Influence of the ionic environment on the in vitro transcription of the spinach plastid DNA by a selectively bound RNA-polymerase DNA complex.

The in vitro transcription of chloroplast DNA (ctDNA) is studied using a DNA-protein complex isolated from spinach plastids. The RNA products are compared to the in vivo synthesized ctRNA by competition for hybridization. At least 80% of the in vitro RNA sequences are present in vivo. Modifications of ionic strength or introduction of heparin in the reaction medium has an important effect on transcriptional activity of the complex. Furthermore, the length of the RNA chains increases ionic strength and amount of heparin. The RNA products are analysed by Southern hybridizations to EcoRI cTDNA fragments. Changes in the ionic strength or in the amount of heparin modify heterogeneously the transcription of the various DNA regions. The quantitative distribution of transcripts among the ctDNA fragments is used as evidence for the selectivity of the transcription. The activity of the DNA-protein complex is much more resistant to high ionic strength than an heterologous transcription system using Escherichia coli RNA polymerase and ctDNA. This latter system transcribes less ctDNA fragments.

In vitro characterization of iron-phytosiderophore interaction with maize root plasma membranes: evidences for slow association kinetics.

As an attempt to characterize iron(III)-phytosiderophore transport across plant membranes in vitro, a rapid filtration approach was set up in which plasma membrane vesicles from maize roots were incubated with 55Fe-labelled deoxymugineic acid (DMA). Fe-DMA, and not Fe-EDTA, could associate with plasma membrane vesicles. The rate of Fe-DMA association decreased with a half time of 15 min. The initial Fe-DMA association rate, estimated from the amount of Fe-DMA associated after 10 min incubation, exhibited a saturation curve as a function of Fe-DMA concentration. This curve could be satisfactorily fitted to the Michaelis-Menten model (KM=600 nM, Vmax=2 nmol min-1 mg-1 protein). The association rate of Fe-DMA with control liposomes remained negligible and constant in a pH range from 4 to 8, whereas it strongly increased at acidic pH with plasma membrane vesicles. However, the specific association of Fe-DMA to root plasma membrane could not be explained by a vesicle-filling process because: (i) lowering the vesicle volume by decreasing the osmotic potential of the assay medium with sorbitol did not decrease 55(Fe) labelling of the vesicles, (ii) creating inside-out vesicles by a Brij-58 treatment had almost no effect on Fe-DMA association to vesicles, (iii) 55(Fe) labelling is reversible by EDTA and excess free DMA, and (iv) 55(Fe) labelling was the same using plasmalemma vesicles prepared either from wild type maize or from the ys1 maize mutant deficient in iron-phytosiderophore transport. A model is proposed to account for the observed Fe-DMA association as the result of very slow binding kinetics onto membrane proteins. This model was validated by its ability to describe quantitatively both Fe-DMA association as a function of time and of substrate concentration. A prediction of the model was that association of Fe-DMA to plasma membranes might overcome a high activation energy barrier. Indeed, the Arrhenius plot for the association rate constant was linear with an activation energy of 64 kJ mol-1.

Structure and composition of ferritin cores from pea seed (Pisum sativum).

Iron cores from native pea seed (Pisum sativum) ferritin have been analysed by electron microscopy and Mössbauer spectroscopy and shown to be amorphous. This correlates with their relatively high phosphate content (Fe: P = 2.83; 1800 Fe, 640 P atoms/molecule). Reconstituted cores obtained by adding iron (2000 Fe atoms/molecule) in the absence of phosphate to pea seed apoferritin were crystalline ferrihydrite. In vitro rates of formation of pea-seed ferritin iron cores were intermediate between those of recombinant human H-chain and horse spleen apoferritin and this may reflect the amino-acid residues of its ferroxidase and putative nucleation centres. The high phosphate content of pea-seed ferritin suggests that this molecule could be involved in both phosphorus and iron storage. The high phosphate concentration found within plastids, from which the molecules were isolated, is a possible source of the ferritin phosphate.

Tau factor from Escherichia coli mediates accurate and efficient termination of transcription at the bacteriophage T3 early termination site in vitro.

The termination signal that limits transcription through the early region of bacteriophage T3 (T3Te) has been cloned and sequenced. The nucleotide sequence of T3Te is identical with that of T7Te, with the exception of a single G to U substitution in the 3' tail of the terminated transcript, and addition of an AC to the loop in the terminator stem-loop, enlarging the loop to six residues. Previous studies of the properties of T3Te have shown that this site is rho independent and is highly efficient for termination in vivo, but is used poorly in vitro during transcription with purified Escherichia coli RNA polymerase. In contrast, the equivalent site in bacteriophage T7 (T7Te) is an efficient termination signal both in vivo and in vitro. However, T3Te becomes an efficient termination site in vitro in the presence of preparations of tau factor. This factor also alters the sites of RNA chain termination found in vitro at T3Te. Transcripts formed in the presence of tau are several nucleotides shorter than those produced with RNA polymerase alone, and have 3' termini that are almost identical with transcripts found in vivo. These latter results are similar to our earlier findings with T7Te, and suggest that other rho independent terminators may act with transcription termination factors in vivo.

Characterization of a tRNA(Lys)(CUU) gene located in the opposite orientation upstream of a ZmFer2 ferritin gene in the maize nuclear genome.

The first evidence for a plant tRNA(Lys)(CUU) gene is reported. This gene is found closely linked 400 bp upstream, and on the complementary strand, of a ZmFer2 ferritin gene in the maize nuclear genome. Southern blot analysis indicates that this tRNA(Lys) is a member of a multigene family. This gene does not contain any intron, and exhibits classical intragenic regulatory elements found in eukaryotic tRNA genes (A and B boxes). Moreover, 5' and 3'-flanking sequences display typical features found in nuclear encoded tRNAs. The deduced mature tRNA sequence is almost identical to the sequence of a cytoplasmic tRNA(Lys)(CUU) from wheat germ. The maize tRNA(Lys) gene is expressed in vivo in maize and in transgenic tobacco, as shown by RT-PCR analysis.

Expression cloning in Fe2+ transport defective yeast of a novel maize MYC transcription factor.

A complementation approach of the yeast fet3fet4 mutant strain, defective in both low- and high-affinity iron transport, was initiated as an attempt to characterize the Fe(III)-mugineic acid (MA) transporter from grasses. A maize cDNA encoding a novel MYC transcription factor, named 7E, was cloned by screening an iron-deficient maize root cDNA expression library on a minimum media containing Fe(III)-deoxyMA as a unique iron source. 7E expression restored growth specifically to the fet3 fet4 mutant strain. It did not affect growth rate of a trk1trk2 potassium transport defective yeast strain or parental W303 strain growth rate. No 55Fe uptake increase was observed in 7E transformed fet3 fet4 yeast during short-term kinetics. However, the iron accumulation in these cells was 1.3-fold higher than in untransformed cells after a 24-h period. The 7E protein contained 694 amino acids and had a predicted molecular mass of 74.2kDa. It had 44% identity with the RAP-1 protein, a 67.9-kDa MYC-like protein from Arabidopsis thaliana which binds the G-box sequence via a basic region helix-loop-helix (bHLH), without requiring heterodimerization with MYB proteins. Phylogenic comparisons revealed that the maize 7E protein was related to the Arabidopsis thaliana RAP-1 protein and to the Phaseolus vulgaris PG1. This similarity was particularly evident for the bHLH domain, which was 95% identical between maize 7E and Arabidopsis thaliana RAP-1. 7E, RAP-1 and PG-1 proteins revealed a plant MYC-like sub-family that was more related to the maize repressor-like IN1 than to maize R proteins. 7E mRNA was detected in both roots and leaves by the Northern analysis. The amount of 7E mRNA increased, in response to iron starvation, by 20 and 40% in roots and leaves, respectively. The relationship between iron metabolism and myc expression in animal cells is discussed.

Post-transcriptional regulation of plant ferritin accumulation in response to iron as observed in the maize mutant ys1.

The maize mutant ys1 accumulates iron in leaves to a lower extent than a Fe-efficient genotype. In this mutant, ferritin mRNA accumulates in response to iron to a similar level as in other genotypes. However, ferritin protein and mRNA abundance does not correlate in ys1 leaves, demonstrating that iron also controls plant ferritin protein accumulation at the post-transcriptional level.

Iron triggers a rapid induction of ascorbate peroxidase gene expression in Brassica napus.

In plants, only ferritin gene expression has been reported to be iron-dependent. Here it is demonstrated that an iron overload of Brassica napus seedlings causes a large and rapid accumulation of ascorbate peroxidase transcripts, a plant-specific hydrogen peroxide-scavenging enzyme. This result documents a novel link between iron metabolism and oxidative stress. The ascorbate peroxidase mRNA abundance was not modified by reducing agents like N-acetyl cysteine, glutathione and ascorbate or by pro-oxidants such as hydrogen peroxide or diamide. Furthermore, the iron-induced ascorbate peroxidase mRNA accumulation was not antagonized by N-acetyl cysteine. Abscisic acid had no effect on the ascorbate peroxidase gene expression. Taken together these results suggest that iron-mediated expression of ascorbate peroxidase gene occurs through a signal transduction pathway apparently different from those already described for plant genes responsive to oxidative stress.

Transcription of the chloroplast DNA: a review.

The transcription systems of chloroplasts and bacteria share different properties. The genetic material of chloroplasts is organized in the same way as bacterial nucleoids. The regulatory DNA sequences for transcription have a strong homology with their E. coli counterparts and some regulatory mechanisms could be conserved. The RNA polymerase subunits and some transcription factors also share similarities with prokaryotes. However, the chloroplast core-enzyme seems to be synthesized in the cytoplasm from nuclear encoded messages.

Evidence for a translation-mediated attenuation of a spinach chloroplast rDNA operon.

The presence of potential hairpin structures H1, H2, H3 in the leader region of a spinach rDNA operon led us to postulate that this operon is regulated by premature termination. The mechanism would be controlled by the presence or absence of ribosomes translating a leader peptide. In vitro synchronized transcription by E. coli RNA polymerase shows that pauses do occur in the leader region. By their sizes, the transient transcripts could correspond to pauses on H1 and H2 as predicted by the model in the absence of ribosomes. The complete leader sequence (pKOPH) and the leader sequence with the hairpin structures deleted (pKOP) have been used to the GalK gene in the pK01 plasmid. The resulting plasmids have been used to transform a GalK- E. coli strain. Measurements of GalK expression show that the promoter region of spinach chloroplast rDNA is neither subjected to the growth rate nor to the stringent control. However, under growth conditions leading to an excess of free ribosomes, the expression of GalK gene appears systematically to be reduced in pKOPH when compared with that of pKOP. These results are consistent with a role of the leader region in a translation-mediated attenuation of the chloroplast rDNA expression.

Structure, function, and evolution of ferritins.

The ferritins of animals and plants and the bacterioferritins (BFRs) have a common iron-storage function in spite of differences in cytological location and biosynthetic regulation. The plant ferritins and BFRs are more similar to the H chains of mammals than to mammalian L chains, with respect to primary structure and conservation of ferroxidase center residues. Hence they probably arose from a common H-type ancestor. The recent discovery in E. coli of a second type of iron-storage protein (FTN) resembling ferritin H chains raises the question of what the relative roles of these two proteins are in this organism. Mammalian L ferritins lack ferroxidase centers and form a distinct group. Comparison of the three-dimensional structures of mammalian and invertebrate ferritins, as well as computer modeling of plant ferritins and of BFR, indicate a well conserved molecular framework. The characterisation of numerous ferritin homopolymer variants has allowed the identification of some of the residues involved in iron uptake and an investigation of some of the functional differences between mammalian H and L chains.

Conformational changes and in vitro core-formation modifications induced by site-directed mutagenesis of the specific N-terminus of pea seed ferritin.

Plant ferritin has a three-dimensional structure predicted to be very similar to that of animal ferritin. It has, however, an additional specific sequence of 24 amino acids at its N-terminus named extension peptide (EP). In order to determine precisely the interactions between EP and other domains of the pea seed ferritin subunit, three point mutations were performed. The mutated residues were chosen by three-dimensional computer modelling of the pea seed ferritin subunit structure [Lobréaux, Yewdall, Briat and Harrison (1992) Biochem. J. 228, 931-939]. The mutant recombinant proteins were expressed in Escherichia coli and purified to homogeneity; all the mutants were found to be assembled as 24-mers. When Ala-13 was replaced by His, as in mammalian ferritins, ferroxidase activity was significantly reduced. Moreover, in vitro iron-core formation in Pro-X-->Ala, Lys-R-->Glu and Ala-13-->His mutants was increased after denaturation by urea followed by renaturation; this was also observed with the EP deletion mutant (r delta TP/EP). The recombinant ferritins were also analysed using tryptophan fluorescence spectra. The r delta TP/EP, Pro-X-->Ala and Lys-R-->Glu mutants were found to be more susceptible to denaturation by urea than the native r delta TP pea seed ferritin.

Ferritin accumulation and degradation in different organs of pea (Pisum sativum) during development.

Iron concentration and ferritin distribution have been determined in different organs of pea (Pisum sativum) during development under conditions of continuous iron supply from hydroponic cultures. No ferritin was detected in total protein extracts from roots or leaves. However, a transient iron accumulation in the roots, which corresponds to an increase in iron uptake, was observed when young fruits started to develop. Ferritin was detectable in total protein extracts of flowers and pods, and it accumulated in seeds. In seeds, the same relative amount of ferritin was detected in cotyledons and in the embryo axis. In cotyledons, ferritin and iron concentration decrease progressively during the first week of germination. Ferritin in the embryo axis was processed, and disappeared, during germination, within the first 4 days of radicle and epicotyl growth. This degradation of ferritin in vivo was marked by a shortening of a 28 kDa subunit, giving 26.5 and 25 kDa polypeptides, reminiscent of the radical damage occurring in pea seed ferritin during iron exchange in vitro [Laulhere, Laboure & Briat (1989) J. Biol. Chem. 264, 3629-3635]. Developmental control of iron concentration and ferritin distribution in different organs of pea is discussed.

Iron release and uptake by plant ferritin: effects of pH, reduction and chelation.

Ferritins are iron-storage proteins that accumulate in plastids during seed formation, and also in leaves during senescence or iron overload. Iron release from ferritins occurs during growth of seedlings and greening of plastids. Depending on the concentration of the reducing agent ascorbate, either an overall iron release or uptake by ferritins from iron(III) citrate may occur. We have designed methods to measure these simultaneous and independent uptake and release fluxes. Each individual step of the exchange was studied using different iron chelates and an excess of ligand. It is shown that: (i) the chelated form of iron, and not ionic Fe3+, is the substrate for iron reduction, which controls the subsequent uptake by ferritin; (ii) iron uptake by ferritins is faster at pH 8.4 than at pH 7 or 6 and is inhibited by an excess of strongly binding free ligands; and (iii) strongly binding free ligands are inhibitory during iron release by ascorbate. When reactions are allowed to proceed simultaneously, the iron chelating power is shown to be a key factor in the overall exchange. The interactions of iron chelating power, reducing capacity and pH are discussed with regard to their influence on the biochemical mobilization of iron.

Uptake of iron from ferric-citrate in the cyanobacteria Synechocystis PCC6803.

Iron is an essential element for bacterial growth. Its intracellular concentration is mainly controlled at the uptake level, which is mediated by iron-dicitrate transport in various prokaryotes. We report here that the cyanobacterium Synechocystis PCC6803 takes up iron from ferric citrate. This process is dependent of the iron:citrate ratio, with a better efficiency for the highest concentration of citrate. The iron-citrate complex is the substrate of this active uptake, inhibited in part by the uncoupler FCCP. Iron starvation activates this uptake. These data suggest the existence of a specific ferric citrate receptor on the cyanobacterial membrane.

Identification and characterization of a new transcriptional termination factor from Escherichia coli.

We have identified and partially purified an activity from Escherichia coli that enhances transcription termination at the bacteriophage T7 early terminator when cloned on the plasmid pAR1707. The factor also causes the transcript to be terminated at a site several nucleotides earlier than in its absence. The resulting 3' OH ends of the transcripts are identical to those found in vivo by S1 nuclease mapping. From this we conclude that the factor we have identified is probably responsible for determination of the 3' OH ends of T7 RNA transcripts in vivo. This factor does not act by processing a preformed RNA transcript, nor is it replaced by rho protein or nusA or nusB proteins. Therefore, it appears to be a new transcription termination factor, and we have designated it "tau factor." Elucidation of its role in transcription in E. coli will depend on its purification to homogeneity and further studies of its properties.

Properties and characterization of a spinach chloroplast RNA polymerase isolated from a tanscriptionally active DNA-protein complex.

A chloroplast RNA polymerase has been isolated from a transcriptionally active spinach plastid DNA-protein complex. The properties of the complex and of the reconstituted system have been compared. The crude enzyme is at least sevenfold less active when compared with the complex. RNA synthesis by the reconstituted system is sensitive to high ionic strength and heparin, contrarily to RNA synthesis by the chloroplast DNA-protein complex. On the other hand, rifampicin has no inhibitory effect whatever on the transcriptional system used. The RNA polymerase isolated is more efficient with denatured DNA than with double-stranded DNA and the best template is chloroplast DNA. The crude RNA polymerase isolated migrates in a peak of 11 S in glycerol gradient centrifugation and is located in a single band in non-denaturing polyacrylamide gel electrophoresis. About 30 polypeptides (Mr 15 000--180 000) are part of the complex and only eight of them are found in the RNA polymerase preparation. Only five polypeptides are always present with the same yield. They are probably the subunits of the RNA polymerase. The molecular weight of these subunits ranged from 15 000--69 000, even if the isolation of the enzyme was performed in the presence of protease inhibitors.