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BIOL 308
Dragana Miskovic

STUDY QUESTIONS: LEC 24-29 1. Describe the role of histone acetylation/deacetylation in regulation of transcription. The N terminal end of histones contain positive lysine groups that tightly bond with DNA and can either acteylate or deacetylate the histones. When acetylated, the positive charge on lysine is neutralized and eliminates interactions with DNA; chromatin is less condensed chromatin (euchromatin)and the promoter regions are accessible for transcription. The acetylation is catalyzed by histone acetyltransferases, also called HATs and this leads to activation of gene expression. Deacetylation (catalysed by deacetylases) increases the positive charge on the lysine residues leading to lesser repulsion between the DNA strands and therefore, a more condensed heterochromatin structure. Binding sites are now inaccessible for TFs and transcription is inhibited and this leads to repression of gene activity. 2. Describe the role of chromatin remodelling complexes in regulation of transcription. Histone acetylation isn’t sufficient for activation; nucleosomes are still intact and have to be repositioned to expose the promoter elements. This requires ATP and a remodelling complex such as the SWI/SNF complex of yeast and the RSC complex. SWI/SNF proteins and RSC remodelling complexes alter the structure of the nucleosomes core. The SWI proteins also move the nucleosomes on DNA resulting in exposing the enhancers. I.e. the SWI/SNF complex helps chromatin remodelling and removal of histone octamers from nucleosomes. 3. Describe an influence of activators and repressors on assembly of initiation complexes. Activators and repressors can help in recruiting coactivator or corepressor complexes that have histone acetylase or deacetylase activity. They also can act with each other and provide a form of fine tuning through their interactions; they can recruit GTFs by interacting with TAFs, etc. Certain TFs need co-activator proteins that do not contact DNA but connect TF with general transcription factors to assemble the initiation complex. Repressors can: - Bind to sites that overlap activator binding sites - Bind at site adjacent to activator site and interfere with activator. - Bind upstream; interact through mediator with GTFs to inhibit interactions - Recruit co-repressors that alter nucleosomes in such a way to inhibit transcription 4. Explain the role of enchancesomes and architectural proteins in regulation of transcription initiation? The combinations of transcription factors binding enhancers and the GTFs form different initiation complexes, or enhanceosomes, that provide combinatorial control of gene expression through the different combinations and concentrations of activators. This allows for specific gene expression in various tissues based on which TFs are present and in what concentration. Architectural proteins bind elements of the DNA and can change DNA shape to allow other TFs and GTFs to interact and affect transcription. Through this, architectural TFs can cause bending or looping of the DNA to stimulate transcription initiation. 5. Explain the role of mediators (or insulators – different question) in regulation of transcription initiation. Mediators: proteins that are not in direct contact with DNA, but links transcription factors with general transcription factors. Without them, transcription factors would have no effect on transcription regulation. Insulators: set up boundaries between DNA domains prevents activation/repression of genes close by but unrelated. Activators and repressors also prevents gene silencing (spreads chromatin modifications control boundaries between hetero and euchromatin different on different tissues) 6. What is the role of DNA methylation in regulation of transcription initiation? Methylation of DNA affects chromatin structure and is a sign of inactive chromatin (heterochromatin). The methylated CpGs is recognized by methyl-binding domain proteins (MBP) that can recruit histone-modification enzymes such as histone deacetylase (HDAC) and histone methyl transferase (HMT), and this leads to histone deactylation and methylation; and the local chromatin will be condensed. Extensive methylation near (or in) a gene sequence generally prevents (or stops) transcription of that gene. 7. Describe how TFs from nuclear receptors superfamily regulate transcription (one example is enough; remember: nuclear receptor could be in cytoplasm, bound to chaperone protein, or in nucleus, already bound to DNA). Transcription of many TF is under regulation of extracellular signals such as hormones. E.g., steroid hormones are small, lipid soluble and can cross the membrane. They bind transcription factors which are in the cytoplasm, bound to heat-shock proteins. The hsp is released, allowing the TF to enter the nucleus and bind a response element on DNA, influencing the transcription of genes. 8. What is (are) the role(s) of transcription factors during development? Regulation of transcription factors is highly/strictly regulated, there is a critical point at which transcription of genes that produce transcription factors begins dependent on where/when needed. Both accessible binding sites and respective binding proteins are necessary for transcription to occur e.g., in Drosophila development, maternal mRNA is already unevenly distributed in embryo when fertilized and cell division occurs, daughter cells inherit different maternal mRNAs. Different poles of the embryo will have different sets of proteins synthesized, resulting in differentiation. Therefore certain cells will have different sets of active proteins (some transcription factors cascade of regulation interactions starts at the very beginning of cell division). 9. Describe the role of TBP during transcription (think about promoters for all three eukaryotic RNAPs and TATA-less RNAPII promoters – how do RNAPs bind to them; also, think about coordination of activities of all three polymerases). TBP (TATA Binding Protein) is a positioning factor for all RNAP that helps initiate transcription. No matter which RNAP is transcribing, TBP allows the polymerase to bind to its promoter through: o SL1 complex for RNAP I o TFIID for RNAP II o TFIIIB for RNAP III o TATA-less promoters through use of TAFII150 and 250 for elements and through Sp1 for GC boxes. When these form the initiator complex, RNAP is recruited so that it may bind to the promoter correctly. TBP helps control how strongly RNAP binds to the promoter and the rate of transcription. #1 commitment factor (once TBP binds, transcription occurs). TBP also helps coordinate activities of all 3 polymerases by binding to other polymerase- specific factors. 10. What is the role of Sp1 protein? What is the role of SL1 protein? What do they have in common? Sp1 protein has a role in TATA-less promoters involving GC boxes – it binds GC boxes and interacts with TAFs which anchor TBP and allow TFIID binding to the promoter. SL1 protein is involved in RNAP I promoters – it is bound by UPE-bound UBF, and allows for RNAPI positioning at the core promoter. They are similar in that they do not bind TATA boxes, but instead bind to upstream elements and provide the basis for RNAP binding. 11. What is unusual about type 1 and 2 promoters for RNAPIII polymerase? These promoters are unusual because they contain promoter elements that are in the transcribed region (internal promoters). They have binding of TFIIIA or TFIIIC (for type 1 and type 2 respectively), which enables binding of TFIIIB, which in turn recruits RNAPIII. Type 3 promoters on the other hand have TATA boxes and do not need TFIIIA or TFIIIC. 12. Describe the mechanism of attenuation of the Trp operon. Explain the importance of this mechanism for a bacterium? Attenuation is the regulated, premature termination of transcription and does not involve Rho, which is usually required for transcription termination. It is at specific site: the attenuator site (DNA sequence where RNAP either terminates/continues transcription). It differs from normal termination, which occurs at the t-site a feedback mechanism that is more directly controlled by molecule of interest (enables the bacteria to conserve resources more strictly) mRNA of trp operon contains 4 regions: region 1 which synthesizes "leader peptide" which is Trp rich, region 2, region 3, and region 4 which is followed by a polyU region; the region 3 and 4 together are called the attenuator chain of events: attenuation (yes or no) depends on formation of the particular stem loop structure in leader sequence. Formation of the particular stem loop structure depends on rate of ribosomal translation of leader sequence mRNA (connection between transcription and translation they are going at the same time). The rate of ribosomal translation of leader sequence mRNA depends on the supply of tRNA for Trp. The supply of tRNA for Trp depends on amount of Trp present in cell. 13. Could you imagine a mechanism similar to the mechanism of attenuation of the Trp operon in Eukaryotes? Explain your reasoning. No, because the attenuation mechanism involves a formation of a stem loop structure that is created by DNA in transition between transcription and translation only in prokaryotes can transcription and translation work at the same time on the same mRNA in eukaryotes, transcription and translation work separately (nucleus vs. cytoplasm) 14. Describe two di
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