RNA Polymerase III-Dependent BoNR8 and AtR8 lncRNAs Contribute to Hypocotyl Elongation in Response to Light and Abscisic Acid.

Hypocotyl elongation is inhibited by light and promoted by darkness. The plant hormone abscisic acid (ABA) also inhibits hypocotyl elongation. However, details of the molecular mechanism that regulates the integrated effects of light and ABA signaling on hypocotyl elongation remain unclear. Long non-coding RNAs (lncRNAs; >200 nt) do not encode proteins but play many physiological roles in organisms. Until now, only a few lncRNAs related to hypocotyl elongation have been reported. The lncRNAs BoNR8 (272 nt) and AtR8 (259 nt), both of which are transcribed by RNA polymerase III, are homologous lncRNAs that are abundantly present in cabbage and Arabidopsis, respectively. These lncRNAs shared 77% sequence identity, and their predicted RNA secondary structures were similar; the non-conserved nucleotides in both sequences were positioned mainly in the stem-loop regions of the secondary structures. A previous study showed that BoNR8 regulated seed germination along with ABA and that AtR8 may be involved in innate immune function in Arabidopsis. Our results show that the expression levels of BoNR8 and AtR8 were differentially affected by light and ABA and that overexpression (OX) of both BoNR8 and AtR8 in Arabidopsis regulated hypocotyl elongation depending on light and ABA.. The expression levels of light-related genes PHYB, COP1, HY5 and PIF4 and ABA-related genes ABI3 and ABI5 were altered in the AtR8-OX and BoNR8-OX lines, and, in an ABI3-defective mutant, hypocotyl elongation was greatly increased under dark condition with the addition of ABA. These results indicate that BoNR8 and AtR8 regulate hypocotyl elongation together with ABI3 and key downstream light signaling genes.

The AtGSTU7 gene influences glutathione-dependent seed germination under ABA and osmotic stress in Arabidopsis.

Glutathione S-transferases (GSTs) play important roles in metabolism and detoxification of plant cells. However, their functions during development are less well understood. Arabidopsis AtGSTU7 (AT2G29420) encodes a Tau class GST. Here we provide the AtGSTU7 was abundantly expressed in seeds and in roots at an early stage of germination. AtGSTU7 expression was repressed by exogenous ABA and promoted by osmotic stress. A null mutant of AtGSTU7 (atgstu7) accumulated higher contents of reduced GSH and decreased amounts of endogenous H2O2 in seedlings. The atgstu7 plants showed decreased osmotic tolerance during seed germination, which was influenced by GSH and ABI3 gene expression. The results suggested that AtGSTU7 involvement in seed germination is mediated by GSH-ROS homeostasis and ABA signaling.

The Arabidopsis Hypoxia Inducible AtR8 Long Non-Coding RNA also Contributes to Plant Defense and Root Elongation Coordinating with WRKY Genes under Low Levels of Salicylic Acid.

AtR8 lncRNA was previously identified in the flowering plant Arabidopsis thaliana as an abundant Pol III-transcribed long non-coding RNA (lncRNA) of approximately 260 nt. AtR8 lncRNA accumulation is responsive to hypoxic stress and salicylic acid (SA) treatment in roots, but its function has not yet been identified. In this study, microarray analysis of an atr8 mutant and wild-type Arabidopsis indicated a strong association of AtR8 lncRNA with the defense response. AtR8 accumulation exhibited an inverse correlation with an accumulation of two WRKY genes (WRKY53/WRKY70) when plants were exposed to exogenous low SA concentrations (20 µM), infected with Pseudomonas syringae, or in the early stage of development. The highest AtR8 accumulation was observed 5 days after germination, at which time no WRKY53 or WRKY70 mRNA was detectable. The presence of low levels of SA resulted in a significant reduction of root length in atr8 seedlings, whereas wrky53 and wrky70 mutants exhibited the opposite phenotype. Taken together, AtR8 lncRNA participates in Pathogenesis-Related Proteins 1 (PR-1)-independent defense and root elongation, which are related to the SA response. The mutual regulation of AtR8 lncRNA and WRKY53/WRKY70 is mediated by Nonexpressor of Pathogenesis-Related Gene 1 (NPR1).

Novel in vivo system to monitor tRNA expression based on the recovery of GFP fluorescence and its application for the determination of plant tRNA expression.

We developed a novel assay system to quantitatively detect amber codon suppression by tRNAs expressed in plant cells. The assay was based on recovery of the expression of the green fluorescent protein (GFP) as a reporter, in which a fourth Lys codon (AAG) was changed to a premature amber codon TAG, designated as GFP/amber. Plasmids carrying GFP/amber, suppressor tRNA, and red fluorescent protein (RFF) as an internal control, respectively, were introduced into onion epidermal cells to monitor cell numbers with GFP and RFP fluorescence. First, an amber suppressor tRNASer from tobacco (NtS2) to suppress a TAG codon in GFP mRNA was examined, leading to the recovery of GFP fluorescence. Second, we used two different tRNAs (i.e., AtY3II-am and AtY3II-amiG7), both of which are intron-containing amber suppressor tRNAsTyr, the former impaired precursor-tRNA splicing but the latter did not, as confirmed previously using two different approaches (Szeykowska-Kulinska and Beier, 1991; Akama and Beier, 2003). As expected, coexpression of GFP/amber with AtY3II-am gave no green fluorescence, but significant fluorescence was observed with AtY3II-amiG7. Then, we applied this system for the analysis of 5'-regulatory sequences of the tRNAGln gene family from Arabidopsis. A 5'-flanking sequence of each of the 17 tRNAGln genes was fused to a coding region of an amber suppressor tRNASer gene (NtS2/amber) and its 3'-flanking sequence. Chimeric tRNASer gene, GFP/amber, and RFP were coexpressed, and the GFP or RFP fluorescence intensity was determined in cells using laser-scanning microscopy. In parallel, 17 kinds of original Arabidopsis tRNAGln genes and their chimeric genes with NtS2/amber were all analyzed in cell-free nuclear extract (Yukawa et al., 1997). Comparison of in vitro and in vivo expression of these chimeric tRNA genes displayed generally similar results, accompanied by a wide range of variance in the expression of each gene. Nevertheless, the expression patterns of several genes were clearly the opposite of each other comparing between the two different system, demonstrating the importance of in vivo systems in the study on tRNA expression in plants.

Pol III-Dependent Cabbage BoNR8 Long ncRNA Affects Seed Germination and Growth in Arabidopsis.

Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nt that are distributed widely in organisms and play many physiological roles. The BoNR8 lncRNA is a 272 nt long transcript yielded by RNA polymerase III in cabbage that was identified as the closest homolog of the AtR8 lncRNA in Arabidopsis. The BoNR8 lncRNA was expressed extensively in the epidermal tissue in the root elongation zone of germinated seeds, and its accumulation was induced by abiotic stresses, auxins and ABA. To investigate the correlation between the BoNR8 lncRNA and germination, BoNR8-overexpressing Arabidopsis plants (BoNR8-AtOX) were prepared. Three independent BoNR8-AtOX lines showed less primary root elongation, incomplete silique development and decreased germination rates. The germination efficiencies were affected strongly by ABA and slightly by salt stress, and ABA-related gene expression was changed in the BoNR8-AtOX lines.

Arabidopsis Pol II-Dependent in Vitro Transcription System Reveals Role of Chromatin for Light-Inducible rbcS Gene Transcription.

In vitro transcription is an essential tool to study the molecular mechanisms of transcription. For over a decade, we have developed an in vitro transcription system from tobacco (Nicotiana tabacum)-cultured cells (BY-2), and this system supported the basic activities of the three RNA polymerases (Pol I, Pol II, and Pol III). However, it was not suitable to study photosynthetic genes, because BY-2 cells have lost their photosynthetic activity. Therefore, Arabidopsis (Arabidopsis thaliana) in vitro transcription systems were developed from green and etiolated suspension cells. Sufficient in vitro Pol II activity was detected after the minor modification of the nuclear soluble extracts preparation method; removal of vacuoles from protoplasts and L-ascorbic acid supplementation in the extraction buffer were particularly effective. Surprisingly, all four Arabidopsis Rubisco small subunit (rbcS-1A, rbcS-1B, rbcS-2B, and rbcS-3B) gene members were in vitro transcribed from the naked DNA templates without any light-dependent manner. However, clear light-inducible transcriptions were observed using chromatin template of rbcS-1A gene, which was prepared with a human nucleosome assembly protein 1 (hNAP1) and HeLa histones. This suggested that a key determinant of light-dependency through the rbcS gene transcription was a higher order of DNA structure (i.e. chromatin).

The context of transcription start site regions is crucial for transcription of a plant tRNA(Lys)(UUU) gene group both in vitro and in vivo.

Nuclear tRNA genes are transcribed by RNA polymerase III. The A- and B-boxes located within the transcribed regions are essential promoter elements for nuclear tRNA gene transcription. The Arabidopsis genome contains ten annotated genes encoding identical tRNA(Lys)(UUU) molecules, which are scattered on the five chromosomes. In this study, we prepared ten tDNA constructs including each of the tRNA(Lys)(UUU) coding sequences with their individual 5' and 3' flanking sequences, and assayed tRNA expression using an in vitro RNA polymerase III-dependent transcription system. Transcription levels differed significantly among the ten genes and two of the tRNA genes were transcribed at a very low level, despite possessing A- and B-boxes identical to those of the other tRNA genes. To examine whether the in vitro results were reproducible in vivo, the 5' flanking sequence of an amber suppressor tRNA gene was then replaced with those of the ten tRNA(Lys) genes. An in vivo experiment based on an amber suppressor tRNA that mediates suppression of a premature amber codon in a ß-glucuronidase (GUS) reporter gene in plant tissues generated nearly identical results to those obtained in vitro. Analysis of mutated versions of the amber suppressor tRNA gene, which contained base substitutions around the transcription start site (TSS), showed that the context around the transcription start sites is a crucial determinant for transcription of plant tRNA(Lys)(UUU) both in vitro and in vivo. The above transcription regulation by context around TSS differed between tRNA genes and other Pol III-dependent genes.

Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA.

A novel hypoxic stress-responsive long non-coding RNA transcribed by RNA polymerase III in Arabidopsis.

Recently, a large number of non-coding RNAs (ncRNAs) have been found in a wide variety of organisms, but their biological functions are poorly understood, except for several tiny RNAs. To identify novel ncRNAs with essential functions in flowering plants, we focused attention on RNA polymerase III (Pol III) and its transcriptional activity, because most Pol III-transcribed RNAs contribute to key processes relating to cell activities, and have highly conserved promoter elements: upstream sequence elements, a TATA-like sequence, and a poly(T) stretch as a transcription terminator. After in silico prediction from the Arabidopsis genome, 20 novel ncRNAs candidates were obtained. AtR8 RNA (approx. 260 nt) and AtR18 RNA (approx. 160 nt) were identified by efficient in vitro transcription by Pol III in tobacco nuclear extracts. AtR8 RNA was conserved among six additional taxa of Brassicaceae, and the secondary structure of the RNA was also conserved among the orthologs. Abundant accumulation of AtR8 RNA was observed in the plant roots and cytosol of cultured cells. The RNA was not processed into a smaller fragment and no short open reading frame was included. Remarkably, expression of the AtR8 RNA responded negatively to hypoxic stress, and this regulation evidently differed from that of U6 snRNA.

ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro.

Chloroplasts possess common biosynthetic pathways for generating guanosine 3',5'-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5'-(ß,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.

Translation of partially overlapping psbD-psbC mRNAs in chloroplasts: the role of 5'-processing and translational coupling.

The chloroplast psbD and psbC genes encode the D2 and CP43 proteins of the photosystem II complex, and they are generally cotranscribed. We report studies on the basic translation process of tobacco psbD-psbC mRNAs using an in vitro translation system from tobacco chloroplasts. The primary transcript has an unusually long 5'-UTR (905 nt). We show that it is translatable. Processing of the 5'-UTR greatly enhances the translation efficiency of the psbD cistron. A striking feature is that psbD and psbC cistrons overlap by 14 nt. Removal of the psbD 5'-UTR plus the start codon and introduction of a premature termination codon in the psbD cistron considerably reduce the translation efficiency of the downstream psbC cistron. These results indicate that translation of the psbC cistron depends largely on that of the upstream psbD cistron and thus shows translational coupling; however, a portion is independently translated. These observations, together with the presence of monocistronic psbC mRNAs, suggest that the psbD and psbC cistrons are translated via multiple processes to produce necessary amounts of D2 and CP43 proteins.

The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB-atpE dicistronic mRNAs in chloroplasts.

The chloroplast atpB and atpE genes encode subunits ß and ε of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the tobacco dicistronic mRNA, and that the efficiency of atpB translation is higher than that of atpE translation. When the atpB 5'-UTR was replaced with lower efficiency 5'-UTRs, atpE translation was higher than atpB translation. Removal of the entire atpB 5'-UTR arrested atpB translation but atpE translation still proceeded. Introduction of a premature stop codon in the atpB cistron did not abolish atpE translation. These results indicate that atpE translation is independent of atpB translation. Mutation analysis showed that the atpE cistron possesses its own cis-element(s) for translation, located ~25 nt upstream from the start codon.

A common sequence motif involved in selection of transcription start sites of Arabidopsis and budding yeast tRNA genes.

The transcription start site (TSS) is useful to predict gene and to understand transcription initiation. Although vast data on mRNA TSSs are available, little is known about tRNA genes because of rapid processing. Using a tobacco in vitro transcription system under conditions of impaired 5' end processing, TSSs were determined for 64 Arabidopsis tRNA genes. This analysis revealed multiple TSSs distributed in a region from 10 to 2bp upstream of the mature tRNA coding sequence (-10 to -2). We also analyzed 31 Saccharomyces cerevisiae tRNA genes that showed a smaller number but a broader distribution (-13 to -1) of TSSs. In both cases, transcription was initiated preferentially at adenosine, and a common 'TCAACA' sequence was found spanning the TSSs. In plant, this motif caused multiple TSSs to converge at one site and enhanced transcription. The TATA-like sequence upstream of Arabidopsis tRNA genes also contributed to TSS selection.

Translation of psbC mRNAs starts from the downstream GUG, not the upstream AUG, and requires the extended Shine-Dalgarno sequence in tobacco chloroplasts.

The plastid gene psbC encodes the CP43 subunit of PSII. Most psbC mRNAs of many organisms possess two possible initiation codons, AUG and GUG, and their coding regions are generally annotated from the upstream AUG. Using a chloroplast in vitro translation system, we show here that translation of the tobacco plastid psbC mRNA initiates from the GUG. This mRNA possesses a long Shine-Dalgarno (SD)-like sequence, GAGGAGGU, nine nucleotides upstream of the GUG. Point mutations in this sequence abolished translation, suggesting that a strong interaction between this extended SD-like sequence and the 3' end of 16S rRNA facilitates translation initiation from the GUG.

The complete structure of the cucumber (Cucumis sativus L.) chloroplast genome: its composition and comparative analysis.

The complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.

A survey of expressed tRNA genes in the chromosome I of Arabidopsis using an RNA polymerase III-dependent in vitro transcription system.

Eukaryotic tRNA genes are transcribed by RNA polymerase III. These tRNA genes are generally predicted using computer programs, and 620 tRNA genes in the Arabidopsis thaliana genome are currently annotated. However, no effort has been made to assay whether these predicted tRNA genes are all expressed, because it has been difficult to assay by routine in vivo methods. We report here a large-scale tRNA expression assay of predicted Arabidopsis tRNA genes using an RNA polymerase III-dependent in vitro transcription system developed by our group. DNA fragments including an annotated tRNA gene each were amplified by PCR and the resulting linear DNA was subjected to in vitro transcription. The addition of poly(dA-dT).poly(dA-dT) enhanced activity significantly and reduced background. The 124 predicted tRNA genes present in the Arabidopsis chromosome I were examined, and transcription activity and transcript stability from individual genes were determined. These results indicated that eight annotated genes are not expressed. Based on previous reports on pseudo-tRNA genes (e.g., Beier and Beier, Mol. Gen. Genet. 1992; 233: 201-208) and the present results, we estimated that 16% or more of the annotated tRNA genes in the chromosome I are not functional.

A simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts.

RNA editing is found in various transcripts from land plant chloroplasts. In tobacco chloroplasts, C-to-U conversion occurs at 36 specific sites including two sites identified in this work. Our RNA editing assay system using chloroplast extracts facilitated biochemical analyses of editing reactions but required mRNAs labeled with (32)P at specific sites. Here, we have improved the in vitro system using fluorescence-labeled chain terminators, ddGTP and ddATP, and have measured the editing activity at 19 sites in ndh transcripts. Editing activities varied from site to site. It has been reported that one editing site in ndhA mRNAs is present in spinach but absent in tobacco, but a corresponding editing capacity had been found in vivo in tobacco using biolistic transformation. We confirmed biochemically the existence of this activity in tobacco extracts. Using the non-radioactive assay, we examined sequences essential for editing within a 50-nt mRNA region encompassing an editing site. Editing of the ndhB-2 site requires a short sequence in front of the editing site, while that of the ndhF mRNA requires two separate regions, a sequence surrounding the editing site and a 5' distal sequence. These results suggest that distinct editing mechanisms are present in chloroplasts.

Distinct modes of TATA box utilization by the RNA polymerase III transcription machineries from budding yeast and higher plants.

The TATA box is a key upstream control element for basal tRNA gene transcription by RNA polymerase III in some eukaryotes, such as the fission yeast (Schizosaccharomyces pombe) and higher plants, but not in others such as the budding yeast (Saccharomyces cerevisiae). To gain information on this differential TATA box requirement, we examined side-by-side the in vitro transcription properties of TATA-containing and TATA-mutated plant and S. cerevisiae tDNAs in homologous in vitro transcription systems from both organisms and in a hybrid system in which yeast TBP was replaced by its plant homologue. The data support the general conclusion that specific features of the plant transcription machinery, rather than upstream region architecture per se, are responsible for the much stronger TATA box dependence of the plant system. In both systems, however, a strong influence of the TATA box on transcription start site selection was observed. This was particularly striking in the case of plant tDNAs, where TATA-rich upstream regions were found to favour the use of alternative initiation sites. Replacement of yeast TBP with its plant counterpart did not confer any general TATA box responsiveness to the yeast transcription machinery. Interactions involving components other than TBP are thus responsible for the strong TATA box requirement of plant tDNA transcription.

Plant 7SL RNA genes belong to type 4 of RNA polymerase III- dependent genes that are composed of mixed promoters.

The genes transcribed by RNA polymerase III (pol III) display a great diversity in terms of promoter structure and are placed in four groups accordingly. Type 3 subset of pol III genes has promoter elements which reside entirely upstream of the coding region of the gene whereas type 4 consists of genes with mixed promoters that enclose intra- and extragenic regulatory sequences. Plant 7SL RNA genes have been previously classified as type 3 of pol III genes requiring an upstream sequence element and a canonical TATA box for transcriptional activity in transfected plant protoplasts. We have identified two novel functional control regions within the coding region of an Arabidopsis 7SL RNA gene (At7SL-1) that resemble tRNA gene-specific A and B boxes with respect to sequence and position. Single and multiple nucleotide substitutions in either of these regions resulted in a pronounced reduction of transcription activity in tobacco nuclear extract that was not caused by a decreased stability as shown by decay kinetics of wild type and mutant RNA transcripts. These findings suggest that plant 7SL RNA genes should be actually placed in type 4 of pol III-transcribed genes. As a consequence of substantially different upstream promoters utilized by plant and human pol III, in vitro transcription of 7SL RNA genes in heterologous systems is severely impaired. A chimeric human 7SL RNA gene that contains the 5' flanking region up to position -300 of At7SL-1 is yet transcribed with a reduced efficiency in tobacco extract when compared with the plant wild-type gene, supporting the notion that internal regulatory elements contribute to full activity.

In vitro analysis of the sequences required for transcription of the Arabidopsis thaliana 5S rRNA genes.

In vivo, we have already shown that only two of the 5S rDNA array blocks of the Arabidopsis thaliana genome produce the mature 5S rRNAs. Deletions and point mutations were introduced in an Arabidopsis 5S rDNA-transcribed region and its 5'- and 3'-flanks in order to analyse their effects on transcription activity. In vitro transcription revealed different transcription control regions. One control region essential for transcription initiation was identified in the 5'-flanking sequence. The major sequence determinants were a TATA-like motif (-28 to -23), a GC dinucleotide (-12 to -11), a 3-bp AT-rich region (-4 to -2) and a C residue at -1. They are important for both accurate transcription initiation and transcription efficiency. Transcription level was regulated by polymerase III (Pol III) re-initiation rate as in tRNA genes in which TATA-like motif is involved. Active 5S rDNA transcription additionally required an intragenic promoter composed of an A-box, an Intermediate Element (IE) and a C-box. Double-stranded oligonucleotides corresponding to different fragments of the transcribed region, used as competitors, revealed the main importance of internal promoter elements. A stretch of four T is sufficient for transcription termination. Transcription of Arabidopsis 5S rDNA requires 30 bp of 5'-flanking region, a promoter internal to the transcribed region, and a stretch of T for transcription termination.