Margaret J. Hollingsworth

Transfer RNA genes of Euglena gracilis chloroplast DNA - A review

SummaryTransfer 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.

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.

The atpF group-II intron-containing gene from spinach chloroplasts is not spliced in transgenic Chlamydomonas chloroplasts.

In order to determine whether the group-II trans-splicing machinery of the chloroplast of Chlamydomonas reinhardtii can splice a heterologous group-II cis intron, the atpF gene of spinach was transferred into the chloroplast genome of C. reinhardtii using the atpX expression vector. The atpF gene contains a group-II intron which, like other higher plant chloroplast introns, does not self-splice in vitro. The chimeric transgene was expressed at high levels, based on the accumulation of the precursor; however, spliced products could not be detected by Northern blotting, or by RT-PCR coupled with Southern-blot hybridization of the amplified products with an exon-junction probe. These results indicate that the spinach atpF intron is not spliced in transgenic C. reinhardtii chloroplasts. Thus, splicing of chloroplast introns mediated by cellular factors may be species-specific; alternately, the group-II splicing machinery of C. reinhardtii is specific for trans-spliced introns.

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.

ATP synthase 5′ untranslated regions are specifically bound by chloroplast polypeptides

Abstract 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.

Expression of the Large ATP Synthase Gene Cluster from Spinach Chloroplasts

Summary We have examined the expression of the spinach ( Spinacia oleracea ) chloroplast large ATP synthase gene cluster. This gene cluster encodes the small ribosomal subunit protein two (rps2), and four proteins of the ATP synthase protein complex: CF 0 -IV (atpI), CF 0 -III (atpH), CF 0 -I (atpF), and CF 1 -α (atpA). Experiments to specifically 5′ end label (cap) primary transcripts revealed two regions of transcription initiation, one located upstream of the rpen gene and the other upstream of atpH. Northern analysis revealed greater than thirty transcripts derived from this gene cluster, the majority of which are processing products derived from the primary transcripts. In addition, two RNA species were identified as containing the atpF intron in a lariat structure. Processing steps include splicing to remove the group II intron found in atpF, as well as endonucleolytic and exonucleolytic cleavages. An ordered series of processing steps is proposed.

Proteins are shared among RNA–protein complexes that form in the 5′ untranslated regions of spinach chloroplast mRNAs

Abstract. Gene expression in chloroplasts is strongly regulated at the post-transcriptional level. Most post-transcriptional mechanisms require RNA–protein complexes. Here we report an analysis of RNA–protein complexes that form in the 5′ untranslated regions (5′UTRs) of spinach chloroplast mRNAs. Previous studies from our laboratory showed that four ATP synthase 5′UTRs were able to compete with each other for binding by proteins in a chloroplast extract. This implied that at least some of the binding proteins recognized all four of those ATP synthase 5′UTRs. Here, we examine whether the binding proteins are ATP synthase-specific by performing competition-binding assays between an ATP synthase 5′UTR and 5′UTRs from other chloroplast genes. Competition substrates were chosen to represent a wide range of chloroplast mRNAs, including those encoding the photosystems, NADH dehydrogenase, cytochromes and ribosomal subunits, and two previously unexamined ATP synthase subunits. Results from these experiments revealed that, although the ATP synthase-binding proteins do not bind universally to every chloroplast 5′UTR, they do bind to the majority (12/14) of those examined. Thus, these RNA-binding proteins are candidates for factors that link the post-transcriptional expression of many chloroplast genes of disparate function.

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.

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.