CHEMICAL BIOLOGY

Transcription-Based Therapeutics

Amberlyn M. Wands and Anna K. Mapp, University of Michigan, Ann Arbor, Michigan

doi: 10.1002/9780470048672.wecb607

Aberrant transcription patterns are associated with most human diseases. Therefore, enormous interest exists in the development of designer molecules that can be used to regulate directly the transcription of predetermined genes for the ultimate treatment of a wide range of disease states. One emerging strategy is to identify molecules that reconstitute one or more functions of the endogenous proteins that upregulate transcription, transcriptional activators. In doing so, they function as either inhibitors or activators of transcription. Using this approach, a variety of protein- and small molecule-based transcriptional regulators have been developed, and at least one has reached clinical trials.

Diseased cells possess different transcription profiles relative to their normal counterparts; therefore, considerable interest exists in the discovery of molecules that correct errant transcription patterns for use as mechanistic tools and as therapeutic agents. One emerging mechanism for accomplishing this task is the use of molecules that mimic key functions of the endogenous proteins that upregulate transcription: transcriptional activators. By doing so, they either inhibit or activate the expression of specifically targeted genes (1). As illustrated in Table 1, exogenous agents that can upregulate or downregulate transcription are being developed for the eventual treatment of such ailments as cancer, inflammation, viral infections, metabolic disorders, and genetic disorders. For example, one method by which apoptosis can be induced in cancer cells is via the modification of the aberrant expression levels of those proteins that regulate cell growth and survival. A molecule that upregulates the proapoptotic bax gene directly could induce apoptosis when introduced into p53-deficient osteosarcoma cells (3). Conversely, a molecule that inhibits transcription of the survivin gene (an inhibitor of apoptosis protein) could induce apoptosis when introduced into lung carcinoma cells (9). In this article, we describe the structure and the function of natural transcriptional activators and outline the most common strategies for designing exogenous molecules that affect their function directly. References for more detailed treatments of the individual topics are provided at the end of the article.

Biological Background

As their name implies, eukaryotic transcriptional activators are responsible for initiating gene-specific transcription. To accomplish this task, activators localize at specific DNA sequences in a signal-responsive manner and facilitate the assembly of the eukaryotic transcriptional machinery (RNA polymerase II and associated factors) (1, 21). This process requires activators to participate in many protein-protein and protein-DNA interactions yet can be accomplished with a fairly simple architecture. Activators are composed minimally of a DNA binding domain (DBD) and a transcriptional activation domain (TAD). The primary function of the DBD is to localize the transcriptional activator to specific sites within genomic DNA. The DBD thus imparts much of the gene-targeting specificity of the activator. In contrast, the TAD participates in many protein-protein interactions that are critical for transcription initiation. By doing so, it dictates the timing and extent of gene activation. The two domains can, in general, function independently. In other words, if the DBD of transcriptional activator A is attached to the TAD of activator B, the new chimeric activator will upregulate transcription of gene A.

The modular character of transcriptional activators facilitates the design and the implementation of non-natural molecules that can affect gene transcription. Designer replacements of each of the two domains can be used individually to inhibit transcription by preventing either activator-DNA interactions or activator-transcriptional machinery interactions (Fig. 1). In contrast, linking a DBD and a TAD either covalently or noncovalently is a common strategy used to create activator artificial transcription factors (ATFs), which are molecules that seek out and upregulate the transcription of specific genes (22). The design of inhibitor and activator molecules relies on understanding the structure and the mechanism of the DBD and the TAD of the natural proteins. As illustrated in the subsequent sections, the DBD is more understood than the TAD; therefore, the development of artificial DBDs is far more advanced than TAD replacements.

Figure 1. Strategies for designing molecules that regulate transcriptional activators. Transcriptional activators initiate transcription by binding to DNA sequence specifically and stimulating the assembly of the transcriptional machinery through one or more protein interactions. Molecules that prevent either the DNA binding of the activator or the interaction of activators with their binding partners within the transcriptional machinery can be used to inhibit transcription. Alternatively, gene-specific transcription can be initiated by a molecule that mimics both key functions of an activator, which is an activator artificial transcription factor (activator ATF).

Activator ATFs

As described above, the function of a transcriptional activator can be reconstituted minimally by an activator ATF that contains a DNA-binding domain and a transcriptional activation domain. The earliest artificial activators were composed of DBDs and TADs taken from naturally occurring proteins (1). Although these activator ATFs are powerful mechanistic tools, their application scope is narrow because they can only target genes that contain the DNA binding sites of the endogenous protein DBDs. In addition, controlling the delivery and the stability of the constructs in vivo can be challenging. The development of non-natural replacements for each of the two key activator ATF domains has been an important goal to address these fundamental limitations.

DBDs

Much work has been done to develop artificial DBDs that can bind with high specificity and affinity to predetermined DNA sequences (22). This binding has been achieved, for example, by mutating amino acids on natural protein scaffolds to recognize novel sequences (zinc fingers), using the hydrogen bonding properties of nucleic acid-like molecules [triplex-forming oligonucleotides (TFOs), peptide-nucleic acids (PNAs)], or tailoring the DNA-binding properties of natural products (polyamides, for example). This section describes only the properties of zinc fingers and polyamides, because TFOs and PNAs are described in depth in related articles. Table 1 summarizes many of the applications of these molecules.

Table 1. Summary of progress toward transcription-targeted therapeutics

Zinc fingers (ZFs)

Protein DNA-binding domains offer several attractive features for activator ATF design; the most important of which is the high affinity and specificity with which they typically recognize their cognate DNA sequence. The Cys₂His₂ ZF fold has proven to be enormously versatile as a DNA-targeting entity. It is composed of ~30 amino acids folded into a PPa structure that is stabilized by hydrophobic interactions and by the coordination of a zinc ion by two conserved cysteine residues in the antiparallel P sheet and two histidine residues in the a helix. The solid-state structure of the 3-finger protein Zif268 in complex with DNA illustrates that each finger makes its primary sidechain-base interactions to three adjacent nucleotides in the sense strand of the DNA duplex (Fig. 2a). It does so by inserting its a helix into the major groove of DNA, on which amino acids at positions -1,3, and 6 of the helix contact the 3', middle, and 5'-nucleotides of the 3-bp subsite, respectively (23). Also, in some ZFs an aspartic acid at position 2 of the helix interacts with a cytosine or adenine base in the antisense strand of the adjacent triplet, which makes these domains seem to recognize a 4-bp subsite instead (23, 24).

One simple method for creating a ZF protein capable of binding to a predetermined DNA sequence is through the “modular assembly” approach. In this approach, pre-existing, single finger ‘modules’ with known specificities are assembled into a multifinger array. To facilitate this, three archives of known zinc finger modules have been created by the Barbas laboratory, Sangamo BioSciences Inc., Richmond, CA and ToolGen Inc. Seoul National University, South Korea. The Barbas modules were developed using a combination of phage display and rational design methods under the assumption that ZF domains function with position independence. They are capable of recognizing all GNN triplets, most ANN and CNN triplets, and a few TNN triplets (N = any base). The Sangamo modules were also developed by phage display but under the assumption of position dependence, and are capable of recognizing all GNN triplets and a smaller number of non-GNN triplets. And finally, the ToolGen modules are naturally occurring human zinc fingers whose nucleotide triplet sequences were identified through a yeast one-hybrid assay. And in collaboration with the Zinc Finger Consortium, these archives are now available through a web-based server called ZiFiT (Zinc Finger Targeter) that facilitates the design of multifinger arrays that bind to your desired DNA sequence (24).

In practice, ZF proteins composed of 3-6 fingers with apparent dissociation constants in the picomolar to nanomolar range have been attached to proteinacious transcriptional activation domains and used successfully to upregulate endogenous genes in mammalian cell culture (Table 1). One such activator ATF that contains a six-finger DBD that binds within the γ-globin promoter can increase fetal hemoglobin levels 7-16 fold in human erythroleukemia cells (2). Additionally, ZF-based activator ATFs that target the VEGF-A (vascular endothelial growth factor) gene have even been shown to function in animal models (4, 5); despite this success, stable delivery remains a challenge because they must be administered by viral vectors (23).

Figure 2. Designer DNA binding domains. (a) Crystal structure of Zif268 in complex with DNA (PDB accession number 1aay)(54). Arg 74, Glu 77, and Arg 80 in positions -1, 3, and 6, respectively, of the recognition helix of finger 3 are shown projected into the major groove. (b) Crystal structure of polyamide (ImHpPyPy)₂ in complex with DNA (PDB accession number 407 d).

Polyamides

Small molecule DBDs represent an attractive choice for activator ATF construction because they may circumvent the delivery limitations of proteins. Toward this end, considerable progress has occurred in developing programmable small molecules that can be designed readily to target a wide range of DNA sequences (22). In particular, the polyamide class of DBDs has been used successfully for the construction of several activator ATFs (25). The inspiration for the polyamides developed from the minor groove-binding natural products distamycin and netropsin. These natural products are composed of pyrrole amino acids linked through amide bonds; they bind to A/T-rich tracts of DNA in the minor groove with moderate affinity through a combination of hydrogen bonds between the amide bonds and the minor groove functional groups, hydrophobic contacts, and electrostatic interactions with the phosphate backbone. Polyamides consist not only of pyrrole amino acids, but also of imidazole, pyrrole, and other heterocyclic amino acids that enable recognition of A●T, T●A, C●G, and G●C base pairs through the formation of specific hydrogen bonds with minor groove functionality, although overall specificity varies with sequence context (Fig. 2b). Although several different polyamide structural motifs exist, the hairpin polyamide in which a flexible amino acid tether connects two polyamide arms is used most commonly. As the name suggests, this molecule folds into a hairpin-like structure in the minor groove such that the arms are side-by-side, which maximizes hydrophobic interactions with the walls of the minor group and facilitates the formation of polyamide-DNA hydrogen bonds. The molecules exhibit greatly enhanced DNA binding affinities relative to distamycin and netropsin, with dissociation constants in the pico- molar to nanomolar range, and they have been shown in several applications to traffic to the nucleus and to interact with their cognate DNA sites. Enhancing their use even more, Dervan et al. (25) have developed a set of “pairing rules” that can be used to design molecules to target specific DNA sequences. In addition, the molecules can be prepared by solid phase synthesis, which makes them accessible to many users.

Polyamides have been used as the basis for several different activator ATF constructs that function in cell-free and in cellular systems. In contrast to protein DBDs, they are synthesized easily to contain both peptidic and nonpeptidic TADs (19-20, 26). However, they often require special modifications to enhance cellular permeability and typically target shorter DNA sequences (6-8 base pairs) relative to proteins (27).

TADs

The most common TADs used in the construction of activator ATFs are derived from the activation domains of natural proteins. In particular, sequences taken from the amphipathic class of activators are composed of hydrophobic amino acids interspersed with polar ones and typically possess robust activity across organisms (1, 22). For example, activating sequences from the viral protein VP16, the yeast activator Gal4, and the p65 subunit of the human activator NF-KB have all been attached to ZF proteins and function as activator ATFs in mammalian cell culture (2-4, 6). However, activator ATFs that contain nonprotein DBDs typically use much smaller sequences to minimize the overall size of the construct. For instance, a monomeric or dimeric repeat of eight residues of VP16 (VP1 and VP2) could upregulate a reporter gene in vitro using yeast nuclear extracts when attached to a polyamide (18), whereas a monomeric or dimeric repeat of an 11 residue sequence taken from VP16 (ATF14 and ATF29, respectively) could upregulate a reporter gene in mammalian cell culture when attached to a TFO (10).

Several strategies have been employed to develop novel peptidic TADs that function similarly to natural TADs. For example, a 20 amino acid peptide sequence designed rationally to form an amphipathic helix (AH) (Fig. 3a) was successful in upregulating a reporter gene in yeast when fused to the Gal4 DBD (28), as well as in vitro with yeast nuclear extracts when fused to a polyamide DBD (18). In addition, another successful approach has been to use phage display peptide libraries to select against the protein targets of natural TADs. For instance, a selection performed against the KIX domain of the mammalian coactivator p300/CBP yielded an 8-amino acid peptide named KBP 2.20 that is capable of upregulating a reporter gene 40-fold in mammalian cells when attached to the Gal4 DBD (29). Additionally, a selection performed against the masking protein (Gal80) of the yeast activator Gal4 yielded a 20-amino acid peptide named G80BP-A that is capable of activating transcription of a reporter gene in both yeast (30) and mammalian (15) cell culture when attached to the Gal4 DBD. This TAD functioned only weakly when a PNA was used as a DBD, however, because of the distortion of the promoter on binding (15).

Finally, nonpeptidic TADs have been developed recently that possess the advantage of increased stability toward proteolytic degradation. Often, these molecules are considerably smaller than protein-derived TADs and thus may exhibit advantageous cell permeability properties. The first small molecule TAD to be reported was an amphipathic isoxazolidine 1 (Fig. 3b). Its ability to upregulate transcription was demonstrated initially in a cell-free assay where it proved to be as active as a larger peptidic activator (MW 290 vs 1674) (31). This amphipathic isoxazolidine also functions in mammalian cell culture in a dose-dependent manner with up to 80-fold activation at 1 μM and an EC₅₀ of 31 nM (32). In addition, a hydrophobic molecule named wrenchnolol designed originally as an inhibitor of the ESX-Sur2 interaction is also capable of functioning as an activation domain on localization to DNA. When conjugated to a polyamide, this molecule upregulated transcription in a cell-free system 3.5-fold over background. This synthetic activator ATF was, however, inactive in mammalian cell culture, which was possibly caused by limited nuclear localization (19). Finally, peptoids (oligo-N-substituted glycines) are emerging as an effective alternative to peptidic TADs. KBPo2, which is a peptoid that was identified from a library screen against the KIX domain of CBP, is capable of activating robustly (up to 1000-fold) transcription of a reporter gene in mammalian cells in a 2-hybrid assay with an EC50 of 10 μM (33). Furthermore, a polyamide-peptoid conjugate is capable of activating transcription of 45 endogenous genes that contain multiple binding sites for the polyamide within their promoters by at least 3-fold in HeLa cells (20). Although this last achievement establishes that purely synthetic activator ATFs are indeed capable of perturbing the expression profiles of cellular systems, one can envision needing an increase in specificity and in potency before their full potential as therapeutic agents can be assessed.

Figure 3. Designer transcriptional activation domains. (a) Sequences of both natural and non-natural peptidic activation domains. Although little sequence homology exists, all domains are amphipathic and thought to interact with target proteins through helix formation. (b) Structures of nonpeptidic transcriptional activation domains.

Passive Intervention

Modulation of transcription by a reconstituted activator ATF proceeds via an “active” mechanism whereby the transcriptional machinery is assembled at the gene of interest. However, modulation of transcription can also be accomplished passively using molecules that block one or more key transcriptional activator binding interactions. As illustrated by the examples below, it can be used either to inhibit or to activate transcription. One advantage of the passive intervention strategy is that it requires the molecules to carry out only a single function—bind to DNA or interact with a protein—whereas activator ATFs are bifunctional molecules and are correspondingly more complex.

DNA-protein interactions

One method by which transcription can be modulated passively is by targeting the promoter of a gene with artificial DBDs. Using this method, inhibition of transcription can be achieved by competing for the binding sites of endogenous transcriptional activators (22). For example, TFOs designed to target a sequence overlapping the Sp1 binding site in the promoters of the MCP-1 (7) and Ets-2 (8) genes can downregulate successfully expression of these proteins in cell culture. A PNA conjugated to the NLS peptide PKKKRKV designed to target a sequence that encompasses the Ets-1 and AML-1 sites in the Ep enhancer downregulates successfully the production of the c-myc translocated oncogene in Burkitt’s Lymphoma cells (11). Furthermore, a polyamide that binds to a sequence within the hypoxic response element (HRE) in the VEGF promoter can compete with HIF1-α/ARNT heterodimer formation at this site, thereby downregulating expression of this targeted protein in hypoxic HeLa cells (17). When used in combination, polyamides designed to target binding sites adjacent to TBP, LEF-1, and Ets-1 in the promoter of HIV-1 could prevent its replication in infected human PBMC culture (16). A less-common approach to prevent an endogenous activator from binding to DNA is to isolate a small molecule that interacts with its DNA-binding surface. For example, an NMR library screen against FBP (involved in c-myc expression) yielded benzoylanthranilic acids that can target a hydrophobic pocket used for ssDNA binding (34). Targeting the promoter of a gene can also lead to the activation of transcription, as demonstrated by the action of small duplex RNAs (35, 36), D-loop forming PNAs (14), and agents that compete with the binding of endogenous transcriptional repressors (37).

Transcriptional inhibition can also be achieved by targeting a sequence located within the gene itself using artificial DBDs to block RNA polymerase II during elongation. For example, a PNA that was designed to target a region encompassing a point mutation in codon 12 of the KRAS proto-oncogene showed a concentration-dependent ability to downregulate expression of the mutant allele over the wild type in pancreatic cancer cells (12). In part because of their lower binding affinities, polyamides must be conjugated to DNA-modifying agents to arrest a transcribing polymerase. For example, a polyamide conjugated to the DNA alkylator chlorambucil exhibited cytostatic activity in colon carcinoma cells; this effect was attributed to the downregulation of the H4c gene~2-fold, most likely because of the alkylation of a G residue located two bases downstream of a target site present in its coding region. This alkylating polyamide was even effective at suppressing tumor growth in a dose-dependent manner in a mouse model with no obvious toxicity (38). On the other hand, unconjugated polyamides have been used successfully to alleviate blockage of an elongating RNA polymerase by targeting expanded intronic repeat sequences and by preventing formation of non-B DNA structures (39). Finally, in addition to inhibiting elongation, PNAs also can block RNA polymerase II at the initiation step of transcription. This action can be achieved by targeting ssDNA sequences in the open complex located at the transcription start site, and it has been applied successfully with PNAs to downregulate expression of the human progesterone receptor (hPR) isoforms A and B in breast cancer cells (13).

Protein-protein interactions

Another method by which transcription can be modulated passively is by inhibiting the interaction of natural activators with their protein targets (Fig. 4). Transcriptional inhibition can be achieved by blocking activator-coactivator interactions. For example, the histone acetyltransferase CBP/p300 is a global coactivator capable of binding to a diverse group of activators including CREB, c-Myb, Jun, and HIF-1α (40). To identify a molecule that can disrupt the interaction of HIF-1α with p300, a high-throughput competition binding screen was performed against the minimal protein complex, and a small-molecule fungal metabolite named chetomin was identified (Fig. 4). Chetomin not only reduced endogenous HIF-1α/p300 complex formation in cells, as demonstrated by coimmunoprecipitaion experiments, but also reduced the activation of hypoxia-responsive reporter genes both in cell culture and in vivo. On probing the specificity of this inhibitor against other p300-dependent activators such as RAR, SREBP2, SRC-1, and STAT2, only the activity of STAT2 was significantly attenuated, which is most likely because it is the only one that, along with HIF-1α, targets the CH1 domain on p300 (41). Overall, this example illustrates the challenges of inhibiting activator function selectively by this mechanism. For instance, although the molecule KG-501, which inhibits the KID/KIX interaction within the CREB/CBP complex, could inhibit significantly transcription of CREB-dependent genes in human cells, preliminary studies indicate that it also impedes activation by another CBP-dependent activator NF-KB (42). In addition, although inhibiting complex formation between the coactivator β-catenin and the activator Tcf4 with the fungal metabolites PKF115-584 and CGP049090 leads to antiproliferative effects in colon cancer cells, these molecules also can prevent complex formation between β-catenin and the tumor suppressor protein APC, which impacts their effectiveness (43). Taken together, these results demonstrate that inhibitors of activator-coactivator interactions have enormous potential and are in need of additional evaluation.

Figure 4. Inhibitors of activator-coactivator and activator-masking protein interactions. Structures of small molecules that compete effectively with transcriptional activators for binding sites within coactivators and within masking proteins.

Inhibiting protein interactions can also lead to the activation of transcription. Activators are often regulated by masking proteins that bind to the TAD, which prevents it from contacting the transcriptional machinery until upregulation is required. Typically, TAD-masking proteins are of higher affinity and specificity than TAD-transcriptional machinery interactions and are therefore more straightforward to target with small molecules. For example, the p53 TAD binds as a helix to a relatively deep hydrophobic cleft in the protein MDM2, and a small molecule library screen for inhibitors of this interaction yielded a series of cis-imidazoline analogs termed Nutlins. The active enantiomer of Nutlin-3 inhibits recombinant p53/MDM2 complex formation in vitro with an IC₅₀ value of 0.09 μM, and activates p53 (thereby inducing apoptosis) in cancer cells that contain wild-type p53. When evaluated in vivo, Nultin-3 treatment of osteosarcoma xenografts established in nude mice results in a 90% inhibition of tumor growth (44). In addition to the Nutlins, many other inhibitors of the p53/MDM2 interaction that exhibit biologic activities have been reported (40). Terphenyl 2 increases p53 activity by 10-fold in colon cancer cells at a concentration of 40 μM (45). In tumor cells that overexpress MDM2, sulfonamide 3 (IC₅₀ value of 32 μM) increases p53 activity by 20% (46). Benzodiazepine 4 binds to MDM2 with an 80 nM K_D and exhibits antiproliferative activity in cancer cells with an IC₅₀ value of 30 μM (47). Finally, an isoindolinone with an IC₅₀ value of 5 μM (5) induces p53-dependent transcription in a dose-dependent manner in MDM2 overexpressing human sarcoma cells (48).

Other Approaches

Additional strategies are available to alter gene expression patterns for therapeutic purposes, many of which are discussed in other articles of this volume. In most cases, these approaches target processes that are upstream or downstream of transcription and can thus influence a wide array of genes both positively and negatively. One strategy involves targeting the ligand-dependent class of transcription factors known as nuclear receptors with small molecule agonists or antagonists to promote their interaction with coactivator or corepressor proteins, respectively. For example, metabolic disorders caused by malfunctioning PPAR nuclear receptors can be alleviated by targeting the a isoform with an agonist class of molecules called fibrates for the treatment of hyperlipidemia and by targeting the y isoform with the agonist class of molecules called thiazolidinediones for the treatment of diabetes (49). Another strategy involves targeting protein kinases with small molecule inhibitors to intervene at some stage in the signaling cascade that precedes the process of transcription. For instance, in the treatment of inflammation, one pathway by which the non-steroidal anti-inflammatory drugs aspirin and sodium salicylate exert their effects is by inhibiting the binding of ATP to the kinase IKK-β, which in turn prevents the phosphorylation (and ultimately the degradation) of the cytoplasmic sequestering protein of the transcription factors NF-KB (50). And finally, the HDAC inhibitor SAHA was approved by the FDA in October 2006 for the treatment of cutaneous T-cell lymphoma, and at least 10 more compounds are currently in clinical trials in hopes of finding inhibitors for market against every major tumor type (51).

Targeting the transcript of a particular gene using the related methods of RNAi and antisense is another powerful strategy for manipulating expression. Currently, two siRNAs that target the VEGF transcript are in clinical trials for the treatment of macular degeneration, as well as one for the treatment of respiratory syncytial virus infection. Although many antisense oligonucleotides are still in various phases of clinical trials, to date only fomivirsen has been approved for the treatment of cytomegalovirus retinitis (52). One disadvantage of this strategy is that by targeting the transcript rather than a sequence within the genome, these methods usually require higher concentrations to produce an efficient outcome (23).

Future Directions

Tremendous progress has been made toward developing activator ATFs as therapeutic agents for the treatment of a variety of disorders. Indeed, ZF-based ATFs are currently in clinical trials (http://www.sangamo.com/index.php) and a purely synthetic activator that upregulates the transcript levels of endogenous genes in cell culture has been developed. The next frontier is the incorporation of additional functionality into activator ATFs that would confer properties such as temporal control and tissue specificity analogous to endogenous activators. The three-hybrid approach in which the TAD and the DBD associate noncovalently only in the presence of a small molecule was the first demonstration that temporal control could be engineered in an ATF, and this pioneering approach has been used in a variety of contexts (53). For entirely synthetic ATFs, one recently reported strategy exploits conformational entropy to create protein-DNA dimerizers that are inactivated at elevated temperatures but function well at a lowered temperature (54). In all examples, delivery of the molecules to the appropriate tissues and to the nuclei of those tissues remains an open challenge. Creation of a fully functional activator ATF will thus stimulate advances on a variety of scientific fronts.

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Further Reading

Ptashne M, Gann A. Genes & Signals. 2002. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Gossen M, Kaufmann J, Triezenberg SJ. Transcription Factors (Handbook of Experimental Pharmacology). 2004. Springer, Berlin.

See Also

Gene Therapy and Cell Therapy

Peptide Nucleic Acids

Protein-Nucleic Acid Interactions

Protein-Protein Interactions

Small Molecules to Disrupt Protein-Protein Interactions

Transcription Factors

Transcription, Activators and Repressors of

Transcription, Initiation of