Sequence and structure of the RNA subunit of RNase P from the cyanobacterium Pseudoanabaena sp. PCC6903.

The catalytic RNA subunit of ribonuclease P (RNase P) from the cyanobacterium Pseudoanabaena sp. PCC6903 has been cloned and sequenced. The RNA has a primary and secondary structure with overall similarity to other cyanobacterial RNase P RNAs characterized so far but contains some peculiarities of its own. A consensus promoter sequence can be identified at the 5' end of the gene.

Cloning and expression in Escherichia coli of the gene coding for phytoene synthase from the cyanobacterium Synechocystis sp. PCC6803.

The gene coding for the carotenoid biosynthesis enzyme phytoene synthase (pys) has been cloned from the unicellular cyanobacterium Synechocystis sp. PCC6803. The gene has been functionally expressed in Escherichia coli, where it directs the biosynthesis of phytoene from geranylgeranyl pyrophosphate (GGPP). Analysis of the sequence of the Synechocystis pys protein deduced from the gene sequence shows that it is highly homologous to the tomato and Synechococcus phytoene synthases and shows conserved domains with other bacterial phytoene synthase enzymes. The pys gene starts 60 nucleotides downstream of the pds gene (which codes for phytoene desaturase). However, it seems to be transcribed mainly from its own promoters, because insertions that disrupt the pds gene do not affect significantly the expression of the pys gene.

Characterization of two carotenoid gene promoters in the cyanobacterium Synechocystis sp. PCC 6803.

The in vivo promoter activity of the 5'-region of the genes encoding the carotenogenic enzymes phytoene desaturase (crtP) and phytoene synthase (crtB) from the cyanobacterium Synechocystis sp. PCC 6803 has been studied by deletion mapping, primer extension, Northern blot, and using the chloramphenicol acetyltransferase (cat) reporter gene. crtP and crtB are closely linked in the Synechocystis genome, but it is shown that both genes are independently transcribed. The level of transcription in both cases is very low. Their expression is affected in a similar way by light intensity: very little or no expression is observed in the dark compared with expression under illumination conditions. High light intensities induce a transient increase in transcription initiation as detected with the cat probe. DNA fragments containing 81 base pairs upstream of the crtP putative transcription initiation site or 192 base pairs upstream of the crtB putative transcription initiation site are enough to confer light sensitive expression to a reporter gene.

Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli.

The genes for the protein (C5 protein) and RNA (M1 RNA) subunits of Escherichia coli RNase P have been subcloned and their products prepared in milligram quantities by rapid procedures. The interactions between the two subunits of the enzyme have been studied in vitro by a filter-binding technique. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme complex is approximately 4 x 10(-10) M. C5 protein also interacts with various RNA molecules in a non-specific manner with a dissociation constant of 2 x 10(-8) to 6 x 10(-8) M. Regions of M1 RNA required for interaction with C5 protein have been defined by deletion analysis and footprinting techniques. These interactions are localized primarily between nucleotides 82 to 96 and 170 to 270 of M1 RNA.

Protein synthesis inhibitors and catalytic RNA. Effect of puromycin on tRNA precursor processing by the RNA component of Escherichia coli RNase P.

RNase P and ribosomes must interact with similar substrate molecules, tRNA precursors in the case of RNase P and aminoacyl-, peptidyl- or free tRNAs in the case of ribosomes. In order to compare the substrate recognition mechanisms between ribosomes and RNase P, protein synthesis inhibitors have been assayed for their effect on the catalytic activity of the RNA component of Escherichia coli RNase P (M1 RNA). Puromycin has an inhibitory effect that could be related to similar substrate recognition mechanisms by rRNA in the ribosome and by M1 RNA in RNase P.

Functional reconstitution of RNase P activity from a plastid RNA subunit and a cyanobacterial protein subunit.

The plastid (cyanelle) from the Glaucocystophyceae alga Cyanophora paradoxa contains an RNase P RNA subunit (P RNA) similar to the cyanobacterial P RNA. We have synthesized this RNA by in vitro transcription and analyzed its activity in the absence or presence of the RNase P protein subunit (P protein) from Escherichia coli and the cyanobacterium Synechocystis sp. PCC 6803. In contrast to the bacterial P RNA, the cyanelle P RNA is not active in the absence of protein in any of the conditions tested. A functional enzyme could be reconstituted with the Synechocystis protein but not with the E. coli protein. This is the first demonstration of RNase P activity reconstitution from organellar and bacterial subunits.

Toxic-oil syndrome: case reports associated with the ITH oil refinery in Sevilla.

Toxic-oil syndrome (TOS), a new disease that occurred in epidemic form in Spain in 1981, has been associated with the ingestion of unlabelled oil bought principally from travelling salesmen. Chemical analysis of oils taken from ill families has shown them to consist of varying proportions of different vegetable oils and animal fats, often showing chemical evidence of prior treatment with aniline. We investigated the unusual circumstances surrounding the reported occurrence of three TOS cases in two families in Sevilla, a city located far away (approximately 300 km) from the group of 14 provinces in central and northwestern Spain where 99% of the TOS cases occurred. Each case we investigated fitted the clinical picture of TOS and was not consistent with any other diagnosis. Illness apparently occurred as a result of ingestion of oil taken from the ITH oil refinery in Sevilla, a plant in which rapeseed and grapeseed oils were refined for the distributing firm through which oil bearing the causative agent of TOS is thought to have entered the market. These data provide further strong support for the hypothesis that food oil was the vehicle by which the aetiological agent of TOS was transmitted. Because ingestion of refined denatured rapeseed oil was most closely associated with the illness in time, the TOS agent was probably contained initially in this type of oil. The agent very probably entered later oil mixtures through such contaminated rapeseed oil.

Gene sll0033 from Synechocystis 6803 encodes a carotene isomerase involved in the biosynthesis of all-E lycopene.

The function of gene sll0033 from Synechocystis 6803 which is homologous to the bacterial crtI-type phytoene desaturase genes was elucidated as a novel carotene isomerase. Escherichia coli transformed with all genes necessary for the formation of zeta-carotene and expressing a zeta-carotene desaturase synthesized the positional isomer prolycopene (7,9,7',9'Z lycopene) which cannot be cyclized in the subsequent reactions to a- and beta-carotene. Upon cotransformation with sll0033, the formation of all-E lycopene is mediated instead.

The RNase P RNA from cyanobacteria: short tandemly repeated repetitive (STRR) sequences are present within the RNase P RNA gene in heterocyst-forming cyanobacteria.

The RNase P RNA gene (rnpB) from 10 cyanobacteria has been characterized. These new RNAs, together with the previously available ones, provide a comprehensive data set of RNase P RNA from diverse cyanobacterial lineages. All heterocystous cyanobacteria, but none of the non-heterocystous strains analyzed, contain short tandemly repeated repetitive (STRR) sequences that increase the length of helix P12. Site-directed mutagenesis experiments indicate that the STRR sequences are not required for catalytic activity in vitro. STRR sequences seem to have recently and independently invaded the RNase P RNA genes in heterocyst-forming cyanobacteria because closely related strains contain unrelated STRR sequences. Most cyanobacteria RNase P RNAs lack the sequence GGU in the loop connecting helices P15 and P16 that has been established to interact with the 3'-end CCA in precursor tRNA substrates in other bacteria. This character is shared with plastid RNase P RNA. Helix P6 is longer than usual in most cyanobacteria as well as in plastid RNase P RNA.

Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria.

The genes encoding the RNA subunit of ribonuclease P from the unicellular cyanobacterium Synechocystis sp. PCC 6803, and from the heterocyst-forming strains Anabaena sp. PCC 7120 and Calothrix sp. PCC 7601 were cloned using the homologous gene from Anacystis nidulans (Synechococcus sp. PCC 6301) as a probe. The genes and the flanking regions were sequenced. The genes from Anabaena and Calothrix are flanked at their 3'-ends by short tandemly repeated repetitive (STRR) sequences. In addition, two other sets of STRR sequences were detected within the transcribed regions of the Anabaena and Calothrix genes, increasing the length of a variable secondary structure element present in many RNA subunits of ribonuclease P from eubacteria. The ends of the mature RNAs were determined by primer extension and RNase protection. The predicted secondary structure of the three RNAs studied is similar to that of Anacystis and although some idiosyncrasies are observed, fits well with the eubacterial consensus.

Modification of 40S ribosomal subunits from yeast with dimethylmaleic anhydride.

Modification of 40S ribosomal subunits from Saccharomyces cerevisiae with dimethylmaleic anhydride (DMMA), a reagent for protein amino groups, is accompanied by loss of polypeptide-synthesizing activity and by dissociation of proteins from the particles. The protein-deficient ribosomal particles, originated from 40S subunits by treatment with dimethylmaleic anhydride at a molar ratio of reagent to particle of 250, can partially reconstitute active subunits upon addition of the corresponding released proteins, and regeneration of the modified amino groups.

Affinity chromatography with an immobilized RNA enzyme.

M1 RNA, the catalytic subunit of Escherichia coli RNase P, has been covalently linked at its 3' terminus to agarose beads. Unlike M1 RNA, which is active in solution in the absence of the protein component (C5) of RNase P, the RNA linked to the beads is active only in the presence of C5 protein. Affinity chromatography of crude extracts of E. coli on a column prepared from the beads to which the RNA has been crosslinked results in the purification of C5 protein in a single step. The protein has been purified in this manner from cells that contain a plasmid, pINIIIR20, which includes the gene that codes for C5 protein. A 6-fold amplification of the expression of C5 protein is found in these cells after induction as compared to cells that do not harbor the plasmid.

Cloning, purification and characterization of the protein subunit of ribonuclease P from the cyanobacterium Synechocystis sp. PCC 6803.

The rnpA gene from the cyanobacterium Synechocystis sp. PCC 6803, which codes for the protein subunit of ribonuclease P (RNase P), has been cloned by functional complementation of an Escherichia coli mutant. This protein had previously been characterized only in proteobacteria and gram-positive bacteria. rnpA and the closely linked rpmH gene, which code for the large subunit ribosomal protein L34, have been sequenced. The Synechocystis 6803 L34 protein is more similar to the homologous protein from some non-green chloroplasts than to the L34 protein from other bacteria. The protein subunit of RNase P from Synechocystis 6803 has been overexpressed in E. coli and purified to homogeneity. Antibodies raised against the Synechocystis 6803 RNase P protein did not recognize the homologous protein from E. coli (C5 protein). Similarly, antibodies raised against the E. coli C5 protein did not recognize significantly the Synechocystis 6803 protein. In spite of the lack of immunological cross-reactivity and the low level of sequence identity, the E. coli and Synechocystis 6803 proteins are functionally interchangeable. In enzymatic assays using either an E. coli precursor tRNA(Tyr) or a Synechocystis 6803 precursor tRNA(Gln) as substrates, we have detected RNase P activity with holoenzymes reconstituted with the RNA subunit from E. coli and the protein subunit from Synechocystis 6803 or with the RNA subunit from Synechocystis 6803 and the protein subunit from E. coli. The relative efficiency of cleavage of the different substrates is dependent on the origin of the protein subunit used to reconstitute the holoenzyme.

Substrate binding and catalysis by ribonuclease P from cyanobacteria and Escherichia coli are affected differently by the 3' terminal CCA in tRNA precursors.

We have studied the effect of the 3' terminal CCA sequence in precursors of tRNAs on catalysis by the RNase P RNA or the holoenzyme from the cyanobacterium Synechocystis sp. PCC 6803 in a completely homologous system. We have found that the absence of the 3' terminal CCA is not detrimental to activity, which is in sharp contrast to what is known in other bacterial systems. We have found that this is also true in other cyanobacteria. This situation correlates with the anomalous structure of the J15/16 loop in cyanobacteria, which is an important loop in the CCA interaction in Escherichia coli RNase P, and with the fact that cyanobacteria do not code the CCA sequence in the genome but add it posttranscriptionally. Modification of nucleotides 330-332 in the J15/16 loop of Synechocystis RNase P RNA from GGU to CCA has a modest effect on kcat for CCA-containing substrates and has no effect on cleavage-site selection. We have developed a direct physical assay of the interaction between RNase P RNA and its substrate, which was immobilized on a filter, and we have determined that Synechocystis RNase P RNA binds with better affinity the substrate lacking CCA than the substrate containing it. Our results indicate a mode of substrate binding in RNase P from cyanobacteria that is different from binding in other eubacteria and in which the 3' terminal CCA is not involved.

Partial reconstitution of 60S ribosomal subunits from yeast.

Ribosomal 60S subunits active in polyphenylalanine synthesis can be reconstituted from core particles lacking 20-40% of the total protein. These core particles were obtained by treatment of yeast 60S subunits with dimethylmaleic anhydride, a reagent for protein amino groups. Upon reconstitution a complementary amount of split proteins is incorporated into the ribosomal particles, which have the sedimentation coefficient of the original subunits. Ribosomal protein fractions obtained by extraction with 1.25 M NH4Cl, 4 M LiCl, 7 M LiCl, or 67% acetic acid, are much less efficient in the reconstitution of active subunits from these core particles than the corresponding released fraction prepared with dimethylmaleic anhydride. Attempts to reconstitute active subunits from protein-deficient particles obtained with 1.25 M NH4Cl plus different preparations of ribosomal proteins, including the fraction released with dimethylmaleic anhydride, were unsuccessful. Therefore, under our conditions, of the disassembly procedures assayed only dimethylmaleic anhydride allows partial reconstitution of active 60S subunits.

Protein-deficient ribosomal particles from yeast 60S subunits obtained by modification with dimethylmaleic anhydride and by treatment with NH4Cl.

The protein-deficient particles prepared from yeast 60S subunits by modification of lysine residues with the reversible reagent dimethylmaleic anhydride are compared with those obtained by treatment with NH4Cl. The two procedures cause selective dissociation of certain proteins. With a few exceptions, the dissociation pattern is similar in both cases. When using dimethylmaleic anhydride, a variation in the protein composition of the ribosomal cores is obtained by modification of the ribosomal subunits in the presence of any of the following ligands: elongation factor-2, ricin A, verrucarine A and puromycine.

RNase P from bacteria. Substrate recognition and function of the protein subunit.

RNase P recognizes many different precursor tRNAs as well as other substrates and cleaves all of them accurately at the expected position. RNase P recognizes the tRNA structure of the precursor tRNA by a set of interactions between the catalytic RNA subunit and the T- and acceptor-stems mainly, although residues in the 5'-leader sequence as well as the 3'-terminal CCA are important. These conclusions have been reached by several studies on mutant precursor tRNAs as well as cross-linking studies between RNase P RNA and precursor tRNAs. The protein subunit of RNase P seems also to affect the way that the substrate is recognized as well as the range of substrates that can be used by RNase P, although the protein does not seem to interact directly with the substrates. The interaction between the protein and RNA subunits of RNase P has been extensively studied in vitro. The protein subunit sequence is not highly conserved among bacteria, however different proteins are functionally equivalent as heterologous reconstitution of the RNase P holoenzyme can be achieved in many cases.

Effects of different amino-group reagents on ribosomal integrity: structural role of lysine residues.

Treatment of 60S subunits from yeast ribosomes with dicarboxylic acid anhydrides (maleic, dimethylmaleic and tetrahydrophtalic), which introduces negatively-charged residues, is accompanied by substantial dissociation of protein components (35-55%). In contrast, acetic anhydride or cyanate, which introduce uncharged groups, cause practically no protein release, even after extensive modification. Therefore, in addition to blocking lysine-RNA interactions, a large change in the electric charge of the proteins appears to be necessary to obtain dissociation. These results seem to indicate that lysine residues are not essential to ribosome integrity, while arginine-RNA interactions should play an important role in the maintenance of ribosomal structure.

Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA.

tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3' end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (deltassrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of deltassrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the deltassrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics.