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Department
Biology
Course
BIOL 308
Professor
Dragana Miskovic
Semester
Summer

Description
N.B: Transcription initiation is controlled by 4 molecular mechanisms: a. Direct influence of TFs on GTFs. b. Chromatin remodelling caused by TFs. c. Regulation of concentration and activity of TFs by hormones and other signals. d. Protection of active gene promoters from methylation. 1. Describe the role of histone acetylation/deacetylation in regulation of transcription. Histones have N terminal tails of 20-40 AA which contain positively charged lysine groups. When Lysine is acetylated (by acetyle transferrase aka histone acetylases HATs), the positive charge is neutralized. Since DNA is negatively charged and the neutralization effect of the positive lysine is now lost, repulsion occurs leading to the formation of a less condensed chromatin (Euchromatin). Binding sites are now exposed and transcription is activated. (GTFs bind followed by RNAP) Deacetylation on the other hand, 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. N.B: Deacetylases are present in co-repressor complexes and coactivators have HAT 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 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. NOT IN NOTES: RSC is a 15-subunit complex with the capacity to remodel the structure of chromatin. It exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to change nucleosome structures. It is at least 10-fold more abundant than the SWI/SNF complex and is essential for mitotic growth. (wiki) 3. Describe an influence of activators and repressors on assembly of initiation complexes. ** Activators/repressors do not bind to promoters but enhancers/silencers. Initiation complexes (ex. TFIID and such) bind to these sequences rather than the promoter to stimulate/inhibit transcription. Competition, inhibition, direct repression. a. Repressors can inhibit gene activation by binding to a site that overlaps an activator binding site (repressor incapable of DNA binding replaces activator → inactive heterodimer) b. Repressor protein binds at site adjacent to activator site and interferes with AD of activator. c. Repressor binds upstream, interacts through mediator with GTFs to inhibit initiation. d. Corepressors recruited that alter nucleosomes in such a way (such as Deacetylation) as to inhibit transcription. 4. Explain the role of enchancesomes and architectural proteins in regulation of transcription initiation? Enhanceosomes are protein complexes that bind to the enhancers. They help initiate specific gene expression by binding tissue specific TFs, non-specific TFs and co- activators along with the GTFs. (combinatorial control) Enhanceosomes work together with TFs forming a DNA loop that helps improve the interaction between initiation complex at enhancer and RNAP at promoter. The looping works better if the distance between enhancer and core promoter isn’t too short. Architectural proteins bind to specific binding sites leading to a change in the shape of a DNA in a control region so other proteins can interact with each other and with GTFs to stimulate transcription. 5. Explain the role of mediators and insulators in regulation of transcription initiation. Mediators and insulators are types of TF. Mediators work with TF of enhancer and core promoter to form the initiator complex and to help RNAP be positioned correctly. Insulators define the effective range of transcription so that effects of silencers or enhancers apply only to the target section of DNA. Details: Mediator complexes help bridge different protein complexes necessary for the assembly of the pre-initiation complex (RNAP + GTFs) in some genes. Also, by allowing the binding of different activators, mediators control the rate of re-entry of RNAP II. (Slow re-entry or fast re-entry) If the enhancer is >50Kb away from the promoter, insulators are required to help control their activity. Insulators setup boundaries between DNA domains preventing activation/repression of genes that are close by but unrelated to that specific activator/repressor. They also prevent gene silencing by controlling the boundaries between Heterochromatin and Euchromatin. 6. What is the role of DNA methylation in regulation of transcription initiation? Both DNA and histones can be methylated leading to formation of inactive chromatin. Methylation leads to change in conformation which prevents binding of TFs to enhancer or promoter. Therefore, to initiate transcription, the promoter has to be un-methylated. However, an un-methylated promoter isn’t necessarily active. (other factors required) Demethylation causes cytosine to become thymine through DNA repair mech. 7. Describe how steroid hormones regulate transcription (one example is enough; remember: nuclear receptor could be in cytoplasm or in nucleus, already bound to DNA). ** Transcription of TF is under external signal regulation and such signal includes steroid. Receptors for steroid on nuclear receptors bind to the ligand and increase abundance of TF. Steroid control TF and TF control transcription. Details: Steroid hormones bind to steroid hormone receptors (aka nuclear receptors = TFs) forming a complex. The complex then binds to another copy of itself forming a homo-dimer. The dimer then binds (may be transported from the cytoplasm to the nucleus) to its response element on DNA which is part of the promoter. Binding of the complex activates or represses transcription. 8. What is (are) the role(s) of transcription factors during development? Different TFs are synthesized at different stages of development. These TFs help control transcription by forming mediators and transcription complexes that bind different GTFs and lead to a more stage specific gene transcription. Therefore, TFs help control the specificity of gene expression. (Cis elements are the same at all times) 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 transcriping, TBP allows the polymerase to bind to its promoter through: a. SL1 complex for RNAP I b. TFIID for RNAP II c. TFIIIB for RNAP III d. 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 help control how strongly RNAP binds to the promoter and the rate of transcription. #1 commitment factor (once TBP binds, trans 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 is a TF involved in gene expression in TATA-less promoters. They help bind TFIID to a promoter containing GC box. SL1 is a complex assembled by UBF – TBP + 3 RNAPI specific TAFs (TBP-associated factors). It is mainly responsible for initiating transcription and trans complex formation Both proteins help position RNAP at the start site of transcription, they both have TBP and TAF parts in common and both bind to upstream elements. Also, they both allow assembly of TAF. N.B: Each RNAP has different promoters, requires polymerase specific GTFs and recognizes different DNA control elements. RNAP I & III do not require ATP hydrolysis to initiate transcription . RNAPI RNAPII RNAPIII Single type of promoter: Discussed in 20-23. 3 types of promoters: 2 core promoter + upstream Include TATA box, Initiator internal (1 for tRNA and 1 control element (Inr), downstream for 5S rRNA) and an elements (DPE) and TF II upstream promoter for B recognition element snRNAs. (BRE) as well as regulatory elements. 11.What is unusual about type 1 and 2 promoters for RNAPIII polymerase? ** Type 1 and 2 are internal promoters that exist near structural genes. Their effect is minimal, the upstream elements are the ones whose change that control the efficiency of transcription. They also form part of the coding sequence. 12.Describe the mechanism of attenuation of the Trp operon. Explain the importance of this mechanism for a bacterium? Attenuation is premature termination of transcription. A Leader sequence is the sequence between the promoter-operator region and the CDS. It consists of a leader RNA (regions 1 & 2) and an attenuator (point of choice between elongation and termination- regions 3&4). Trp leader sequence contains 2 Trp residues. In excess Trp, nascent mRNA is quickly formed. Regions 1 and 2 may form a loop (without polyU) pausing transcription transiently only. Ribosome initiates translation at the start codon near the 5’end. The Trp leader peptide (contains 2 Trp residues) is translated easily due to the abundant Trp available. Finally, the loop involving regions 1 and 2 melts, a new loop involving the attenuator sequence (region 3 and 4) is formed. This loop includes a PolyU region and RNAP terminates transcription before reaching the Trp structural genes (Rho independent termination). This mechanism is important to save energy. If Trp is present in excess, formation of structural Trp genes will be a waste of resources and vice versa. N.B: the mechanism above only occurs in prokaryotes as it involved transcription and translation occurring at the same site which is only valid for prokaryotes. 13.Could you imagine a mechanism similar to the mechanism of attenuation of the Trp operon in Eukaryotes? Explain your reasoning. No, because transcription and translation are conducted at different parts in eukaryotes, mRNA must be modified (ex. spliced) before it can be translated and the slow mechanism (relative) of eukaryotes would not allow this process to be effective. 14.Describe two distinctly different ways in which the trp operon is controlled by the overall availability of tryptophan. ** 1. The mechanism described in 12. Also, in low Trp conditions, nascent mRNA is quickly formed. Ribosome initiates translation but stalls at Trp codons in region 1 until some Trp charged tRNA is formed. Region 1 can’t bind to region 2. However, region 2 forms a loop with region 3 (without polyU). RNAP continues transcription and passes the attenuator. Structural genes of Trp operon are expressed. 2. The aporepressor-corepressor mechanism: An aporepressor for Trp is transcriped by a separate promoter from that of the Trp structural gene. However, the aporepressor can’t bind to the operator of the Trp gene due to differences in conformation. In excess of Trp, Trp acts as a corepressor, binds to the aporepressor causing a change in its conformation and forming the repressor. The repressor has the correct conformation and can bind to the operator and inhibit further transcription. 15.Describe the mechanism responsible for shutdown of the trp operon when a plentiful supply of free tryptophan is available. Same as 14. Attenuation and repressor. 16.Describe the mechanism by which the leader-attenuator region fine tunes the extent of transcription of the structural genes in the trp operon when Trp is available (but not to the point to activate the repressor). When tryptophan is available, tRNA becomes more available as well. This influences the rate of leader sequence, which controls the loop formation. Thus, when there is tryptophan, the leader behaves in a way that creates region 3-4 loop to cause attenuation. 17.Describe rho-dependant transcription termination Rho is a hexameric RNA helicase. It has an RNA binding domain (N-term) and ATPase (C-term) activity. It binds to rut site (rho utilization site) on mRNA and tracks along the RNA until it catches up with the RNAP (requires hairpin or stem loop to stop RNAP until rho catches up). Rho unwinds DNA-RNA hybrid using ATP. mRNA, Rho and RNAP are all released. N.B: Rho has no affinity for RNAP 18.Describe rho-independent transcription termination They occur at self complementary GC rich sites (dyad symmetry). Transcription of these sites lead to the formation of a stem loop structure followed by series of U residues. The loop interacts with RNAP causing it to pause. The AU bonds at the 3’ end of the new RNA are very weak and melt during the RNAP pause on the loop. RNA chain is released and DNA strands re-anneal. RNAP dissociates from the DNA. 1. Describe the process of mRNA cleavage and poly-adenylation. (Don’t forget the role of CTD tail) – not on Version 2- Q19B replaces it. mRNA Cleavage: Group I and II are self-splicing introns. i.e. RNA functions as a ribozyme and splices itself. Some group II organellar introns code for proteins maturases which increases the rate of self-splicing by stabilizing 3D lariat conformation. Group III can’t splice thems
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