl radical RNA cleavage is a very powerful method and has been applied to studies of stable RNAs for the first time.     

Chemical cross-linking of protein to RNA within intact ribosomal subunits from Escherichia coli.

Summary Bifunctional reagents, namely bis-(2-chloroethyl)-amine (nitrogen mustard) and activated esters of 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid (bromo-ketone reagent) are used to cross-link protein to RNA within intact ribosomal subunits. The cross-linked proteins are analysed on two different two-dimensional gel electrophoresis systems, and the existence of a stable cross-linkage is demonstrated by isolating cross-linked protein-oligonucleotide complexes from subunits containing ³²P-labelled RNA. Proteins S3, S4, S5, S9/S11 and S13 from the 30S subunit, and proteins L1 and L2 from the 50S subunit were cross-linked to RNA by the nitrogen mustard, together with a number of other so far unresolved proteins. Correspondingly S3, S4, S7, S9/S11, and L2 were cross-linked by the bromoketone reagent, although in lower yield. The reagents should prove useful for topographical studies on ribosomal subunits, and arguments are presented favouring the use of non-cleavable and relatively non-specific RNA-protein cross-linking reagents for such studies.     

Interaction of Human Ribosomal Protein S26 with Fragments of Its Own mRNA and Pre-mRNA in Nuclear Extract of HeLa Cells

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, recombinant rpS26 binds to the first intron of the rpS26 pre-mRNA (apparent association constant (K _a) 5.0 · 10⁷ M^–1) and, to a lesser extent, to the rpS26 mRNA (K _a 2.0 · 10⁷ M^–1). The binding was specific, since human rpS19 had an order of magnitude lower K _a with the first intron and did not bind with the rpS26 mRNA. Immunoassays with specific antibodies showed that rpS26 contained in the nuclear extract of HeLa cells binds to the first intron of its pre-mRNA and, less efficiently, to its mRNA. In either case, RNA binding substantially increased in the presence of recombinant rpS26. Along with other (48K, 59K) nuclear proteins, rpS26 was assumed to form complexes, the functional role of which is storage of pre-mRNAs inactive in splicing.     

Recruitment of beta-2-glycoprotein 1 to cell surfaces in extrinsic and intrinsic apoptosis

Apoptotic cells and phagocytes have developed a diverse array of distinct ligand-receptor systems that drive the recognition and uptake of dying cells. Phagocytes recognize apoptotic cells either directly, by binding to specific ligands at their cell surface, or indirectly, by binding to bridging proteins that bind these ligands. Previous observations showed that the plasma bridging protein 2GP1, binds PS containing vesicles, and enhances their binding and engulfment by phagocytes in vitro. In this study we show that apoptotic cells injected intravenously and intraperitonealy into syngeneic mice recruited the PS binding protein, 2GP1. Examination of peritoneal exudates and spleen thin sections showed that only the injected apoptotic cells picked up endogenous 2GP1. Recovery of cells from the peritoneum showed that apoptotic cells bearing 2GP1 were clustered around host peritoneal phagocytes. In addition, tissue sections from mice injected with Fas antibody showed colocalization of 2GP1 with TUNEL-positive apoptotic cells. These results provide evidence that endogenous 2GP1 binds apoptotic cells in vivo, suggesting that the protein plays an important physiologic role in the recognition of dying cells.     

The effect of the ribosomal protein S1 from Escherichia coli on the synthesis in vitro of bacterial-, DNA phage- and RNA phage proteins.

Summary Using an in vitro preparation for protein synthesis, we have studied the effect of the ribosomal protein S1 fromEscherichia coli on the synthesis of the coat protein of the RNA-containing phages Q and MS2, on that of an early and a ate enzyme encoded by the DNA containing phage T7, and on that of anthranilate synthetase, an enzyme encoded by the bacterial tryptophan operon. Our results indicated that for the synthesis of these five proteins the presence of S1 is required. From these results we conclude that S1 is an essential protein for the translation of bacterial and bacteriophage messenger RNA.     

The accessibility of proteins of the Escherichia coli 30S ribosomal subunit to antibody binding

Summary The accessibility of each of the proteins on the E. coli 30S ribosomal subunit was established by investigating whether or not immunoglobulins (IgG's) and their monovalent papain fragments (Fab's), specific for each of the 21 single ribosomal proteins, bind to the 30S subunit. The interpretation of the results of five different experimental approaches, namely Ouchterlony double diffusion and immunological sandwich methods, sucrose gradient and analytical ultracentrifugation, and functional inhibition tests, indicate that all 21 proteins of the 30S subunit have determinants available for antibody binding. There were quantitative differences between the degree of accessibility of the different ribosomal proteins. An attempt was made to correlate the results with the protein stoichiometric data of the small subunit proteins.     

Characteristics of ribonucleic acids isolated from Botryodiplodia theobromae pycnidiospores

Nucleic acids isolated from dormant and germinated Botryodiplodia theobromae pycnidiospores contain five distinct species of RNA. They include two ribosomal species, two ribosomal-associated species and transfer RNAs. Sedimentation coefficients of 25.1S and 18S were obtained for the two ribosomal RNA species and 5.8S and 5S for the two ribosomal-associated RNA components. Molecular weights of 1.20, 0.67, 0.054 and 0.035x10⁶ daltons were obtained after formaldehyde treatment and electrophoresis on polyacrylamide gels for these same four RNAs. Methylated nucleotides were present in the transfer RNAs and large and small ribosomal RNAs; in contrast 5.8S and 5S RNAs contained few methylated nucleotides. In addition to the 5 distinct RNA species, polyadenylate-containing RNA was isolated from both dormant and germinated spores.Published with the approval of the Director as paper no. 5006, Journal Series, Nebraska Agricultural Experiment Station. The work was conducted under Nebraska Agricultural Experiment Station Project no. 21-17.     

Characterization of an RNA "binding site" for a specific ribosomal protein of Escherichia coli

Summary A portion of the 16S ribosomal RNA that binds specifically to, and is protected from nucleolytic attack by, ribosomal protein S4 has been characterized in terms of its partial primary structure. The specific RNA (S4aR) in question comprises slightly more than onefourth of the full 16S molecule, and appears to be located (at least in part) in the 5-proximal half of the molecule. The functional significance of S4aR is discussed.     

Studies on the interaction between ribosomes and 14 CH 3 -F 3 initation factor

Summary Initiation factor F₃ has been purified fromEscherichia coli and labelledin vitro by reductive alkylation. The¹⁴CH₃–F₃ so obtained had a specific activity of about 1 000 cpm/g and was shown to have retained its biological activity. Labelled F₃ binds to 30S ribosomal subunits ofEscherichia coli andBacillus stearothermophilus, but does not bind to either 70S ribosomes or 50S ribosomal subunits. The stoichiometry of the binding indicates that one molecule of¹⁴CH₃–F₃ is bound to each 30S ribosomal subunit. Several antibiotics, known to interact with 30S subunits, inhibit the binding. Functional studies indicate that F₃ is released from 30S ribosomes as a result of the formation of the 70S initiation complex.     

Structure of the cDNA coding for conglutin γ, a sulphur-rich protein from Lupinus angustifolius

The sequence of cDNA coding for a sulphur-rich storage protein from Lupinus angustifolius L., conglutin , was determined. The coding region contained an N-terminal leader peptide of 28 amino acids which directly preceded subunits of M _r 28 239 and 16 517. Extensive sequence homology between the protein encoded by conglutin cDNA and basic 7S globulin from soybean was observed. Sequence homology to proteins from other classes of storage proteins, 11S, 7S and 2S, was limited to short and highly fragmented sequences. The amino acid sequence, Asn-Gly-Leu-Glu-Glu-Thr, characteristic of the primary site for post-translational cleavage of the precursors of 11S proteins, was absent from the sequence predicted for prepro-conglutin . It is concluded that conglutin is a representative of a fourth type of storage protein in legumes, distinct from the 11S, 7S and 2S storage protein families.     

Optimal conditions for the interaction of ribosomal protein S8 and 16S RNA and studies on the reaction mechanism

Summary Protein S8 occurred in two conformations. A minor component, constituting up to about 30% of the total protein, which bound to 16S RNA at 0°, and a major component which did not bind to the 16S RNA at 0°. The latter could be converted to a binding conformation by heating (E12 kcal/mole). The RNA binding site, for most of the RNA population, was stable at 0°, but a minimum Mg⁺⁺ concentration of 5×10^-3 M, and an optimum concentration of 3×10^-2 M or higher, was necessary for this stability. Specific binding of protein S8 to 16S RNA occurred over a wide range of K⁺ concentrations, but below 0.25 M KCl there was some additional non-specific binding, and above 0.35 M KCl there was a gradual decrease in the binding affinity of S8 for 16S RNA. A maximum in the pH dependence binding curve was at pH 7.4 to 8.0.Paper No. 41 on Ribosomal Proteins. Preceding paper is in press in Mol. Gen. Genetics (1972).