Download Control of Gene Expression 3 - Dr. Kordula

Control of Gene Expression III Tomasz Kordula, Ph.D. Resource: Lehninger et al., Principles of Biochemistry, Chapter 28; Lodish et al., Molecular Cell Biology, Chapter 11. Learning Objectives: 1. Understand the global purpose of transcriptional control in multicellular organisms. 2. Identify and describe the general promoter elements near the transcription start site. 3. Understand the cell­specific promoter elements and enhancers and the action of their protein activating factors. 4. Understand the combinatorial complexity of transcriptional control. 3. Describe the steps in initiation by Pol II. 4. Know the role of chromatin remodeling in genetic expression. 5. Describe the activity of steroid hormones and nuclear receptors. I. Transcriptional Control in Eucaryotes – The primary purpose of this kind of control in multicellular organisms is to orchestrate the up and down modulation of specific genes during development and differentiation. Most eukaryotic genes are positively regulated. We will concentrate on Pol II genes since these are the controlled switches, or the finely­tuned elements, that dictate protein concentrations in different cell types. A. Some common, general promoter elements lie near the transcription start site. 1. The “TATA Box” has the consensus sequence TATAAA, is located at about ­25 to –30 and acts as an attractor and measuring device for Pol II. It is brought into play by TATA­binding protein (TBP) that affects polymerase II binding affinity and promoter strength. Its other role is to designate the start site for transcription initiation., based upon its positioning relative to +1. The TATA box is a prominent feature of rapidly transcribed genes. 2. The initiator is common in TATA­lacking genes and has the consensus C ­1 A +1 . It is thought that the initiator is also important in designating the transcription start site. However, mutation of this region around +1 can substantially alter promoter strength as well. 3. CG Island is located at –20 to –50 in lowly expressed, constitutive (housekeeping) genes that lack a TATA and initiator. The sequences vary in length, but are CG­rich compared to the bulk of the genome.
Figure 1. Taken from Lehninger et al. “Principles of Biochemistry” copyright © 2000 B. Other proximal promoter elements that are more cell­specific­ These sequences are 8­20 bp long and lie within 200 bp of the +1 site. They serve as binding sites for various cell­specific transactivator proteins that form part of the landing port for RNA polymerase II. These elements include, for example, the CCAAT Box (TGGN6GCCAA bound by CTF1), the GC box (GGGCGG bound by Sp1), the NRF1 sequence (a 12­bp palindrome bound by nuclear respiratory factor) and the yeast UASG (a 17­bp palindrome bound by the Gal4p). The protein transcription factors that have special affinity for these promoter elements typically have a DNA­binding domain and a second domain that is directly involved in transcription activation or in binding other transcription factors. Examples of the secondary domains are the acidic domain of Gal4p, the glutamine­rich domain of Sp1 and the proline­rich domain of CTF1. See Figure 3 below. C. Enhancers­ These DNA elements, located 200 bp to 50 kb from the +1, affect gene expression despite their distance from the promoter region. Enhancers can be located upstream, downstream, or perhaps in an intron and have been shown to work in either orientation. This long­distance effect suggests that the intervening DNA exists as a large loop that brings the enhancer into proximity with the promoter. This looping, or bending, is mediated by protein factors whose expression confers cell­type specificity for a particular enhancer. For example, the immunoglobulin enhancer is active only in B lymphocytes where the relevant protein factors are expressed. Enhancers become activated by the binding of often large numbers of protein factors (the enhancesome) that bend the DNA to maximize cooperative protein­ protein and protein­DNA interactions. Figure 2. Taken from Lodish et al. “Molecular Cell Biology” copyright © 2004
In Figure 2 (a) above, it is worth noting that a single gene may have multiple promoter elements, functioning coordinately, depending on the relative concentrations of transcription factors. This permits sensitive tuning of their expression. In Figure 3 below, the binding of multi­domain transactivating factors associated with their DNA elements is shown with the RNA Pol II complex at the initiation site. The proline­rich, the acidic, and the glutamine­rich activation domains of the factors are designated by PPP, ­­­, and QQQ, respectively. Figure 3. Taken from Lehninger et al. “Principles of Biochemistry” copyright © 2000 D. The combinatorial complexity of transcriptional control. The human genome encodes about 2000 transcription factors. Because of the (1) multiplicity of promoter elements, (2) the ability of the group of transcription factors to work cooperatively, (3) the possibility for various combinations of homodimers and heterodimers of transcription factors, and (4) the cooperative interactions between factors binding nearby or distal elements, the expression of the all the 30,000+ genes of the genome can be regulated. For example, consider that four similar, but not identical, transcription factor subunits can form a total of 10 different types of dimers (4 homodimers and 6 heterodimers). Each set of dimers might have different affinities for the same or different promoter elements, hence the breadth of their composite activities can be dramatically amplified and diversified. E. The assembly of the Pol II complex, and the steps of the initiation and elongation of transcription (Refer to Figure 4). The process starts with the binding of the TATA box by TATA binding protein (TBP), often with the help of TFIID and TFIIA, followed by TFIIB binding. A Pol II­TFIIF complex is guided to this partially decorated promoter by the TFIIF subunit, which has high affinity for the already­bound TFIIB and low affinity for non­promoter DNA. TFIIE and TFIIH complete the formation of the closed complex. The multifunctional TFIIH
(helicase + phosphorylating activity) goes into action to separate the template and phosphorylate the C terminus of the largest Pol II subunit. Transcription begins and the elongation complex emerges from the promoter site. Note that the phosphorylated C terminus is eventually dephosphorylated after release of the nascent transcript. Figure 4. Taken from Lehninger et al. “Principles of Biochemistry” copyright © 2005 F. The regulation of transcription factors and the activities of nuclear receptors (a superfamily). In general, the expression of a gene is controlled by the accessibility of the promoter, the presence of various DNA regulatory sequences, and the kinds and concentrations of transcription factors present. However the activation of the transcription factors can also depend on their response to intra­ and extracellular signaling molecules like growth factors and hormones. As an example, consider the nuclear receptor superfamily proteins that act as transcription activators, but only in response to the binding of some incoming ligand (e.g.; estrogen, progesterone, glucocorticoid, thyroxine, retinoic acid). The protein receptors exhibit common features that include a variable
transcription activation region, a DNA­binding domain, and a ligand binding domain. Figure 5. Taken from Lehninger et al. “Principles of Biochemistry” copyright © 2005 The DNA regulatory element bound by these receptors typically contains two adjacent 6­bp repeats (direct or inverted), and the receptor lies dormant until bound by the ligand (such as a lipid­soluble steroid hormone). The complex then assumes a conformation that promotes dimerization and the dimer translocates to the chromatin where it binds the appropriate DNA hormone response element, leading to transcription activation. See the cartoon Figure 6. Figure 6. Taken from Lodish et al. “Molecular Cell Biology” copyright © 2004