**5. Transcription machine and transcription regulation methods in chloroplasts**

Chloroplasts are believed to have emerged as a result of the coexistence of photosynthetic cyanobacteria and the ancestors of modern eukaryotic plant cells following various genomic rearrangements. Compared to the genome of cyanobacteria, which is 3Mpz, the genome of terrestrial chloroplasts is 20 times smaller. Despite such a size difference between the two genomes, the expression of chloroplast genes is regulated by more complex systems than cyanobacterial genes. Most importantly, the expression of chloroplast genes strongly depends on post-transcriptional regulation, which includes polystyrene mRNA processing, intron binding, and RNA editing. Chloroplast genes are transcribed in flowering plants by two types of RNA polymerase. Multisubunit bacterial species (PEP) RNA is encoded by the chloroplast genome and Phage Polymerase T3/T7 RNA (NEP) is encoded by the nuclear genome. In adult chloroplasts, PEP represents the primary transcription machine, which transcribes more than 80% of the original chloroplast transcripts. NEP, on the other hand, transcribes chloroplast housekeeping genes. NEP is a phage-type RNA polymerase enzyme with a single subunit [15].

Both PEP and NEP are essential for the transcription of chloroplast proteins. Even though NEP and PEP identify different promoters, many chloroplast genes have promoters that are detected by both PEP and NEP. Promoters detected by NEP can be divided into three groups—2,1a, 1b. All promoters belonging to species 1a are identified by a protected nuclear motif called YRTa. Promoters of this type are several nucleotides higher than the transcription initiation sequence. Type 1b promoters, in addition to the protected motif in their structure called the GAAbox, are located between the 18th and 20th nucleotides above the YRTa motif.

Experiments on mutant tobacco have shown an essential role of the GAA motif for proper recognition of the promoter performed by NEP.

*DOI: http://dx.doi.org/10.5772/intechopen.105125 Transcription Flexibility of* Dunaliella *Chloroplast Genome*

In contrast, type 2 promoters all consist of NEP promoters without the YRTa motif. Many promoters have been identified by PEP similar to bacterial G70 promoters and are identified by two motifs of normal sequences spaced from the transcription start site by −10 and −35 nucleotides. The first motif of 10 nucleotides that is farther from the transcription site is the TATAAT sequence. Whereas, the second motif of 35 nucleotides that is farther away from the transcription site is the TTG ACT sequence. Due to the great diversity among plants, the position of conventional sequences of specific PEE promoters may be different. For example, in the barley chloroplast genome, the TATAAT 3–9 nucleotide sequence is located above the transcription start site, while the TTG ACT 15–21 nucleotide sequence is located upstream of the transcription start site.

PEP is mainly responsible for the transcription of chloroplast genes, which are protein products that are related to photosynthesis in various ways. However, some genes encoding proteins involved in photosynthesis are encoded by NEP. There is a small group of chloroplast genes that are not related to photosynthesis and are specifically transcribed by NEP. It includes the ACCD gene encoding acetyl-CoA carboxylase subunit in dicotyledonous plants, RPL23 gene encoding ribosomal protein L23, CLPP gene encoding ATP-dependent proteolytic subunit in monocotyledonous plants, and RPOB gene encoding all major subunits in PE. Therefore, chloroplast genes with their promoters can be divided into three categories, which are as follows:


In the case of dicotyledonous plants where two different NEPs have been detected, an extended portion of the promoters can be suggested. The activity of RPOTp and RPOTmp in tissues is different at different stages of plant development. In *Arabidopsis taliana*, increased RPOTmp activity has been observed mainly in young dividing cells and photosynthetic inactive tissues, whereas enhanced RPOTp activity has been observed in photosynthetic green tissues. Differences in the structure of known promoters were identified by the two types of NEP. Interestingly, both NEP and PEP are active at all stages of plant development in plastids of unbleached tissues, including roots, fruits, and seeds. The persistent activity of these two types of polymerases is related to their involvement in the transcription of housekeeping genes, such as tRNA-encoding genes [17].

Regulation of transcription of chloroplast genes is essential for the proper functioning of chloroplasts and overall plant growth under normal and adverse conditions.

#### **5.1 RNA polymerase types**

It is an enzyme that consists of several subunits. Although most of the genes in the PEP subunit have been transferred to the nuclear genome, the genes encoding the primary and nuclear PEP subunits (α, β, β′, and β′′) have been preserved in the chloroplast genome. One of the main differences between the central subunits of PEP is their molecular weight. The alpha subunit has a molecular weight of 38 kDa, the beta subunit has a molecular weight of 120 kDa, the beta subunit has a molecular weight of 85 kDa, and the beta subunit has a molecular weight of 185 kDa. Similarly, in bacteria and most land plants, the RPOA gene encodes the alpha major subunit and with the ribosomal protein genes, organizes into an operon under the control of the same promoter. In contrast, the RPOC2, RPOC1, and RPOB genes encode the major and central subunits of β, β′, and β′′, respectively, and form a separate operon designated as RPOBC. In addition to the major subunits, PEP is composed of additional protein factors encoded by the nucleus genome, including sigma factors (SIGs) and PAPs, polymerase-related proteins. Sigma chloroplast factors, which are stations for the bacterial transcription initiation factor, play an essential role in the transcription of the chloroplast encoded gene. Sigma factors regulate transcription at different developmental stages by identifying different promoters and allowing a complete set of PEP to begin to polymerize. The highlighted secondary PEP factor appears to be PAPs that are involved in almost every stage of transcription [17].

PEP has a promoter-identifier subunit called the sigma factor. The major PEP enzyme subunits are encoded by a set of genes located in the plastid genome: rpoA, rpoB, rpoC1, and rpoC2. Conversely, during evolution, the sigma factor genes, which specifically provide the promoter needed for PEP, have been transferred to the nucleus genome to possibly allow the nucleus to regulate the expression of the chloroplast gene in response to environmental and developmental signals. PEP and a set of proteins associated with polymerase PAPs constitute a massive protein complex required for transcription. All PAPs are encoded by genes in the nucleus, and most are components of the active pTAC transcription chromosome. These proteins are predicted to be involved in DNA and RNA metabolism, regulate redox from photosynthesis, and protect the PEP complex from reactive oxygen species (ROS).

#### **5.2 Sigma factors**

Chloroplasts are the cytoplasmic organs in which photosynthesis takes place in plants and algae. Due to their cyanobacterial strain, chloroplasts contain a small transcriptional active genome and a bacterial gene expression machine.

Sigma factors are separable subunits of bacterial RNA polymerases that ensure effective transcription initiation of gene promoters. Chloroplasts together have a type of bacterial RNA polymerase with a sigma factor subunit due to their prokaryotic origin. The excellent plant *A. taliana* contains six sigma factors (SIGs) for hundreds of its chloroplast genes. The role of this relatively large number of transcription initiation factors for the small chloroplast genome is not fully understood.

Sigma factors are bacterial RNA polymerase subunits. They are capable of effective transcription of bacterial genes with their three distinct activities—transferring the promoter recognition property to RNA polymerase, melting two strands of the promoter region into single single-stranded open complexes capable of transcription, and interacting with other DNA-binding transcription factors that are regulated for gene expression.

The genome of chloroplasts usually contains 100–300 genes that are mostly organized in polystyrene operons, such as bacteria. Most chloroplast genes contain promoter elements of bacterial types 10 and 35 that are identified and transcribed by a multi-subunit bacterial RNA polymerase. The major subunits of the bacterial RNA polymerase are encoded in the plastid genome and are called PEP. Like bacterial polymerase, the original PEP enzyme requires reversible binding to the sigma factor subunit encoded in the nucleus for effective transcription [18].

Is a single subunit enzyme that performs a single transcription protein; from the identification of the promoter to the end of the process, regardless of the structural

pattern of DNA. This enzyme evolved through replication of the mitochondrial RNA polymerase encoding nuclear gene and bears a strong resemblance to phage species RNA polymerase and is also made of a subunit. Three types of NEP are detectable, and all three are encoded by RPOT genes. RPOTp occurs in monocotyledonous plants, while RPOTp and RPOTmp have been identified in dicotyledonous plants. In addition, the third form of the NEP family enzyme, RPOTm, has occurred in mitochondria [17]. In *A. taliana*, NEP is encoded by the two nuclear genes rpoTp, and romp [15].

In flowering plants and mosses, one or more single subunits of phage RNA polymerase, known as NEP, transcribe a small subset of chloroplast genes from distinct promoter elements. Genes transcribed by NEP include rpoB, the PEP beta subunit encoder, and several tRNA genes. Some chloroplast genes include NEP and PEP promoters, which are transcribed by two RNA polymerases at all stages of plastid development and in all plant tissues [18].

Conversely, SIGs (sigma factors) are important for PEP binding to promoters of relevant genes. Six different SIGs are involved in the transcription of *A. thaliana* genes, bacterial-derived sigma factors that show great similarity to their ancestors only through the conservative region at the end of their molecules. The nonconservative region seems to be crucial for the functioning of certain sigma factors. SIGs sigma factors are regulated by phosphorylation of specific sequences in the aforementioned nonconservative region. Research on the activation of sigma factors has often been conducted exclusively in SIG1 and SIG6. Phosphorylation of SIGs appears to be a complex process performed by various enzymes. In 1996, Baginsky and his team proposed that SIG6 phosphorylation was mediated by a PEP associated with the serine–threonine protein kinase called plastid transcription kinase. In addition, in the literature, PTK has been abbreviated as cpck2 due to the high similarity between the catalytic components of PTK and the casein kinase2 (ck2) subunit. However, cpck2 has been shown to use SIG6 as a substrate for regulatory phosphorylation and is not the most sensitive site for SIG6 phosphorylation for conventional cpck2. Therefore, this hypothesis requires the presence of other kinases involved in this process. Phosphorylation of SIGs can both initiate and stop the transcription of genes identified by the PEP complex. A type of promoter known to be determined by a specific factor in this process appears to be determined, for example, SIG6 factor phosphorylation is necessary for transcription of the ATPB gene, but does not affect transcription of the PSBA gene. In turn, the lack of phosphorylation of SIG1 reduces the transcription of two DOGA and PSBA genes. The transcription of genes encoded by the chloroplast genome can be affected by smaller molecules, such as rare nucleotides. Recent studies show an interest in these molecules, they are considered new signal molecules that stimulate the response of plants to various types of biotic and abiotic stresses, and they are called Alarmones. Laboratory studies have shown that under different stress conditions, guanosine tetraphosphate is produced in plastids and then binds to the beta subunit of the PEP enzyme and inhibits RNA synthesis [19].

Large-scale prediction and analysis of primary operons in plastids reveal unique genetic features in the evolution of chloroplasts. While bacterial operons have been thoroughly studied, therefore, there is little analysis of chloroplast operons (limited ability to study the basic elements of these structures and apply them to synthetic biology).

Plastids are cellular organelles found mainly in a diverse group of photosynthetic organisms. The endosymbiotic theory explains the origin of plastids (Section 2). Since the beginning of this common evolutionary interaction, most cyanobacterial genes have been lost or transferred horizontally to the host nucleus genome, while the plastid genome has largely retained photosynthesis-related genes and conservative

genes. As a result, the plastid is heavily dependent on encoded nuclear proteins for basic operations, making it a non-autonomous organelle. Nevertheless, it retains many of its ancestral characteristics and genomic traits, such as the cyclic structure of the genome, bacterial 70S ribosomes, and PEP or the organization of genes in operon transcription units such as bacteria. Operons are DNA units made up of several genes controlled by a single promoter that often share a common function.

Unlike bacteria, plastid operons are not available in databases and are only examined by a small number of studies focused on higher plant model organisms, in which the entire operon map was revealed in the atmosphere by a differential RNA sequence. In tobacco, part of the polystyrene transcripts was detected using the northern stain. In spinach, psbB and rpoBC operons were detected using Northern blot, while ATP synthase operons were proposed in Escherichia coli by comparing their gene content and ordering their homology to gene clusters.

In algae, part of the operons of *C. reinhardtii* was studied, and several operons were detected using the northern spot. Whereas, two recent reports identified 16 and 22 polystyrene subunits by research for compatibility in RNA sequence overlap, called intergenic regions of adjacent genes. However, there is no extensive analysis of chloroplast operons. The ability to identify them, identify their features, and use this data for synthetic biology purposes remained limited.

The expression of the plastid gene differs from the bacterial model in terms of different characteristics. Chloroplast transcripts are often edited and bound by RNA. The role of transcription termination is significantly reduced, many noncoding RNAs are replicated repeatedly, and the plastid genome is suggested to be completely transcribed. In addition, gene expression often relies on RNA-binding proteins (Pentatricopeptide repeat family) that bind Cis elements upstream of the starting codon. Thus, it inhibits the activity of exoribonucleases and stimulates translation by suppressing the stem-loop, which inhibits ribosome binding. In addition, polystyrenes are regulated by several promoters and are widely processed, thus contributing to the formation of various transcript isoforms derived from a single primary transcription unit. Thus, the structure of plastid operons has evolved significantly compared to classical bacterial operons. This difference likely affected the composition and properties of chloroplast operons and gave rise to unique properties. Consequently, the ability to convert synthetic and synthetic genes into plastids has had a major impact on plant biotechnology, when it points to significant advantages over nuclear deformation. These benefits include uniform composition based on specific coalition location, no gene silencing, relatively high expression of dissimilar genes, and long-term deformation in most crops due to maternal inheritance.

One obvious advantage specifically for this study is the use of the ability of natural plastids to express polystyrenes and to design vectors with multiple genes under the control of a single promoter, thus minimizing plasmid sizes and allowing the introduction of multiple metabolites to the cells. Associated with transgenes in a single deformation. Because both basic scientific questions and biological ideas are hampered by the lack of extensive information on plastid operons.

#### **5.3 Transcription factors**

Transcription factors are proteins that bind to DNA regulatory sequences (amplifiers, suppressors, or extinguishers), which are usually located in the 5′ region of target genes, to adjust the speed of transcription and the number of transcripts. This may lead to enhancing or reducing gene transcription and protein synthesis,

consequently altering cell function. There are several families of transcription factors, and members of each family may have common structural characteristics. These families include:


Many transcription factors are common to several cell types (ubiquitous), while others are cell-specific and may specify the phenotypic characteristics of a cell. Transcription factors may be activated directly by ligands, such as glucocorticoids and vitamins A and D. Also, stimulation of cell surface receptors launches multiple intracellular signal transduction pathways, including MAPK, PKA, JAK, and PKC that lead to indirect activation of transcription factors. Transcription factors may act as the nucleus messenger and translate transient peripheral signals at the cell surface into long-term changes in gene transcription. Transcription factors may be activated within the nucleus, often with a transcription factor already attached to DNA, or within the cytoplasm, leading to exposure to nuclear localization signals and targeting the nucleus. Phosphorylation, acetylation, and nitration in transcription factors as post-translational changes can affect the DNA binding quality or transcriptional activity [20].

Membrane-transcription factors are transcription factors that are anchored in the membranes in a passive state and are activated by external or internal stimuli. These transcription factors are released from the maternal membranes and transported to the nucleus. Research shows that some proteins attached to the cytoplasmic membrane (PM) and some proteins attached to the endoplasmic reticulum can enter the nucleus. Based on specific signal recognition signals, some transcription factors attached to membrane-bound proteins undergo proteolytic cleavage to release intracellular fragments that enter the nucleus to control gene transcription. In addition, some transcription factors bind to membrane proteins as integral proteins in the cell nucleus through smuggling into the Golgi and endoplasmic reticulum, where membrane-releasing mechanisms rely on endocytosis. In contrast, transcription factors attached to the membrane of the endoplasmic reticulum are transmitted directly to the nucleus or by transfer to the Golgi. In both pathways, only fragments of transcription factors attached to the membrane of the endoplasmic reticulum are transported to the nucleus. Most transcription factors are located in the cytoplasm. After receiving a signal from the transmembrane signal transduction, the transcription factors are activated and transported to the nucleus after the cytoplasm, where they interact with the corresponding DNA framework (cis active elements) [21]. But keep in mind that transcription factors originate from the nucleus and target the promoter of nuclear and chloroplast genes.

Transcription factors are proteins involved in the process of converting DNA to RNA. They contain a large number of proteins, except RNA polymerase, which initiates and regulates gene transcription. One of the hallmarks of transcription factors is that they have DNA-binding domains that give them the ability to bind to specific DNA sequences called amplifier sequences or promoter sequences. Other transcription factors bind to regulatory sequences, such as activating and suppressing sequences, which can stimulate or suppress transcription of the relevant gene [22].

The task of transcription factors is to regulate and turn off genes to ensure that they are expressed in the cell at the right time and in the right amount throughout the life of the cell. The transcription factor group acts in concert to guide cell division, cell growth, and cell death throughout life. Transcription factors are members of the proteome as well as the regulome. Transcription factors alone or with other proteins in a complex act as activators or inhibitors of RNA polymerase affinity to specific genes [23]. They are classified into different classes based on their DNA-binding domains [24].

## *5.3.1 Family classification of transcription factors belonging to D. salina species*

*D. salina* transcription factors include a total of 31 different transcription factors, presented in **Table 1**, each of which is classified into specific families of transcription factors. In *D. salina CCAP 19/18* TF involved in nuclear and chloroplast gene transcription is a few differences.

Out of a total of 31 types of transcription factors related to *D. salina*, 10 types of transcription factors are involved in the regulation of the nucleus genome and are not involved in the regulation of the chloroplast genome. The 21 other types are involved in the regulation of the chloroplast genome and are located on the promoter regions of 66 chloroplastic proteins encoding gens.

In this way can be arranged *D. salina* 31TFs in 23 families according to **Table 2**.

The GARP transcription factor family (made up of G2-like and ARR-B) (family number 11 in **Table 2**) has a structural but distant relationship with the MYB transcription factor (family number 12 in **Table 2**).

The SBP transcription factor family (family number 20 in **Table 2**) interacts with the C3H transcription factor (family number 7 in **Table 2**).

Homeobox encodes a DNA helix-turn-helix binding motif called the homeodomain. The second DNA binding is a second independent folded protein that contains at least one structural motif that recognizes dual or single-stranded DNA. A second DNA binding can identify a specific DNA sequence (a recognition sequence) or have a general tendency for DNA [7].

Transcription-activating domains are regions of transcription factors that, in conjunction with a DNA-binding domain, can activate transcription from the promoter by direct contact with the transcription machine (general transcription factor and


#### **Table 1.**

*D. salina CCAP 19/18 TF involved in nuclear and chloroplast gene transcription.*
