Martin R. Hartley

Single‐chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA

The rRNA N‐glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single‐chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single‐chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A2660, which lies in a sequence that is highly conserved in all species.

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

Abstract The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [ 35 S]methionine-containing chymotryptic peptides of the 52,000 M r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [ 35 S]methionine in vivo . From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.

The structure and function of ribosome-inactivating proteins

Many plants produce ribosome-inactivating proteins (RIPs) — enzymes that act on ribosomes in a highly specific way, thereby inhibiting protein synthesis. Some RIPs can bind to and enter cells, making them among the most toxic substances known. More commonly, however, RIPs are unable to enter healthy cells, and are therefore poorly cytotoxic. Their role in plants is currently a matter of debate, although it has been suggested that they may have defensive functions, or an involvement in programmed senescence, in certain organs. The RIPs are frequently produced in large amounts — up to 10% of total protein — that are far in excess of the amounts required to inhibit protein synthesis. In most RIP-producing plants, the ribosomes are sensitive to the enzyme, and the two are physically separated.

Pea chloroplast ribosomal proteins: Characterization and site of synthesis

SummaryThe characterization of pea chloroplast ribosomal proteins has been carried out. The 30 S and 50 S subunits contain 24 and 32 ribosomal proteins respectively. These numbers are very similar to the numbers previously found for spinach chloroplast (Mache et al. 1980).Chloroplast-synthesized ribosomal proteins have been determined using the light driven system of protein synthesis of Ellis and Hartley (1981). It is concluded that at least 6 ribosomal proteins of the 30 S and 5 proteins of the 50 S subunits are made in chloroplasts. The possibility that 4 additional ribosomal proteins (3 of the 30 S and 1 of the 50 S subunit) are chloroplastmade, is discussed.

Wheat ribosome-inactivating proteins : seed and leaf forms with different specificities and cofactor requirements

Distinct forms of ribosome-inactivating proteins were purified from wheat (Triticum aestivum L.) germ and leaves and termed tritin-S and tritin-L, respectively. These differ in size and charge and are antigenically unrelated. They are both RNA N-glycosidases which act on 26S rRNA in native yeast (Saccharomyces cerevisiae) ribosomes by the removal of A3024 located in a universally conserved sequence in domain VII which has previously been identified as the site of action of ricin A-chain. Tritin-S and tritin-L differ in both their ribosome substrate specificities and cofactor requirements. Tritin-S shows only barely detectable activity on ribosomes from the endosperm, its tissue of synthesis, whereas tritin-L is highly active on leaf ribosomes. Additionally, tritin-S is inactive on wheat germ, tobacco leaf and Escherichia coli ribosomes but active on rabbit reticulocyte and yeast ribosomes. Tritin-L is active on ribosomes from all of the above sources. Tritin-S, unlike tritin-L shows a marked requirement for ATP in its action.

Toxic plant proteins

Evolution of Plant Ribosome-Inactivating Proteins.- RNA N-Glycosidase Activity of Ribosome-Inactivating Proteins.- Enzymatic Activities of Ribosome-Inactivating Proteins.- Type I Ribosome-Inactivating Proteins from Saponaria officinalis.- Type 1 Ribosome-Inactivating Proteins from the Ombu Tree (Phytolacca dioica L.).- Sambucus Ribosome-Inactivating Proteins and Lectins.- Ribosome-Inactivating Proteins from Abrus pulchellus.- Ribosome-Inactivating Proteins in Cereals.- Ribosome Inactivating Proteins and Apoptosis.- The Synthesis of Ricinus communis Lectins.- How Ricin Reaches its Target in the Cytosol of Mammalian Cells.- Ribosome-Inactivating Protein-Containing Conjugates for Therapeutic Use.

Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes

SummaryYeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants. Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains analysed transcribed full-length RTA RNA. The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alterations within the RTA-encoding DNA causing production of mutant toxin. Ribosomes isolated from mutant strains were shown to be susceptible to RTA modification in vitro suggesting that the strains contain alterations in RTA. This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein. Although serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modifying activity. This decrease in activity apparently allows yeast cells to survive expression of RTA as only a proportion of the ribosomes become modified. We demonstrate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activity compared with native RTA when produced in Escherichia coli. Such mutations provide a valuable means of identifying residues important in RTA catalysis and of further understanding the precise mechanism of action of RTA.

Photoregulation of Gene Expression during Chloroplast Biogenesis

Chloroplast biogenesis in higher plants is light dependent. The mature organelles differentiate either from rudimentary proplastids present in light-grown plants or from etioplasts which develop in plants maintained in darkness. Both types of transition are characterized by major changes in the structural organization, molecular composition and photosynthetic activity of the plastids, as discussed in a. recent review (Bradbeer 1981).

Binding Sites of E. coli DNA-dependent RNA polymerase on spinach chloroplast DNA.

SummaryUnder stringent conditions E. coli DNA-dependent RNA polymerase holoenzyme binds selectively to some spinach chloroplast DNA fragments generated by restriction endonucleases. The strongest of these binding sites, as judged by the initial rate of complex formation, are located in the large single-copy DNA region (Crouse et al. 1978) of this molecule and correspond in map location with known protein coding sequences. Some of these binding sites have characteristics of complex formation comparable with those of the PR and PL promoters of phage lambda DNA.Binding sites located close to the rRNA operons on the chloroplast DNA bind polymerase less strongly than those described above. Since the rRNAs are the most abundant transcription products in vivo and in isolated chloroplasts (Hartley and Head 1979; Bohnert et al. 1977) this suggests that the E. coli and chloroplast enzymes do not recognize all of the major promoters in chloroplast DNA with the same efficiency of binding.We have investigated in detail one region of the chloroplast DNA from spinach which contains three strong binding sites. This region has been shown to contain at least the gene for a 32,000 dalton protein (Driesel et al. 1980) which is most probably the so-called photogene (Bedbrook et al. 1978).One of these three E. coli RNA polymerase binding sites is not more than approximately 150 by apart from what, by hybridization studies using isolated mRNA, we know to be the coding sequence for this protein.The results suggest that for some genes on the chloroplast DNA the bacterial RNA polymerase may be used to search for transcription initiation sites.

Enzymatic Activities of Ribosome-Inactivating Proteins

Ribosome-inactivating proteins (RIPs) constitute a diverse group of proteins that share an RNA N-glycosidase activity that acts very specifically on the ribosomal RNA of the 50S/60S ribosomal subunit to inhibit protein synthesis. Additionally, the majority of RIPs act on non-ribosomal RNA and DNA in a sequence context-independent fashion, releasing multiple adenines and sometimes guanines. One such activity depends on the presence of a 5′ cap structure, and may be responsible for the anti-viral properties of some RIPs. In addition to their N-glycosidase activity on nucleic acids, some ribosome-inactivating enzymes have been reported to be bifunctional with another, unrelated activity. No active sites for these unrelated activities have been found, and their presence in preparations of RIPs may be due to contamination.