ATP synthase 5' untranslated regions are specifically bound by chloroplast polypeptides.

Expression of the large ATP synthase gene cluster in spinach (Spinacia oleracea) chloroplasts is regulated at the post-transcriptional level. RNA stability and the translational efficiency of some chloroplast transcripts have been shown to be regulated through RNA-protein interactions in the 5' untranslated region (5' UTR). In this report we show that spinach chloroplast extracts contain polypeptides that specifically interact with the 5' UTRs of three of the four genes in the large ATP synthase gene cluster. A subset of binding polypeptides may be gene-specific, although at least one appears to be a more general chloroplast RNA-binding protein. We hypothesize that these RNA-protein interactions may affect the expression of this gene cluster from two perspectives. The first would be at a gene-specific level, which could serve to control the stoichiometry of ATP synthase subunits. The second would be a more global effect, which may adjust the abundance of the entire ATP synthase complex in response to environmental or developmental cues.

RNA processing alters open reading frame stoichiometry from the large ATP synthase gene cluster of spinach chloroplasts.

The large ATP synthase gene cluster of spinach chloroplasts is a multigenic cluster that encodes the small ribosomal subunit 2 followed by four ATP synthase subunits. The stoichiometry of the ATP synthase gene products from this cluster changes markedly between transcription and assembly of the complex. The two primary transcripts from this gene cluster undergo a complex series of RNA processing steps. Here we show that the extensive RNA processing that the large ATP synthase gene cluster transcripts undergo results in a substantial change in the stoichiometry of complete open reading frames (ORFs) of the four ATP synthase genes. Processing directly affects the stoichiometry of open reading frames from this gene cluster by intragenic cleavage. It may also affect open reading frame stoichiometry more indirectly, but equally significantly, by cleavage-induced alteration of stability of some of the processed transcripts relative to the others.

Factors in a chloroplast extract specifically bind to the 5' untranslated regions of chloroplast mRNAs.

The spinach chloroplast large ATP synthase cluster encodes five genes, the last four of which encode chloroplast ATP synthase subunits. Because the stoichiometry of the ATP synthase polypeptide products differs substantially from that of the primary transcripts, we have chosen this system as a model to study posttranscriptional mechanisms that regulate chloroplast gene expression. Here we present data on a chloroplast extract containing factors that bind to the 5' untranslated regions of chloroplast ATP synthase transcripts. The binding is transcript-specific. This binding may influence translational efficiency of chloroplast transcripts.

Ribosomes pause during the expression of the large ATP synthase gene cluster in spinach chloroplasts.

The large ATP synthase gene cluster from spinach (Spinacia oleracea) chloroplasts encodes five genes, the last four of which encode subunits of the ATP synthase complex. In preliminary experiments (J.K. Kim, M.J. Hollingsworth [1992] Anal Biochem 206: 183-188) it was shown that ribosomes pause during translation of these open reading frames. We have examined ribosome pausing in the four ATP synthase open reading frames of this gene cluster to determine whether it could affect the final ratio of the ATP synthase polypeptides derived from the cluster. Ribosome pauses were mapped and found to be distributed in a nonuniform manner. We have quantitated the relative extent of ribosome pausing within each open reading frame. There is a general but not absolute correlation between the extent of ribosomal pausing and the protein levels found within the ATP synthase complex. We conclude that although it is not the sole factor, ribosome pausing may be a significant posttranscriptional mechanism affecting the expression of the large ATP synthase gene cluster in spinach chloroplasts.

Tissue-specific expression of the large ATP synthase gene cluster in spinach plastids.

Plastids present in different tissues may vary morphologically and functionally, despite the fact that all plastids within the same plant contain identical genomes. This is achieved by regulation of expression of the plastid genome by tissue-specific factors, the mechanisms of which are not fully understood. The proton translocating ATP synthase/ATPase is a multisubunit complex composed of nine subunits, six encoded in the plastid and three in the nucleus. We have investigated the tissue-specific expression of the large ATP synthase gene cluster in spinach (Spinacia oleracea). This gene cluster encodes four of the six plastid-encoded ATP synthase genes. Transcript abundance, transcriptional activity, and transcript stability were investigated relative to gene dosage in root plastids and in stem, leaf, and flower chloroplasts. All three of these factors display significant tissue-specific variation. It was intriguing to discover that, although transcript abundance normalized to gene dosage varies in each tissue, transcript abundance as a proportion of the entire plastid RNA population in each tissue is not significantly different. Thus it appears that in these tissues the variation in transcription and stability of transcripts derived from the large ATP synthase gene cluster balances to yield an equivalent proportion of these transcripts in the plastid RNA population. Expression of this gene cluster in photosynthetic as well as non-photosynthetic tissues may facilitate the plasticity of structure and function which is characteristic of plastids.

Splicing of group II introns in spinach chloroplasts (in vivo): analysis of lariat formation.

To investigate the mechanism of chloroplast mRNA splicing in vivo, RNAs from four spinach chloroplast group II intron-containing genes were analyzed. For each of these genes, atpF, rpoC1, petD, and petB, Northern analysis of chloroplast RNAs detected putative lariat-intron/3' exon-splicing intermediates. Treatment of these RNAs with HeLa cell-debranching extract caused the putative splicing intermediates to disappear, thereby confirming their identities. The lariat-splicing intermediates were further examined by reverse transcriptase extension to determine the branch point location. The in vivo branch points of the atpF and petD introns were found to be eight bases upstream of their respective 3' intron/exon boundaries. In contrast, no splicing intermediates could be detected by primer-extension analysis of petB and rpoC1. This unexpected result served to demonstrate that the quantity of lariat-intron/3' exon-splicing intermediates present in the chloroplast RNA population is considerably less in the cases of rpoC1 and petB compared to atpF and petD. The steady-state level of any splicing intermediate is the result of a balance between the splicing kinetics of a particular RNA and the susceptibility of the splicing intermediate to degradation. We conclude that the balance between these two factors varies significantly for chloroplast introns, even for those, such as petB and petD, that are transcribed from the same promoter.

Expression of the Large ATP Synthase Gene Cluster in Spinach Plastids during Light-Induced Development.

The large ATP synthase gene cluster in spinach (Spinacia oleracea) plastids encodes four of the six chloroplast-encoded ATP synthase subunits. Expression of this cluster was examined to determine its response to light-induced plastid development. Spinach plastid transcripts were isolated from etiolated tissues, etiolated tissues exposed to 24 h of light, young (1-3 cm) leaves, and mature (8-10 cm) leaves. Transcript levels were examined from each developmental stage as a function of either the quantity of total RNA or gene dosage. The relative transcriptional activity of this gene cluster at each of the four developmental stages was also investigated. The stability of these transcripts was deduced by comparing the transcriptional activity with steady-state transcript levels. During the initial 24 h of light-induced development of an etioplast to a chloroplast, transcription decreases in conjunction with increased transcript stability. Transcriptional activity of this cluster per genome then increases between the 24-h and young stages, with a concomitant decrease in the stability of the transcripts. As the young chloroplast matures, the transcripts from this cluster again become markedly more stable, and the transcription of this set of genes declines. Therefore, the regulation of the expression of this cluster is dependent upon a complex interaction between transcriptional and posttranscriptional factors throughout light-induced plastid development.

Localization of in vivo ribosome pause sites.

A protocol for the localization of the 5' boundaries of in vivo ribosomal pausing sites has been developed. These mapping experiments combine two basic techniques. The first is the isolation of polysomal transcripts via centrifugation of tissue extracts through a sucrose cushion in the presence of translational elongation inhibitors. The second technique involves a micrococcal nuclease protection assay first developed by Wolin and Walter for in vitro-bound ribosomes (EMBO J. 7, 3559-3569, 1988). Using this method, the 5' boundaries of in vivo ribosomal pause sites were localized on spinach chloroplast mRNAs derived from the atpA gene. This method is easily adaptable to the identification of in vivo ribosomal pause sites from any organism. It could also be adapted to the localization of in vivo binding sites for other nucleic acid binding proteins.

Characterization of yeast mitochondrial RNase P: an intact RNA subunit is not essential for activity in vitro.

We have previously described a mitochondrial activity that removes 5' leaders from yeast mitochondrial precursor tRNAs and suggested that it is a mitochondrial RNase P. Here we demonstrate that the cleavage reaction results in a 5' phosphate on the tRNA product and thus the activity is analogous to that of other RNase Ps. A mitochondrial gene called the tRNA synthesis locus encodes an A + U-rich RNA required for this activity in vivo. Two regions of this RNA display sequence similarity to conserved sequences in bacterial RNase P RNAs. This sequence similarity coupled with the analogous activities of the enzymes has led us to conclude that the RNAs are homologous and that the tRNA synthesis locus does code for the mitochondrial RNase P RNA subunit. The smallest and most abundant transcript of the tRNA synthesis locus is 490 nucleotides long. However, during purification of the holoenzyme, RNA is degraded and pieces of the original RNA are sufficient to support RNase P activity in vitro.

Alteration of a mitochondrial tRNA precursor 5' leader abolishes its cleavage by yeast mitochondrial RNase P.

A mitochondrial specific RNase P is required to process 5' leaders from mitochondrial tRNA precursors in Saccharomyces cerevisiae. Experiments with a pair of mitochondrial pretRNAs(Asp) having leaders of different base composition suggest that this enzyme is unexpectedly sensitive to leader sequence or structure. Asp-AU (75% AU leader) is cleaved by the mitochondrial RNase P while Asp-GC (39% AU) is not. Both are substrates for E. coli RNase P. Partial nuclease digestions show that the tRNA portions of the two precursors differ in tertiary structure, while their 5' leaders differ in secondary structure. It is unusual for an RNaseP to have substrate specificity requirements which preclude processing of a pretRNA known to be a suitable substrate for an RNaseP from another species.

RNase P activity in the mitochondria of Saccharomyces cerevisiae depends on both mitochondrion and nucleus-encoded components.

A requisite step in the biosynthesis of tRNA is the removal of 5' leader sequences from tRNA precursors. We have detected an RNase P activity in yeast mitochondrial extracts that can carry out this reaction on a homologous precursor tRNA. This mitochondrial RNase P was sensitive to both micrococcal nuclease and protease, demonstrating that it requires both a nucleic acid and protein for activity. The presence of RNase P activity in vitro directly correlated with the presence of a locus on yeast mitochondrial DNA previously shown by genetic and biochemical studies to be required for tRNA maturation. The product of the locus, the 9S RNA, and this newly described mitochondrial RNase P activity cofractionated, providing further evidence that the 9S RNA is the RNA component of yeast mitochondrial RNase P.

Detection of multiple, unspliced precursor mRNA transcripts for the Mr 32,000 thylakoid membrane protein from Euglena gracilis chloroplasts.

The psbA gene is the coding locus for a polypeptide of 32 kilodaltons that is involved in electron transport through photosystem II. The 4.9 kilobasepair (kbp) EcoRI restriction endonuclease fragment EcoI from the 145 kbp Euglena gracilis chloroplast DNA was shown to encode psbA. Five transcripts of size 3.1, 2.8, 2.3, 1.8, and 1.2 kilobases were detected by hybridization of psbA probes to nitrocellulose filter blots of electrophoretically separated RNAs. This same pattern was observed when the hybridization probe consisted of only exon sequences from this split gene. A synthetic, intron specific probe hybridized to all RNA precursors except the 1.2 kb mature RNA. These results and psbA DNA sequence data lead to the conclusion that the four higher molecular weight transcripts are unprocessed precursors of the 1.2 kilobase RNA, some of which contain unspliced intervening sequences. There is an increase in psbA transcripts during light induced maturation of the chloroplasts.

Transfer RNA genes of Euglena gracilis chloroplast DNA : A review.

Transfer RNA genes have been mapped to at least nine different loci on the physical map of the Euglena gracilis chloroplast genome. One of these loci in the ribosomal RNA operons is present three times per genome. The DNA sequences of six of the nine different loci, containing 21 different tRNA genes, have been determined. Genes corresponding to the amino acids Ala, Arg, Asn, Cys, Gln, Gly (2), Glu, His, Ile, Leu (2), Met (2), Phe, Ser, Thr, Trp, Tyr, Val, and one unassigned species have been identified. All genes except one are found in clusters of 2-6 genes. None of the known genes contains introns, nor codes for the 3'-CCA terminus. In addition to these genes, two pseudo tRNA genes are present in the rDNA leader region.

Euglena gracilis chloroplast transfer RNA transcription units. Nucleotide sequence analysis of a tRNATyr-tRNAHis-tRNAMet-tRNATrp-tRNAGlu-tRNAGly gene cluster.

A tRNA coding locus in the Bam-Sal 9 region of Euglena gracilis Pringsheim strain Z chloroplast DNA was chosen for detailed study. This DNA contains the previously mapped tRNA coding sequences of the adjacent Euglena chloroplast EcoRI products of EcoV and EcoH (Orozco, E. M., Jr., and Hallick, R. B. (1982) J. Biol. Chem. 257, 3258-3264). The 3.2-kilobase pair Bam-Sal 9 fragment was cloned into the BamHI and SalI cut plasmid vector pBR322, resulting in the recombined plasmid pPG76. The tRNA coding locus was mapped to a region of Bam-Sal 9 that contains portions of both EcoH and EcoV. The DNA sequence of 1-kilobase pair Bam-Sal 9, containing the entire tRNA coding locus, was determined. A cluster of six tRNA genes was found. The gene organization is as follows, where bp is base pair: tRNATyrGUA-64 bp spacer-tRNAHisGUG-14 bp spacer-tRNAMetCAU-4 bp spacer-tRNATrpCCA-27 bp spacer-tRNAGluUUC-6 bp spacer-tRNAGlyUCC. The tRNAMetCAU is believed to be an elongator tRNA. The first four genes are within EcoV. The EcoRI cleavage site that separates EcoV and EcoH is in the tRNAGlu gene. The tRNAGly gene is in EcoH. This is the largest known chloroplast tRNA gene cluster.