Variations in ppGpp levels in Rhodopseudomonas spheroides during adaptation to decreased light intensity

The guanosine nucleotides ppGpp and pppGpp have been found in the photosynthetic bacterium $\text{Rhodopseudomonas spheroides}$ supporting a suggestion that these are ubiquitous compounds in bacteria. ppGpp levels undergo an abrupt rise coincident with the cessation of RNA accumulation which occurs in this bacterium following a down-shift in incident light intensity. An abrupt decrease in the rate of [³H] uridine uptake into acid-precipitable material characteristic of light shift-down is also coincident with the rise in ppGpp levels. A regulatory role for this nucleotide in $\text{R. spheroides}$ similar to that proposed in other bacteria is suggested.     

Altered metabolism of ppGpp in quinone-treated E. coli: Possible role of aminoacyl-tRNA synthetases

The bacteriostatic quinone 6-amino-7-chloro-5,8-dioxoquinoline inhibits leucyl-tRNA synthetase in vivo and in vitro (Ogilvie et al. Biochim. Biophys. Acta $\text{407}$, 357–364; 1975). In this report it is shown that the quinone also interferes with the metabolism of ppGpp. Quinone treatment of E. coli MRE 600 causes the same phenotypic pattern as found in spoT^? mutants: overproduction of ppGpp and a drastic increase of its half-life; the formation of pppGpp, the possible degradation product of ppGpp, is blocked. A model is discussed to explain how the inhibition of leucyl-tRNA synthetase could account for the altered metabolism of ppGpp.     

Preparation of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) from Escherichia coli ribosomes

A means of preparative enzymatic synthesis of guanosine tetraphosphate (ppGpp), guanosine pentaphosphate (pppGpp), and related derivatives is deseribed. The Escherichia coli ribosomes can be recovered, stored, and used repeatedly as a source of synthetic activity. The procedure described affords a relatively simple means of synthesizing gram amounts of these nucleotides as well as some derivatives such as the β-γ methylenyl derivative of guanosine pentaphosphate (peppGpp).     

Ribosome specificity for the formation of guanosine polyphosphates

Ribosomes obtained from $\text{Bacillus brevis}$ (ATCC 8185) were slightly active in synthesizing guanosine polyphosphates, which activity was greatly stimulated by addition of $\text{Escherichia coli}$ stringent factor. $\text{Chlamydomonas reinhardtii}$ chloroplast ribosomes did not produce guanosine polyphosphates on incubation but responded with abundant synthesis to addition of the stringent factor from $\text{E. coli}$. In contrast, cytoplasmic ribosomes from the same organism did not respond. Interchange experiments between either subunit from chloroplasts with the $\text{E. coli}$ counterparts showed good activity. When the small subunit of cytoplasmic $\text{Chlamydomonas}$ ribosomes was combined with the large subunit of $\text{E. coli}$ or of chloroplasts, a small but definite response was obtained.     

Synthesis of pppGpp by ribosomes from an Escherichia coli spoT mutant and the metabolic relationship between pppGpp and ppGpp.

Both ribosomes and a cell-free extract (S-30) prepared from an Escherichia coli spoT mutant catalyzed the synthesis of guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) as efficiently as did ribosomes and S-30 from a spoT+ strain. In both cases, the level of pppGpp reached its maximum before ppGpp maximally accumulated. pppGpp added to the ribosome system was rapidly converted to ppGpp. These results indicate that the spoT+ gene product may not have a direct role in the synthesis of pppGpp and that pppGpp is a precursor of ppGpp.     

Induction of translational errors (misreading) by tuberactinomycins and capreomycins

The well characterized translocation inhibitor viomycin (=tuberactinomycin B) promotes translational errors (misreading) in an in vitro system from Escherichia coli. It strongly stimulates both the binding of noncognate Tyr-tRNA to poly(U)-programmed ribosomes and the subsequent synthesis of acPhe(Tyr)_n-tRNA ($\text{n}\text{?20}$). The closely related antibiotics capreomycin and tuberactinomycins A,N and O also inhibit translocation and induce misreading.     

Extracellular ribosomes during metamorphosis of the blowfly Calliphora vicina R. D. -- A. reappraisal of their authenticity.

Extracellular ribosomes during adult development of the blowfly $\text{Calliphora}\text{vicina}$ were previously considered to occur naturally $\text{in}\text{vivo}$. A variety of radioisotopic experiments performed at different stages of metamorphosis now demonstrates that the specific activities of extra- and intracellular ribosomes are consistently very similar. Further, in addition to previously described physico-chemical similarities, extra- and intra-cellular ribosomal protein profiles are now shown to be essentially identical. These results vitiate the notion of a natural pool of extracellular ribosomes, the occurrence of which is now ascribed to an experimental artifact, resulting from unusual cell fragility.     

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 extraction of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) from Escherichia coli using low pH reagents: a reevaluation.

It has been found that the most widely used method for the extraction of guanosine 5′-diphosphate, 3′-diphosphate (ppGpp) from $\text{E. coli}$ (1 M formic acid at 0°) results in its $\text{in vitro}$ degradation to ppGp and GDP. A comparison with several other extraction procedures indicated that this breakdown is due to the low pH of the reagents used during extraction. This degradation can largely be prevented by using a new extraction technique which involves freezing and thawing of the cells in the presence of lysozyme at a neutral pH followed by treatment with deoxycholate. With this method it is possible to recover from three to five times as much ppGpp from both unstarved and amino acid starved stringent strains of $\text{E. coli}$ as compared with the most widely used formic acid procedure. Consequently, it will be necessary to reevaluate the ppGpp values obtained from cells when formic acid or other low pH reagents were used during extraction.     

ppGpp cycle in Escherichia coli.

Summary Kinetics of accumulation and degradation of ppGpp and pppGpp were analysed in spoT ⁺ and spoT strains of Escherichia coli. The experimental data in this paper indicate that on degradation ppGpp is not converted to pppGpp but instead is converted to GDP which is in turn phosphorylated to GTP. In addition the data are consistent with the idea the pppGpp is a direct precursor of ppGpp. We propose that ppGpp is metabolised according to the following pathway: GTP-pppGpp-ppGpp-GDP-GTP, which we call the ppGpp cycle. Coupled with the observations in spoT strains we assume that ppGpp blocks its own synthesis by inhibiting the synthesis of pppGpp but not the interconversion of the two nucleotides.     

Increased de novo phospholipid biosynthesis in the stimulated rat exocrine pancreas

The $\text{hisU1820}$ mutant (TA799) of $\text{Salmonella}\text{,}\text{typhimurium}$ shows a substantial increase in the levels of ppGpp (MSI) and of pppGpp (MSII) during several types of metabolic shifts. Noticeable amounts of ppGp (MSIII) are also present post-carbon/energy source downshifts and temperature up-shifts. The increased levels of these guanosine polyphosphates were observed despite the absence of the expected reduction in RNA synthesis upon a nutritional downshift. We, therefore, suggest that the $\text{hisU}$ mutation causes an increase in the accumulation of MSI and MSII; and that ppGpp alone is not sufficient to promote restriction of RNA synthesis during a nutritional transition.     

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.     

The effect of stratification on in vitro protein synthesis in seeds of Pinus lambertiana

Incorporation of ¹⁴C-phenylalanine in $\text{in vitro}$ systems from sugar pine ($\text{Pinus lambertiana}$) seeds was studied. Embryo ribosomes from both dry and stratified seeds supported incorporation (431 and 326 pmoles, respectively, of phenylalanine per mg ribosome) when combined with an embryo pH 5 fraction from stratified seeds. Female gametophyte ribosomes from dry seeds were active (302 pmoles phenylalanine incorporated per mg ribosome) but lost 61 percent of their capacity to support protein synthesis after 35 hours' stratification. The pH 5 fraction from embryos increased in capacity to support incorporation as stratification progressed up to 60 days (398 pmoles phenylalanine per mg ribosome when ribosomes were from 90-day stratified embryos) while the pH 5 fraction from female gametophytes was never active.     

Initiation complex formation with double stranded RNA

Binding of formylmethionyl tRNA and ribosomes to double stranded RNA has been obtained under conditions identical to those required for initiation complex formation with single stranded RNA. While natural double stranded RNAs from Penicillium $\text{chrysogenum}$ virus and Penicillium $\text{stoloniferum}$ virus were efficient in forming initiation complexes, the synthetic polynucleotide poly(I).poly (C) was inactive. This suggests that ribosomes can recognize initiation sequences even if these are present in base-paired form.     

Synthesis of guanosine tetraphosphate (magic spot I) in Saccharomyces cerevisiae.

Guanosine tetraphosphate (Magic Spot I) has been found in formic acid extracts of $\text{Saccharomyces cerevisiae}$ subjected to heat shock. The yeast compound comigrates with authentic ppGpp in several chromatographic systems, and further tests confirm its identity with the guanosine-5′-diphosphate-3′-diphosphate found in bacteria. Oxytetracycline inhibits its formation in yeast cells, suggesting that it is produced on the mitochondrial ribosomes.     

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.     

A method for extracting intact chloroplast and cytoplasmic ribosomal RNA from leaves

A method is described for extracting intact chloroplast and cytoplasmic ribosomal RNA from leaves of two higher plant species. Sodium dodecyl sulfate (1%) and 25 mM magnesium ions are required to inhibit ribonuclease action during RNA purification by phenol deproteinization. The ethanol-precipitated RNA product, including 23s chloroplast ribosomal RNA, is completely stable during electrophoresis in the absence of magnesium ions, even in the presence of EDTA. The $\text{in}$$\text{vivo}$ mole fraction of chloroplast ribosomes relative to cytoplasmic ribosomes is estimated. Bentonite is shown to cause preferential losses of chloroplast RNA during extraction.     

Acidic proteins from Saccharomyces cerevisiae ribosomes.

Two dimensional gel electrophoresis of ribosomal proteins from $\text{Saccharomyces}\text{cerevisiae}$ reveals the presence of three spots in the region corresponding to proteins of high acidic character. Washing the ribosomes with 0.4 M NH₄Cl and 50% ethanol, followed by chromatography of the extracted proteins on DEAE-cellulose, indicated the presence of two fractions of acidic proteins; (A and Ax), having very similar molecular weights (12.000–13.000), but phosphorylated to different extents. Fractions A and Ax are immunologically distinct and their immunologic properties differ from acidic proteins found in $\text{Escherichia}\text{coli}$, rat liver, and $\text{Artemia}\text{salina}$ ribosomes.Protein A can be resolved into two bands by electrofocusing, and two dimensional gel electrophoresis. The two components correspond to proteins L44 and L45 according to the standard nomenclature. Proteins Ax seems to correspond to the spot that moves above and to the left of L44 and L45 and is at least three times more phosphorylated than these two proteins.