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.

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.

One major 5S RNA, 120 bases long, was revealed by an analysis of mature 5S RNA from tissues, developmental stages, and polysomes inArabidopsis thaliana. Minor 5S RNA were also found, varying from the major one by one or two base substitutions; 5S rDNA units from each 5S array of the Arabidopsis genome were isolated by PCR using CIC yeast artificial chromosomes (YACs) mapped on the different loci. By using a comparison of the 5S DNA and RNA sequences, we could show that both major and minor 5S transcripts come from only two of the genomic 5S loci: chromosome 4 and chromosome 5 major block. Other 5S loci are either not transcribed or produce rapidly degraded 5S transcripts. Analysis of the 5′- and 3′-DNA flanking sequence has permitted the definition of specific signatures for each 5S rDNA array.[EMBL accession nos: AF330825-AF331032;AF335777-AF335873.]

Summary: The effect of alteration of 5′ and 3′ flanking sequences on the transcription of plant tRNA genes was analysed using an RNA polymerase III‐dependent in vitro transcription system derived from nuclei of cultured tobacco cells. A TATA‐like sequence and the CAA motif frequently observed upstream of plant tRNA genes, and the poly(T) stretch usually present downstream, were shown to be necessary for efficient re‐initiation of transcription. The CAA motif was shown to be a transcription initiation site. Introduction of the CAA and TATA‐like motifs into a gene naturally lacking them greatly enhanced transcription by promoting efficient re‐initiation.

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.