There are two types of TSS proximal signals in DNA that control transcription in eukaryotic cells. One of these, the promoter, defines where transcription initiation will occur on the DNA template; the other set of DNA signals serve to either stimulate and repress transcription, and thus contribute to the control of how frequently transcription occurs. For example, in the thymidine kinase (tk) gene of the herpes simplex virus (HSV), which utilizes transcription factors of its mammalian host for its early gene expression program, there is a single unique TSS, and accurate transcription initiation from this site depends on a nucleotide sequence located about 25 nucleotides upstream from the start site (ie, at −25) (Figure 1). This region has the sequence of TATAAAAG and bears remarkable similarity to the functionally related TATA box that is located about 10-bp upstream from the prokaryotic RNA TSS. Mutation of the TATA box markedly reduces transcription of the HSV tk gene. Other cellular genes contain this consensus cis-active element (Figures 1 and 2). The TATA box is located 25 bp upstream from the TSS in mammalian genes that contain it. The consensus sequence for a TATA box is TATAAA, though numerous variations have been characterized. The human TATA box is bound by the 34-kDa TATA-binding protein (TBP), a subunit in an essential multi subunit complex termed TFIID. The non-TBP subunits of TFIID are proteins called TBP-associated factors (TAFs). Binding of the TBP-TAF TFIID complex to the TATA box sequence is thought to represent a first step in the formation of the transcription complex on the promoter.

Fig1. Transcription elements and binding factors in the herpes simplex virus thymidine kinase (tk) gene. DNA-dependent RNA polymerase II (not shown) binds to the region encompassing the TATA box (which is shown here bound by transcription factorTFIID) and TSS at +1 (see also Figure 3) to form a multicomponent PIC capable of initiating transcription at a single nucleotide (+1 TSS). The frequency of this event is increased by the presence of upstream cis-acting elements (the GC and CAAT boxes) located either near to the promoter (promoter proximal) or distant from the promoter (distal elements; see Figure 2). Proximal and distal DNA cis-elements are bound by trans-acting transcriptional activating factors, in this example Sp1 and CTF (also called C/EBP, NF1, NFY) bound to proximal elements. Most proximal and distal cis-elements can function independently of orientation (arrows; see also Figure 2).

Fig2. Schematic showing the transcription control regions in a hypothetical mRNA-producing eukaryotic gene transcribed by RNA polymerase II. Such a gene can be divided into its coding and regulatory regions, as defined by the transcription start site (arrow; +1). The coding region contains the DNA sequence that is transcribed into mRNA, which is ultimately translated into protein, typically after extensive mRNA processing via splicing. The regulatory region consists of two classes of elements. One is responsible for ensuring basal expression. The “promoter,” often composed of the TATA box and/or Inr and/or DPE elements, directs the RNA polymerase II transcription machinery to the correct site (fidelity). However, in certain genes that lack a consensus TATA, the so-called TATA-less promoters, an initiator (Inr) and/or DPE element(s) may direct the polymerase to this site. Another component, the upstream elements, specifies the frequency of initiation; such elements can either be proximal (50–200 bp) or distal (1000–105 bp) to the promoter as shown. Among the best studied of the proximal elements is the CAAT box, but several other elements (bound by the transactivator proteins Sp1, NF1, AP1, etc.) may be used in various genes. The distal elements enhance or repress expression, several of which mediate the response to various signals, including hormones, heat shock, heavy metals, and chemicals. Tissue-specific expression also involves specific sequences of this sort. The orientation dependence of all the elements is indicated by the arrows within the boxes. For example, the proximal promoter elements (TATA box, INR, DPE) must be in the 5′→3′ orientation, while the proximal upstream elements often work best in the 5′→3′ orientation but, most can be reversed. The locations of some elements are not fixed with respect to the transcription start site. Indeed, some elements responsible for regulated expression can be located interspersed with the upstream elements or can be located downstream from the start site, within, or even downstream of the regulated gene itself.

A large number of eukaryotic mRNA-encoding genes lack a consensus TATA box. In such instances, additional DNA cis-elements, an initiator (Inr) sequence and/or the down stream promoter element (DPE) among others, direct the RNA polymerase II transcription machinery to the promoter and serve to direct RNA pol II to start transcription from the correct site. The hexameric Inr element spans the start site (from −3 to +5) and consists of the general consensus sequence TCA+1 g/t T t/c (A+1 indicates the first nucleotide transcribed, ie, TSS). The proteins that bind to Inr in order to direct pol II binding include TFIID. Promoters that have both a TATA box and an Inr may be “stronger,” or more frequently transcribed, than those that have just one of these elements. The DPE has the consensus sequence a/gGa/tCGTG and is localized about 25-bp downstream of the +1 TSS. Like the Inr, DPE sequences are also bound by the TAF subunits of TFIID. In a survey of hundreds of thousands of eukaryotic protein coding genes, roughly 30% contained a TATA box and Inr, 25% contained Inr and DPE, 15% contained all three elements, whereas ~30% contained just the Inr.

Sequences generally, though not always, just upstream from the start site contribute importantly to how frequently transcription occurs. Not surprisingly, mutations in these regions reduce the frequency of transcription initiation 10-fold to 20-fold. Typical of these DNA elements are the GC and CAAT boxes, so named because of the DNA sequences involved. As illustrated in Figure 36–7, each of these DNA elements are bound by a specific protein, Sp1 in the case of the GC box and CTF by the CAAT box; both bind through their distinct DNA-binding domains (DBD). The frequency of transcription initiation is a consequence of these protein-DNA interactions and complex interactions between particular domains of the transcription factors (distinct from the DBD domains—so-called activation domains; AD) and the rest of the transcription machinery (RNA polymerase II, the basal, or general factors, GTFs, TFIIA, B, D, E, F, H, and other coregulatory factors such as media tor, chromatin remodelers, and chromatin modifying factors). (Figures 3 and 4) The protein-DNA interactions at the TATA box involving RNA polymerase II and other components of the basal transcription machinery ensures the fidelity of initiation.

Fig3. The eukaryotic basal transcription complex. Formation of the basal transcription complex begins when TFIID binds, via its TATA binding protein (TBP) subunit and several of its 14 TBP-associated factor (TAF) subunits, to the TATA box. TFIID then directs the assembly of several other components by protein-DNA and protein–protein interactions: TFIIA, B, E, F, H, and polymerase II (pol II). The entire complex spans DNA from about positions −30 to +30 relative to the TSS at +1 (marked by bent arrow). The atomic level, X-ray–derived structures of RNA polymerase II alone and of the TBP subunit of TFIID bound to TATA promoter DNA in the presence of either TFIIB or TFIIA have all been solved at 3-Å resolution. The structures of mammalian and yeast basal transcription complexes (aka preinitiation complexes, or PICs) have also recently been determined at 10-Å resolution by electron microscopy. Thus, the molecular structures of the transcription machinery in action are beginning to be elucidated. Much of this structural information is consistent with the models presented here.

Fig4. Nucleosome covalent modifications, remodeling, and eviction by chromatin-active coregulators modulate PIC formation and transcription. Shown in (A), is an inactive mRNA encoding gene (see X over the +1 transcription start site/TSS) with a single dimeric transcription factor (Activator-1; violet ovals) bound to its cognate enhancer binding site (Activator-1). This particular enhancer element was nucleosome-free and hence available for interaction with its cognate activator binding protein. However, this gene is still inactive (X over TSS) due to the fact that a portion of its enhancer (in this illustration the enhancer is bipartite and composed of Activator-1 and Activator-2, DNA binding sites) and the promoter are covered by nucleosomes. Recall that nucleosomes have ~150 bp of DNA wound around the histone octamer. Hence, the single nucleosome over the promoter will occlude access of the transcription machinery (pol II + GTFs) to the TATA, Inr and/or DPE promoter elements. (B) Enhancer DNA-bound Activator-1 interacts with any of a number of distinct ATP-dependent chromatin remodelers and chromatin-modifying coregulator complexes. These coregulators together have the ability to both move, or remodel (ie, change the octameric histone content, and/or remove nucleosomes) through the action of various ATP-dependent remodelers as well as to covalently modify nucleosomal histones using intrinsic acetylases (HAT; resulting in acetylation [Ac]) and methylases (SET; resulting in methylation [Me], among other PTMs) carried by subunits of these complexes. (C) The resulting changes in nucleosome position and nucleosome occupancy (ie, nucleosomes −4, 0 and +1), composition (nucleosome −1 and nucleosome +2; replacement of nucleosomal H2A with histone H2AX[Z]) thus allows for the binding of the second Activator-2 dimer to Activator-2 DNA sequences, which ultimately allows for the binding of the transcription machinery (TFIIA, B, D, E, F, H; polymerase II and mediator) to the promoter (TATA-INR-DPE) and the formation of an active PIC, which leads to activated transcription (large arrow at TSS).

Together, the promoter plus promoter-proximal cis-active upstream elements confer fidelity and modulate the frequency of initiation on a gene respectively. The TATA box has a particularly rigid requirement for both position and orientation. As with bacterial promoters, single-base changes in any of these cis-elements can have dramatic effects on function by reducing the binding affinity of the cognate trans-factors (either TFIID/TBP or Sp1, CTF, and similar factors). The spacing of the TATA box, Inr, and DPE is also critical.

A third class of sequence elements also increase or decrease the rate of transcription of eukaryotic genes. These elements are called either enhancers or repressors/silencers, depending on how they affect transcription. They have been found in a variety of locations, both upstream and down stream of the TSS, and even within the transcribed protein coding portions of some genes. Enhancers and silencers can exert their effects when located thousands or even many tens of thousands of bases away from transcription units located on the same chromosome. Surprisingly, enhancers and silencers can function in an orientation-independent fashion. Literally, hundreds of these elements have been described. In some cases, the sequence requirements for binding are rigidly constrained; in others, considerable sequence variation is allowed. Some sequences bind only a single protein; however, the majority of these regulatory sequences are bound by several different proteins. communication between drastically separated regions of genomic chromatin regularly occur. Indeed, recent studies indicate that specific patterns of long-range chromatin–chromatin interactions play key roles in the regulation of gene transcription in eukaryotes. Such interactions are likely mediated through the actions of specific nucleosomal, specific chromosomal structural/regulatory proteins, as well as transfactor-transfactor interactions. Thus, interactions between the many transfactors binding to promoter distal and proximal cis-elements, regulate transcription in response to a vast array of biologic signals.

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مواضيع ذات صلة

Bacterial Promoters Are Relatively Simple

RNA Synthesis is A Cyclical Process that Involves RNA Chain Initiation, Elongation, & Termination

RNA is Synthesized From A DNA Template by RNA Polymerases

DNA & Chromosome Integrity Is Monitored Throughout the Cell Cycle

All Organisms Contain Elaborate Evolutionarily Conserved Mechanisms to Repair Damaged DNA

Micro-RNA and Long Noncoding RNA

Histone Organization

Noncoding DNA

DNA Synthesis Occurs During the S Phase of the Cell Cycle

Formation of Replication Bubbles

Initiation & Elongation of DNA Synthesis

The Exact Function of Much of the Mammalian Genome is Not Well Understood