Chloroplast tRNA gene contains a long intron in the D stem: Nucleotide sequences of tobacco chloroplast genes for tRNA (UCC) and tRNA (UCU)

The nucleotide sequences of tobacco chloroplast genes for tRNA^Gly (UCC) and tRNA^Arg (UCU) have been determined. The tRNA^Gly gene has a 691-base-pair intron located in the D stem while the tRNA^Arg gene does not have any intron. The tRNA^Gly and tRNA^Arg genes are encoded on the same strand and separated by a 169-base-pair spacer. The tRNA^Gly gene is transcribed as a 900-base precursor RNA molecule. The tRNA^Gly and tRNA^Arg deduced from the DNA sequences show 84% and 55% sequence homologies with Escherichia coli tRNA^Gly (UCC) and phage T4 tRNA^Arg (UCU), respectively.     

Binding and transcription of relaxed DNA templates by fractions of maize chloroplast extracts

Preparations of partially purified chloroplast DNA-dependent RNA polymerase from maize and some other plants transcribe cloned chloroplast genes preferentially and much more actively from appropriately negatively supercoiled templates than from relaxed templates. We have found that the polymerase in such fractions does not bind to promoter regions of the maize chloroplast genes psbA and rbcL on small linear DNA fragments but that some protein(s) in unfractionated chloroplast extracts does bind. DEAE chromatography of the extracts has permitted the separation of a DNA-binding fraction from the bulk of the RNA polymerase activity. The binding fraction contains plastid RNA polymerase activity that is relatively independent of template topology.     

Nucleotide sequence of a preferred maize chloroplast genome template for in vitro DNA synthesis.

Maize chloroplast DNA sequences representing 94% of the chromosome have been surveyed for their activity as autonomously replicating sequences in yeast and as templates for DNA synthesis in vitro by a partially purified chloroplast DNA polymerase. A maize chloroplast DNA region extending over about 9 kilobase pairs is especially active as a template for the DNA synthesis reaction. Fragments from within this region are much more active than DNA from elsewhere in the chromosome and 50- to 100-fold more active than DNA of the cloning vector pBR322. The smallest of the strongly active subfragments that we have studied, the 1368-base-pair EcoRI fragment x, has been sequenced and found to contain the coding region of chloroplast ribosomal protein L16. EcoRI fragment x shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that forms a displacement loop [Wang, X.-M., Chang, C.H., Waddell, J. & Wu, M. (1984) Nucleic Acids Res. 12, 3857-3872]. Maize chloroplast DNA fragments that permit autonomous replication of DNA in yeast are not active as templates for DNA synthesis in the in vitro assay. The template active region we have identified may represent one of the origins of replication of maize chloroplast DNA.     

A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl–tRNA synthetase in Arabidopsis thaliana

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl–tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an Arabidopsis thaliana cDNA coding for a methionyl–tRNA synthetase. This enzyme was chosen because chloroplast-like elongator tRNA^Met genes have been described in several plant species, including A. thaliana. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl–tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between A. thaliana and Synechocystis MetRS strongly suggests that the A. thaliana MetRS gene described here is of chloroplast origin.     

Identification of spacer tRNA genes in individual ribosomal RNA transcription units of Escherichia coli.

Transfer RNA genes ("spacer tRNA genes") are present in the spacer region between 16S and 23S rRNA genes in Escherichia coli. We have analyzed spacer tRNA genes carried by seven rRNA operons with different chromosomal locations. Six of these were isolated on plasmids and one on a transducing phage. We found that, in addition to the two previously identified genes for tRNA2Glu and tRNAIIle, there is a spacer tRNA gene which codes for tRNAIBAla. Of the seven rRNA operons studied, three had both tRNAIBAla and tRNAIIle genes, and the remaining four had the tRNA2Glu gene in their spacers. In addition, genes for tRNAIAsp were found near the distal ends of two different rRNA operons.     

Mutations in the DET1 gene affect cell-type-specific expression of light-regulated genes and chloroplast development in Arabidopsis

When grown in the absence of light, the det1 mutant of Arabidopsis thaliana develops characteristics of a light-grown plant by morphological, cellular, and molecular criteria. Here, we show that recessive mutations at the DET1 locus also result in cell-type inappropriate accumulation of RNAs for light-regulated nuclear and chloroplast genes. det1 root plastids are differentiated into chloroplasts and are present in very high numbers in root cortex cells in contrast to the few starch-containing amyloplasts normally found in Arabidopsis roots. To assay the effect of the det1 mutation on the expression of photoregulated promoters, we used fusion constructs to stably transform wild-type and det1 mutants. We show that the three red-light-regulated chlorophyll a/b binding protein promoters are inappropriately expressed in the roots of det1 seedlings and the blue-light-controlled anthocyanin biosynthetic gene, chalcone synthase, is expressed ectopically in leaf mesophyll cells. These results, together with out previous findings, suggest that the DET1 gene product is a negatively acting regulatory molecule that is used in common by the light stimulus transduction pathway and by temporal or spatial regulatory signals in plants.     

Circadian rhythms in gene transcription imparted by chromosome compaction in the cyanobacterium Synechococcus elongatus

In the cyanobacterium Synechococcus elongatus (PCC 7942) the kai genes A, B, and C and the sasA gene encode the functional protein core of the timing mechanism essential for circadian clock regulation of global gene expression. The Kai proteins comprise the central timing mechanism, and the sensor kinase SasA is a primary transducer of temporal information. We demonstrate that the circadian clock also regulates a chromosome compaction rhythm. This chromosome compaction rhythm is both circadian clock-controlled and kai-dependent. Although sasA is required for global gene expression rhythmicity, it is not required for these chromosome compaction rhythms. We also demonstrate direct control by the Kai proteins on the rate at which the SasA protein autophosphorylates. Thus, to generate and maintain circadian rhythms in gene expression, the Kai proteins keep relative time, communicate temporal information to SasA, and may control access to promoter elements by imparting rhythmic chromosome compaction.