Transcriptionally Active Chromosome Research Articles

Rice TCM1 Encoding a Component of the TAC Complex is Required for Chloroplast Development under Cold Stress.

Transcriptionally active chromosome (TAC) is a component of protein-DNA complexes with RNA polymerase activity, expressed in the plastid. However, the function of rice TAC proteins is still poorly understood. In this paper, we first report the identification of a new rice ( L.) mutant () in the gene encoding TAC. The mutant displayed an albino phenotype and malformed chloroplasts before the three-leaf stage when grown at low temperatures (20°C) and a normal phenotype at higher temperatures (>28°C). Map-based cloning revealed that encodes a novel chloroplast-targeted TAC protein in rice. In addition, the transcript levels of all examined plastid-encoded polymerase (PEP)-dependent genes were clearly downregulated in mutants at low temperatures, although partially recovering levels were obtained at high temperatures, comparable to wild-type plants. Furthermore, the transcripts were ubiquitously expressed in all examined tissues, with high expression levels in green tissues. The data suggest that the rice nuclear-encoded TAC protein TCM1 is essential for proper chloroplast development and maintaining PEP activity under cold stress.

Global RNA association with the transcriptionally active chromosome of chloroplasts.

Processed chloroplast RNAs are co-enriched with preparations of the chloroplast transcriptionally active chromosome. Chloroplast genomes are organized as a polyploid DNA-protein structure called the nucleoid. Transcriptionally active chloroplast DNA together with tightly bound protein factors can be purified by gel filtration as a functional entity called the transcriptionally active chromosome (TAC). Previous proteomics analyses of nucleoids and of TACs demonstrated a considerable overlap in protein composition including RNA binding proteins. Therefore the RNA content of TAC preparations from Nicotiana tabacum was determined using whole genome tiling arrays. A large number of chloroplast RNAs was found to be associated with the TAC. The pattern of RNAs attached to the TAC consists of RNAs produced by different chloroplast RNA polymerases and differs from the pattern of RNA found in input controls. An analysis of RNA splicing and RNA editing of selected RNA species demonstrated that TAC-associated RNAs are processed to a similar extent as the RNA in input controls. Thus, TAC fractions contain a specific subset of the processed chloroplast transcriptome.

Chloroplast transcription, untangling the Gordian Knot.

Chloroplast transcription, untangling the Gordian Knot.

Identification and characterization of chloroplast casein kinase II from Oryza sativa (rice).

Plastid casein kinase II is an important regulator of transcription, posttranscriptional processes, and, most likely, different metabolic functions in dicotyledonous species. Here we report the identification and characterization of pCKII from the monocotyledonous species Oryza sativa. OspCKII activity was enriched from isolated rice chloroplasts using heparin-Sepharose chromatography, in which it co-elutes with the transcriptionally active chromosome (TAC) and several ribosomal proteins. Inclusion mass scanning of the kinase-active fraction identified the gene model for OspCKII. Transient expression of GFP fused to the 184 N-terminal amino acids of the OspCKII sequence in rice confirmed the chloroplastic localization of the kinase. OspCKII activity shows the characteristic features of casein kinase II, such as the utilization of GTP as phosphate donor, inhibition by low concentrations of heparin and poly-lysine, and utilization of the canonical pCKII motif E-S-E-G-E in the model substrate RNP29. Phosphoproteome analysis of a protein extract from rice leaves combined with a meta-analysis with published phosphoproteomics data revealed differences in the target protein spectrum between rice and Arabidopsis. Consistently, several pCKII phosphorylation sites in dicotyledonous plants are not conserved in monocots and algae, suggesting that details of pCKII regulation in plastids have changed during evolution.

SVR4 (suppressor of variegation 4) and SVR4‐like: two proteins with a role in proper organization of the chloroplast genetic machinery

SUPPRESSOR OF VARIEGATION 4 (SVR4, also called MRL7) and its homolog SVR4-like (also called MRL7-Like) were originally identified as important proteins for proper function of the chloroplast in Arabidopsis. Both are nuclear-encoded chloroplast-located proteins, and knockout mutants of either gene result in seedling lethality. Transmission electron microscopy analysis revealed that chloroplast development is arrested at an early developmental stage in both mutants. Accordingly, in the mutant plants severely decreased levels of photosynthetic pigments as well as subunits of the photosynthetic complexes could be detected. In absence of either of the two proteins chloroplast DNA organization was clearly affected. Immunological analysis revealed that SVR4 is a component of the transcriptionally active chromosome (TAC) from barley chloroplasts. Analyses of gene expression indicate that SVR4 and SVR4-like are required for proper function of the plastid transcriptional machinery. We propose that SVR4 and SVR4-like function as molecular chaperones ensuring proper organization of the nucleoids in chloroplasts.

The Reduced Plastid-Encoded Polymerase-Dependent Plastid Gene Expression Leads to the Delayed Greening of the Arabidopsis fln2 Mutant

In Arabidopsis leaf coloration mutants, the delayed greening phenomenon is common. Nonetheless, the mechanism remains largely elusive. Here, a delayed greening mutant fln2–4 of FLN2 (Fructokinase-Like Protein2) was studied. FLN2 is one component of Transcriptionally Active Chromosome (TAC) complex which is thought to contain the complete plastid-encoded polymerase (PEP). fln2–4 displayed albino phenotype on medium without sucrose. The PEP-dependent plastid gene expression and chloroplast development were inhibited in fln2–4. Besides interacting with thioredoxin z (TRX z), we identified that FLN2 interacted with another two members of TAC complex in yeast including its homologous protein FLN1 (Fructokinase-Like Protein1) and pTAC5. This indicates that FLN2 functions in regulation of PEP activity associated with these TAC components. fln2–4 exhibited delayed greening on sucrose-containing medium. Comparison of the PEP-dependent gene expression among two complete albino mutants (trx z and ptac14), two yellow mutants (ecb2–2 and ys1) and the fln2–4 showed that fln2–4 remains partial PEP activity. FLN2 and FLN1 are the target proteins of TRX z involved in affecting the PEP activity. Together with the data that FLN1 could interact with itself in yeast, FLN1 may form a homodimer to replace FLN1–FLN2 as the TRX z target in redox pathway for maintaining partial PEP activity in fln2–4. We proposed the partial PEP activity in the fln2 mutant allowed plastids to develop into fully functional chloroplasts when exogenous sucrose was supplied, and finally the mutants exhibited green phenotype.

TAC7, an essential component of the plastid transcriptionally active chromosome complex, interacts with FLN1, TAC10, TAC12 and TAC14 to regulate chloroplast gene expression in Arabidopsis thaliana

Transcriptionally active chromosome (TAC) is a fraction of protein/DNA complexes with RNA polymerase activity in the plastid. However, the function of most TAC proteins remains unknown. Here, we isolated two allelic mutants of the gene for a TAC component, TAC7, and performed functional analysis in plastid gene expression and chloroplast development in Arabidopsis. tac7-1 is a mutant with a premature translation termination isolated from a population treated with ethyl methane sulfonate, and tac7-2 is a transfer-DNA tagging mutant. Both of them showed an albino phenotype when grown under normal light conditions, and a few appressed membranes were observed inside the defective chloroplasts. These data indicate that TAC7 is important for thylakoid biogenesis. The TAC7 gene encodes an uncharacterized 161 amino acids polypeptide localized in chloroplast. The transcriptional levels of plastid-encoded polymerase (PEP)-dependent genes were downregulated in tac7-2, suggesting that PEP activity was decreased in the mutant. Yeast two-hybrid assay shows that TAC7 can interact with the four TAC components including FLN1, TAC10, TAC12 and TAC14 which are involved in redox state changes, phosphorylation processes and phytochrome-dependent light signaling, respectively, These data indicate that TAC7 plays an important role for TAC to regulate PEP-dependent chloroplast gene expression and chloroplast development.

Regulatory role of Arabidopsis pTAC14 in chloroplast development and plastid gene expression

Transcriptionally active chromosome (TAC) is a fraction of protein/DNA complexes with RNA polymerase activity in the plastid. The function of most TAC proteins is not well known. We isolated a mutant gene encoding a plastid TAC component, pTAC14, and performed functional analysis of plastid gene expression and chloroplast development in Arabidopsis. We found that knockout of pTAC14 led to the blockage of thylakoid formation in the initial process of chloroplast development. Furthermore, the transcript levels of plastid-encoded polymerase (PEP)-dependent genes were downregulated in ptac14, suggesting that PEP activity was decreased in the mutant. On the basis of these results, we briefly review the available evidence and highlight the interaction between pTAC14 and pTAC12 that could help us understand the regulatory role of pTAC14 in chloroplast development and plastid gene expression.

A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression

The SET domain-containing protein, pTAC14, was previously identified as a component of the transcriptionally active chromosome (TAC) complexes. Here, we investigated the function of pTAC14 in the regulation of plastid-encoded bacterial-type RNA polymerase (PEP) activity and chloroplast development. The knockout of pTAC14 led to the blockage of thylakoid formation in Arabidopsis (Arabidopsis thaliana), and ptac14 was seedling lethal. Sequence and transcriptional analysis showed that pTAC14 encodes a specific protein in plants that is located in the chloroplast associated with the thylakoid and that its expression depends on light. In addition, the transcript levels of all investigated PEP-dependent genes were clearly reduced in the ptac14-1 mutants, while the accumulation of nucleus-encoded phage-type RNA polymerase-dependent transcripts was increased, indicating an important role of pTAC14 in maintaining PEP activity. pTAC14 was found to interact with pTAC12/HEMERA, another component of TACs that is involved in phytochrome signaling. The data suggest that pTAC14 is essential for proper chloroplast development, most likely by affecting PEP activity and regulating PEP-dependent plastid gene transcription in Arabidopsis together with pTAC12.

Whirly1 in chloroplasts associates with intron containing RNAs and rarely co-localizes with nucleoids

The nucleic acid binding protein Whirly1 of barley has been located to both chloroplasts and the nucleus of the same cell. Immunogold labelling furthermore showed that in vivo Whirly1 does not strictly co-localize with DNA in chloroplasts, while it is closely associated with DNA in the nucleus. High-resolution imaging of Whirly1-GFP and PEND-RFP fusion proteins revealed that only a minor part of Whirly1 co-localizes with nucleoids. The co-localization with nucleoids is in accordance with the detection of Whirly1 in a conventionally prepared fraction of the transcriptionally active chromosome (TAC). By further purification and enrichment of transcriptional activity Whirly1, however, was lost from the TAC fraction. Knockdown of Whirly1 in transgenic barley plants had neither impact on transcription of selected protein coding genes nor on genes coding for ribosomal RNAs or tRNAs. The results of RIP-chip experiments showed that barley Whirly1 as its maize orthologue associates with a set of intron containing plastid RNAs. Taken together, the results suggest that plastid-located Whirly1 functions primarily in RNA metabolism rather than as a DNA binding protein.

The Etched1 gene of Zea mays (L.) encodes a zinc ribbon protein that belongs to the transcriptionally active chromosome (TAC) of plastids and is similar to the transcription factor TFIIS.

Etched1 (et1) is a pleiotropic, recessive mutation of maize that causes fissured and cracked mature kernels and virescent seedlings. Microscopic examinations of the et1 phenotype revealed an aberrant plastid development in mutant kernels and mutant leaves. Here, we report on the cloning of the et1 gene by transposon tagging, the localization of the gene product in chloroplasts, and its putative function in the plastid transcriptional apparatus. Several alleles of Mutator (Mu)-induced et1 mutants, the et1-reference (et1-R) mutant, and Et1 wild-type were cloned and analyzed at the molecular level. Northern analyses with wild-type plants revealed that Et1 transcripts are present in kernels, leaves, and other types of tissue, and no Et1 expression could be detected in the et1 mutants analyzed. The ET1 protein is imported by chloroplasts and has been immunologically detected in transcriptionally active chromosome (TAC) fractions derived from chloroplasts. Accordingly, the relative transcriptional activity of TAC fractions was significantly reduced in chloroplasts of et1-R plants. ET1 is the first zinc ribbon (ZR) protein shown to be targeted to plastids. With regard to its localization and its striking structural similarity to the eukaryotic transcription elongation factor TFIIS, it is feasible that ET1 functions in plastid transcription elongation by reactivation of arrested RNA polymerases.

Changes in RNA-Polymerase Activity during Biogenesis, Maturation and Senescence of Barley Chloroplasts. Comparative Analysis of Transcripts Synthesized Either in Run-on Assays or by Transcriptionally Active Chromosomes

Summary The relative transcription rates of plastid genes were analyzed by hybridization of both run-on transcripts and TAC (transcriptionally active chromosome) derived transcripts to filter bound barley plastid DNA fragments. The results clearly demonstrate that TACs are able to transcribe all classes of genes. Development related changes in transcription were characterized by hybridization to 16 specific barley plastid DNA fragments of the transcripts derived from proplastids and from four physiologically well characterized stages of chloroplast biogenesis and senescence. Both in vitro transcription with TAC and run-on assays revealed a differential transcription of plastid genes. During biogenesis and maturation of chloroplasts relative transcription of rDNA decreases while relative transcription of psbA , psbD and psaB increases in parallel. Senescence is accompanied by converse changes in transcription. Though the main development related changes in plastid DNA transcription observed in run-on assays are retained by TAC, transcription of some genes ( atpA , rbcL , rpl , rps , rpoB ) is less pronounced in TAC preparations than in run-on assays. Differences between the two transcription systems are particularly obvious in the case of photosynthetically competent chloroplasts and might indicate that parts of the plastid transcriptional apparatus are not represented by TAC.

Nascent transcript-binding protein of the pea chloroplast transcriptionally active chromosome.

This study describes the nascent RNA-binding protein of the pea chloroplast transcriptional complex. The protein has been identified by photoaffinity labelling of the transcriptionally active chromosome (TAC) which utilizes the endogenous plastid DNA as template. UV irradiation of lysed chloroplast or the isolated TAC under conditions optimized for transcription photocross-links nascent radiolabelled transcripts (up to 250 nucleotides in length) to a 48 kDa protein. The photoaffinity labelling of the transcript-binding protein is dependent on UV irradiation, is maximal after about 30 min of irradiation, and is completely dependent on transcriptional activity; no cross-linkage has been observed with pre-synthesized RNA. Cross-linkage is influenced by salts and inhibitors in accordance with their effects on transcription. The photoconjugate is composed of protein and RNA moieties, and can be hydrolysed by several proteases. However, the cross-linked transcript is protected from nucleases until the protein is removed. Manganese enhances photoaffinity labelling of the transcript-binding protein, and this is paralleled by an increase in total transcriptional activity of TAC. This protein was isolated by 2-dimensional polyacrylamide gel electrophoresis and the sequence of 15 amino acid residues at the amino terminus was determined. The nascent transcript-binding protein appears to be involved in the transcription of all three classes of chloroplast genes. We also found a polypeptide of identical molecular weight to get cross-linked to nascent transcripts in chloroplasts isolated from other legumes such as Cicer arietenum, Vigna radiata and Phaseolus vulgaris, and monocots like Zea mays, Oryza sativa and Pennisetum americanum.

Characterization and properties of the spinach chloroplast transcriptionally active chromosome isolated at high ionic strength

The transcriptionally active chromosome (TAC) of spinach (Spinacia oleracea L.) chloroplasts has been isolated at a high ionic strength, with low mechanical shearing, by glycerol gradient centrifugation. The properties of the TAC differ from those previously reported for the TAC isolated either from Euglena chloroplasts or from spinach using a low-ionic-strength solubilization medium and gel filtration. The high-salt-isolated TAC is homogenous in density but not in size and contains fewer weakly bound proteins than its lowsalt-isolated homologue. In vitro, it promotes elongation of the RNA chains previously initiated in vivo. Transcription is not limited to the ribosomal DNA. The transcriptional pattern is not strongly affected by the high-salt preparation. Ribonuclease pretreatment of the TAC, prior to the in-vitro transcription, leads to a more than tenfold increase of the transcription activity. These properties are discussed in relation to the structure of the spinach chloroplast chromosome.

Evidence for two RNA polymerase activities in Euglena gracilis chloroplasts.

Two types of RNA polymerase activity were isolated from Euglena gracilis chloroplasts and compared. One polymerase is tightly bound to chloroplast DNA; this complex is called the transcriptionally active chromosome (TAC) (Rushlow, K. E., Orozco, E. M., Jr., Lipper, C., and Hallick, R. B. (1980) J. Biol. Chem. 255, 3786-3792). The other activity is found in a soluble extract of Euglena chloroplasts. The soluble extract is dependent upon an exogenous DNA template for activity. The two activities can be isolated in two distinct subchloroplast fractions from a single chloroplast preparation. The soluble extract is selective for transcription of transfer RNA genes, whereas the TAC is selective for ribosomal RNA genes. TAC and the soluble extract respond differently to KCl and Mg2+. The soluble extract is sensitive to heparin, and TAC is resistant. The two activities have different temperature optima. Based on this evidence, we conclude that Euglena chloroplasts have at least two distinct RNA polymerase activities.