Nondiscrimination of RNA viral message in binding to 30S ribosomes derived from T4 phage infected Escherichia coli

The effect of T4 phage on ribosomes in terms of their ability to bind RNA viral template is examined. It is found that the 30S subunits of T4 ribosomes bind MS2 RNA as efficiently as do the subunits of uninfected $\text{E. coli}$ ribosomes. On the other hand, analyses of the formation of 70S initiation complex, presumably from MS2 RNA-30S ribosome complex, using both labeled MS2 RNA and initiator tRNA, reveal that T4 ribosomes are only about half as active as $\text{E. coli}$ ribosomes. The latter phenomenon has been reported previously. These results suggest that, following T4 infection, ribosomes are modified in such a way that the attachment of fMet-tRNA_f to MS2 RNA-30S subunit complex is impaired.     

Action of methanol on the assocation of ribosomal subunits and its effect on the GTPase activity of elongation factor G

Methanol causes association of 30S and 50S ribosomal subunits from $\text{E. coli}$ at MgCl₂ concentrations in which they are normally completely dissociated. The 70S ribosome formed under these conditions shows a lower sedimentation velocity and is functionally active in the EF-G GTPase. Association of ribosomal subunits in the presence as well as absence of methanol is affected by washing the ribosomes with 0.5 M NH₄Cl. Methanol reduces the Mg²⁺ concentration required for subunit association as well as for EF-G GTPase activity. The basic requirement for EF-G GTPase activity both with and without alcohol is shown to be the association of 30S and 50S subunits.     

Structural changes in the cell membrane of lambda-lysogenic Escherichia coli induced by colicin E2.

The structural changes in the cell membrane of λ-lysogenic $\text{Escherichia coli}$ induced by colicin E₂ were examined. The addition of colicin E₂ made the cells susceptible to various detergents and the transport rate of o-nitrophenyl-β-D-galactoside into the colicin-treated cells was stimulated markedly by adding a low concentration of sodium dodecyl sulfate. The fluorescence intensity of 8-anilino-1-naphthalenesulfonate bound to the cells was markedly increased by adding colicin E₂. Colicin E₂ stimulated the incorporation of ${}^{32}\text{P}$ from prelabeled phosphatidylglycerol to cardiolipin. All these changes probably suggesting the structural alteration of the cell membrane were dependent on the presence of the $\text{rex}$ gene of λ prophage in the cells.     

Determination of the distribution of protein and nucleic acid in the 70 S ribosomes of Escherichia coli and their 30 S subunits by neutron scattering.

Neutron small-angle scattering of the 70 S Escherichia coli ribosomes and of its smaller 30 S subunit has been measured in $\text{H}{}_2\text{O}{}^2\text{H}{}_2\text{O}$ mixtures. A linear dependence of the square of the radius of gyration on the reciprocal of the contrast is found, which is qualitatively similar to the results from contrast variation with the larger 50 S subunit. The slope α in this plot is a measure of radial segregation of RNA and proteins. It is most pronounced with the 50 S subunit. The 30 S particle appears to be more homogeneous, whereas the 70 S ribosome assumes an intermediate value of α. Neither the 30 S and 50 S subunits nor the 70 S ribosome show a significant separation of the centres of mass of their RNA part and proteins. A quantitative comparison of the parameters obtained suggest that the interaction between the two subunits and the 70 S ribosomes does not involve any major change in the latter.     

Analysis of kethoxal bound to ribosomal proteins from Escherichia coli 70S reacted ribosomes

To investigate ribosome topography and possible function, 70S ribosomes of $\text{Escherichia coli}$ were reacted with the dicarbonyl compound kethoxal. Ribosomal protein was extracted after reaction, and through two dimensional gel electrophoresis, the reactive proteins of the two subunits were identified. From the 30S subunit, the most reacted proteins were S2, S3, S4, S5 and S7 and from the 50S subunit, L1, L5, L16, L17, L18 and L27. The results with kethoxal are compared with other modifiers of ribosomal proteins.     

Binding of [14C]tuberactinomycin O, an antibiotic closely related to viomycin, to the bacterial ribosome.

The binding of [¹⁴C]tuberactinomycin O, an antibiotic closely related to viomycin, to $\text{E. coli}$ ribosomes has been examined by equilibrium dialysis method. The antibiotic has been observed to bind to the 70S ribosome, which possesses two binding sites: one on the 30S ribosomal subunit and another on the 50S subunit. The affinity for the large subunit is greater than that for the small subunit. The binding to both ribosomal subunits is reversed by viomycin, indicating that tuberactinomycin O and viomycin have the same binding sites on the ribosome. The results seem to be in accordance with the previous finding that viomycin exhibits dual actions on ribosomal function: the inhibition of fMet-tRNA_F (initiation) and inhibition of translocation of peptidyl-tRNA.     

The mechanism of action of initiaton factor 3 in protein synthesis,I. Studies on ribosomes crosslinked with dimethylsuberimidate

The mechanism of action of chain initiation factor 3 in translation was examined by using $\text{E. coli}$ 70S ribosomes which were covalently crosslinked with dimethylsuberimidate. Crosslinked ribosomes were inactive in AUG-dependent fMet-tRNA binding, and were not stimulated by IF-3 in poly(U) translation. IF-3 is known to be required for maximal rates of amino acid incorporation with synthetic polynucleotides at 18 mM Mg²⁺. A direct interaction of IF-3 with 70S ribosomes was demonstrated by crosslinking ¹⁴C-labeled IF-3 to 70S ribosomes. The labeled factor was also crosslinked to 30S and 50S ribosomal subunits. A model is presented proposing the mechanism of action of IF-3 on 70S ribosomes.     

In vitro inactivation of ascites ribosomes by colicin E 3

Colicin E 3 treatment of 80 S ribosomes from mouse ascites cells completely arrests in vitro protein synthesis. Isolated 40 S subunits are resistant to the colicin action while the larger subunit becomes inactivated after treatment with this protein. 40 S subunits derived from colicin E 3 treated 80 S ribosomes lose their ability to participate in polyphenylalanine synthesis. Colicin E 3 damaged 80 S ribosomes appear to be functional with regard to Met-tRNA_f^Met binding while they fail to attach Phe-tRNA to the A-site. Thus, except for the susceptibility of their larger subunits to colicin, the inactivation mechanism of 80 S particles resembles the process which alters the bacterial ribosome.     

Effect of a bifunctional imidoester on dissociation of 70S ribosomes in Escherichia coli

Treatment of the 70S ribosome from $\text{Escherichia coli}$ with diethyl malonimidate dihydrochloride, a bifunctional imidoester, was found to result in the formation of crosslinkage between the two subunits. The 70S complex thus obtained no longer dissociates into 50S and 30S particles at 0.5mM Mg²⁺ concentration, but do so at lower concentrations (0.1mM), suggesting the release of protein(s) involved in the inter-particle cross-linkage from one or both ribosomal subunits.     

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.     

A Comparison of the Native and Derived 30S and 50S Ribosomes of Escherichia coli

Four types of ribosomes occurring in E. coli have been separated by sucrose gradient centrifugation. These are the 30S and 50S particles occurring in E. coli extracts (native particles), and the 30S and 50S particles which are the subunits of 70S ribosomes (derived particles). Two criteria were used in comparing these particles: (1) The type of RNA contained in each, as determined by sedimentation velocity in the analytical ultracentrifuge. (2) The ability of mixtures of 30S and 50S ribosomes (derived 30S + derived 50S, native 30S + native 50S) to undergo the reaction: [Formula: see text] Native and derived 30S particles were found to contain 16S RNA. Derived 50S particles contained 23S RNA and a small amount of 15 to 20S RNA, whereas native 50S ribosomes contained only 16S RNA. Derived 30S and 50S particles combined to form 70S particles. However, under identical conditions, native 30S and 50S particles did not form 70S ribosomes.     

Responsibility of 16S RNA for the stimulation of polypeptide synthesis by spermidine.

From the studies on the spermidine stimulation of polyphenylalanine synthesis catalyzed by $\text{E. coli}$ 50S and reconstituted 30S particles containing 16S RNA and 30S ribosomal proteins from $\text{E. coli}$ and $\text{B. thuringiensis}$ in different kinds of combinations, it is concluded that 16S RNA is mainly responsible for the stimulation of polypeptide synthesis by spermidine.     

Gel electrophoretic studies on ribosomal proteins L7L12 and the Escherichia coli 50 s subunit

Three forms of the 50 S ribosomal subunit of Escherichia coli have been separated by agarose/acrylamide gel electrophoresis. The slowest migrating form, S-50 S, corresponded to native 50 S subunits and contained four copies of proteins $\text{L}\text{7}\text{L}\text{12}$. Removal of the four copies of this protein produced a more rapidly migrating form, M-50 S. The M-50 S form was then converted to the fastest migrating form, F-50 S, by removal of additional proteins, including L10 and L11. A one-step removal of a pentameric complex of four copies of $\text{L}\text{7}\text{L}\text{12}$ plus L10 converted the S-50 S subunit directly to the F-50 S subunit. These proteins recombined specifically with the appropriate protein-deficient 50 S subunit at 3 °C to reform the S-50 S subunit, i.e. the M-50 S subunit was converted back to the S-50 S form by the addition of purified proteins $\text{L}\text{7}\text{L}\text{12}$; and the F-50 S subunit bound the pentameric complex of $\text{L}\text{7}\text{L}\text{12}$ and L10 to form S-50 S. The binding of the pentameric complex, isolated by glycerol gradient centrifugation, supports the model that all four copies of proteins $\text{L}\text{7}\text{L}\text{12}$ are together in one part of the ribosome called the “$\text{L}\text{7}\text{L}\text{12}$ stalk”. Only the four copies of $\text{L}\text{7}\text{L}\text{12}$ were removed from the 50 S subunit in low salt (0.125 m-NH₄Cl) plus 50% ethanol at 0 °C. These ribosomes (in the M-50 S form) had less than 5% of the peptide-synthesizing activity of untreated control ribosomes as measured by a poly(U) translation system in vitro. Peptide-synthesizing activity was restored, upon addition of $\text{L}\text{7}\text{L}\text{12}$, back to the treated ribosomes to give 50 S subunits (S-50 S) with a full complement of four copies of $\text{L}\text{7}\text{L}\text{12}$. Antibody to proteins $\text{L}\text{7}\text{L}\text{12}$ bound only to the S-50 S subunits, producing four new bands separated by gel electrophoresis. The bands represented complexes of one, two, three and four antibodies bound to a 50 S subunit. This result was obtained using either 50 S subunits or 70 S tight couples and indicated that all four copies of $\text{L}\text{7}\text{L}\text{12}$ are either located at a single site in the $\text{L}\text{7}\text{L}\text{12}$ stalk or, much less likely, are divided between two symmetrical sites. Proteins $\text{L}\text{7}\text{L}\text{12}$ were not only accessible to their specific antibody but could also be removed from 70 S ribosomes and polyribosomes without causing their dissociation into subunits. The ribosomes and polyribosomes had an increased gel electrophoretic mobility which was reversed by addition of proteins $\text{L}\text{7}\text{L}\text{12}$.     

A complex of acidic ribosomal proteins. Evidence of a four-to-one complex of proteins in the Bacillus stearothermophilus ribosome

Two acidic proteins from the 50 S subunit of Bacillus stearothermophilus ribosomes, namely B-L13 (homologous to Escherichia coli protein $\text{L}\text{7}\text{L}\text{12}$) and B-L8, form a complex. Radioactive B-L13, added to ribosomes before dissociation, does not appear in the complex after electrophoresis, so the (B-L13 · B-L8) complex must exist in the ribosome before dissociation. Digestion of B. stearothermophilus ribosomes with polyacrylamide-bound trypsin causes the appearance of new B-L8 and B-L13 spots on two-dimensional polyacrylamide gel electrophoresis, in a pattern which suggests that single molecules of B-L13 are being sequentially cleaved from a four-to-one complex of B-L13 and B-L8.     

Structural alteration of rRNA in the L7/L12 region of 50s ribosome on removal of L7/L12 proteins

Core particles of 50S ribosomes depleted of $\text{L7}\text{L12}$ proteins are degraded by RNase I at a considerably slower rate than intact 50S ribosomes. The normal rate is restored on incorporating $\text{L7}\text{L12}$ proteins into the core particles. The capacity of the core particles to inhibit the RNase I-catalyzed hydrolysis of poly A and to bind ethidium bromide is also greater with core particles than with intact 50S ribosomes. It appears from these results that the region(s) of rRNA in the vicinity of $\text{L7}\text{L12}$ proteins has less ordered structure which, on removal of $\text{L7}\text{L12}$ proteins, becomes more organized. Apparently, binding of $\text{L7}\text{L12}$ proteins to the 50S core leads to the destabilization of double-stranded regions of rRNA.     

The role of ribosomes in stabilizing bacteriophage T4 deoxynucleotide kinase mRNA.

In the present investigation, an approach toward defining the role of ribosomes in stabilizing functional messenger RNA in cell-free extracts is described. The data presented show that initiation of protein synthesis is necessary for maximal functional stability of bacteriophage T4 deoxynucleotide kinase mRNA $\text{in vitro}$ and suggest that much of the stability is attained by interaction of the deoxynucleotide kinase mRNA initiation site with a 30S ribosomal subunit. Data is also presented which suggest that any of several $\text{E}$. $\text{coli}$ ribonucleases could serve as a messenger ribonuclease $\text{in vivo}$.     

Dual actions of viomycin on the ribosomal functions.

Viomycin was observed to inhibit poly[U]- or f2 RNA-directed protein synthesis in an $\text{E. coli}$ cell-free system. The former was more profoundly affected than the latter. Both initiation complex formation on the 30S ribosomal subunit and on 70S ribosomes were prevented by the antibiotic. In the peptide chain elongation process, viomycin did not significantly affect aminoacyl-tRNA binding to ribosomes and the peptidyl transferase reaction, but markedly inhibit translocation of peptidyl-tRNA from the acceptor site to the donor site. The mechanism of action of the drug appeared to be unique.     

Euglena gracilis chloroplast ribosomes: improved isolation procedure and comparison of elongation factor specificity with prokaryotic and eukaryotic ribosomes

An improved method for the isolation of Euglena chloroplast ribosomes is described which presents a number of advantages over past procedures. First, ribosomes are prepared from whole cell extracts, thus bypassing the need to isolate intact chloroplasts and resulting in a 10-fold improvement in yield. Second, the inclusion of 40 mm Mg²⁺ in the preparation buffers, while stabilizing the chloroplast ribosomes, precipitates and, thereby, virtually eliminates the cytoplasmic 89 S ribosomes. Third, greater than 95% of the chloroplast ribosomes sediment at 68 S rather than as the damaged 53 S particle frequently generated in other preparation procedures. Fourth, even with a high-salt wash to remove endogenous factors, the chloroplast ribosomes still sediment at 68 S and are just as active in in vitro protein synthesis as are E. coli ribosomes. These ribosomes have been tested for activity with elongation factors from prokaryotes, eukaryotes, and the chloroplast itself, and the results have been compared to those obtained with E. coli and wheat germ ribosomes. The data may be summarized as follows: (a) Chloroplast ribosomes use E. coli$\text{EF}\text{-}\text{Tu}\text{Ts}$ and EF-G with the same efficiency as do E. coli ribosomes in protein synthesis, (b) E. coli and chloroplast ribosomes can use Euglena chloroplast EF-G to catalyze translocation, but wheat germ ribosomes cannot, (c) Wheat germ EF-1_H and EF-2 are highly active in polymerization with wheat germ ribosomes, but ribosomes from neither E. coli nor the chloroplast are able to recognize these factors, (d) All three types of ribosomes accept Phe-tRNA from E. coli EF-Tu although to differing degrees. However, neither chloroplast nor E. coli ribosomes recognize wheat germ EF-1_H for the binding of Phe-tRNA.     

U.V. induced covalent crosslinking of E.coli ribosomal RNA to specific proteins

$\text{E.}\text{coli}$ 70S ribosomes uniformly labeled $\text{in}\text{vivo}$ with ³²PO₄ were subjected to varying doses of u.v. radiation and then to the combined action of the RNases A and T₁. Following these treatments the ribosomal proteins were separated by trichloroacetic acid precipitation from the noncovalently attached RNA degradation fragments. Subsequent two-dimensional gel electrophoresis and autoradiography of these proteins revealed that significant ³²PO₄ was associated with unique ribosomal proteins, L2 was among these.     

Evidence that Escherichia coli 30 S ribosomal subunit populations are conformationally heterogeneous.

We have estimated the number of sites on each protein of the 30 S ribosome which are accessible to chemical iodination. First, the total number of iodinatable sites was determined for the intact 30 S ribosome. The proteins were extracted, separated and the relative distribution of iodine in each protein determined. This distribution of iodine divided into the total sites per ribosome gave an estimate of the number of sites per individual protein.Second, the iodinated proteins were purified and their trypsin digestion products separated. The number of radioactive peptides was taken as a measure of the number of sites on that protein open to the iodination reaction. The number of iodinatable sites for each protein was found to be radically different by the two methods. In almost all cases, the number of unique, radioactively labeled peptides, derived from a given 30 S protein, far exceeded the total incorporation into that protein. We suggest that the best explanation for this unexpected discrepancy is that the 30 S ribosome population we used in these experiments is heterogeneous in its topography.In addition we have compared the topography by the chemical iodination procedure for ribosomes in two different conformations: active and inactive (see Zamir et al., 1971). We have found very little change in the chemical reactivity of the proteins when the ribosomes are in the two different conformations. The most notable changes involve proteins S10, $\text{S}\text{18}\text{S}\text{19}$ and especially $\text{S}\text{12}\text{S}\text{13}$.