BCIII-08_SN2024_Handouts PDF Lecture Notes

Transcription regulation https://dav.lbl.gov/archive/Events/SC08/Drosophila/ Biochemie III DCBP UNIVERSITÄT BERN Sofia Nasif [email protected]  Module Date Topic...

Transcription regulation https://dav.lbl.gov/archive/Events/SC08/Drosophila/ Biochemie III DCBP UNIVERSITÄT BERN Sofia Nasif [email protected]  Module Date Topic Lecturer 16.09.24 The role of proteins in DNA and RNA metabolism. DNA Structure Evan Karousis 23.09.24 DNA damage, mutations. DNA Repair Evan Karousis Genes and 30.09.24 Chromatin Structure Sofia Nasif chromosomes 07.10.24 DNA Replication Sofia Nasif 14.10.24 DNA Recombination Evan Karousis RNA Polymerase: Structure and Function 21.10.24 Sofia Nasif Prokaryotic and eukaryotic transcription, including control sequences Transcription: from Transcriptional Regulation in Prokaryotes DNA to RNA 28.10.24 Sofia Nasif Transcriptional Regulation in Eukaryotes (Part 1) 04.11.24 Transcriptional regulation in eukaryotes (Part 2) Sofia Nasif 11.11.24 Eukaryotic mRNA splicing, capping, polyadenylation (Part 1) Oliver Mühlemann Post- Eukaryotic mRNA splicing, capping polyadenylation (Part 2) 18.11.24 Oliver Mühlemann transcriptional RNA Editing RNA processing Elements of RNA degradation: mRNA degradation (general, small RNAs) 25.11.24 Oliver Mühlemann rRNA and tRNA processing Translation I: Elements of protein synthesis 02.12.24 Evan Karousis Structure of tRNAs and ribosomes Translation II: Initiation, Elongation and Termination Protein synthesis 09.12.24 Translation Regulation Evan Karousis Translation-dependent mRNA degradation 16.12.24 Gene expression regulation, examples from health and disease Evan Karousis Final exam 13.01.25  Learning outcomes 1. List the cis-and trans-acting regulators required for eukaryotic transcription and order the series of events required for transcription initiation at eukaryotic promoters. 2. Explain gene reporter assays and design experiments to analyze cis- and trans- acting regulatory elements. 3. Explain how cellular asymmetry can be achieved. 4. Explain the concept of “combinatorial control” and describe examples.  Transcriptional ground state The default transcriptional state of a gene, on or off, is dictated in part by the size and complexity of the genome. ✓ In bacteria, where genomes are relatively small and DNA is readily accessible, the default state of genes is generally “on”. Transcription of each gene or gene cluster is usually limited by a specific protein repressor. ✓ In eukaryotes, where genomes are larger and genes are encapsulated in chromatin, the default state of most genes is “off”. Gene transcription requires chromatin modification followed by the action of transcription activators. A typical eukaryotic promoter Transcription activators bind to: Enhancers (in higher eukaryotes) Upstream activator sequences (UAS) in yeast The promoter regions of multicellular organisms, such as mammals, contain more control elements than those of unicellular eukaryotes, such as yeast. This reflects the need in higher eukaryotes for changes in gene expression during development and for intercellular communication. Pol II Holoenzyme Promoter Binding Requires Five Types of Proteins 1. Transcription activators = bind to enhancers or UASs and facilitate transcription 2. Architectural regulators = facilitate DNA looping 3. Chromatin modification and remodeling proteins = change chromatin structure, to make promoters accessible to the transcription machinery 4. Coactivators = facilitate communication between activators and the complex of Pol II and general transcription factors play a direct role in assembly of the preinitiation complex (PIC) 5. General transcription factors (basal transcription factors)= required at every Pol II promoter  Transcription Activators (bind to enhancers or UASs and facilitate transcription) Activator requirements vary greatly between promoters: – may activate transcription at 100s of promoters or be specific for a few promoters – may be sensitive to signal molecule binding – may bind to enhancers that are distant from the TATA box – may bind to both DNA and RNA – bind DNA in a sequence specific manner (motifs: HTH, homedomain, Zn fingers, etc) – function may be affected by lncRNAs The DNA binding site for regulatory proteins are often short inverted nucleotide repeats where multiple (usually two) subunits of the regulatory protein bind cooperatively.  Architectural Regulators: high mobility group (HMG) proteins Architectural regulators = facilitate DNA looping to allow activators to function at a distance – Nonhistone proteins – abundant in chromatin – bind to DNA with limited specificity high mobility group (HMG) proteins = play an important structural role in chromatin remodeling and transcriptional activation  Chromatin modification and remodeling proteins change chromatin structure, to make promoters accessible to the transcription machinery Chromatin remodeling complexes Histone movement/replacement (enzymes that require ATP) Histone modifying enzymes Histone modifications  Coactivator Protein Complexes (facilitate communication between activators and the complex of Pol II and general transcription factors) Mediator complex = a major eukaryotic coactivator – consisting of 25 (yeast) to 30 (human) polypeptides, many of which are highly conserved – binds to the CTD of the largest subunit of RNAP – required for basal and regulated transcription – facilitates the effects of transcription activators on Pol II stimulates phosphorylation of the Pol II CTD by TFIIH Keep in mind: also, corepressors exist!  TATA-Binding Protein and Basal Transcription Factors required for recruiting RNApol II to promoter regions TATA-binding protein (TBP) = the first component to bind in the assembly of a preinitiation complex (PIC) at the TATA box of a typical Pol II promoter TBP is delivered as part of the TFIID complex. basal transcription factors (TFIIB, TFIIE, TFIIF, TFIIH) make up the minimal PIC Sequence of transcriptional activation events 1. activators bind the DNA first 2. activators recruit remodeling complexes and a coactivator (Mediator) 3. Mediator facilitates the binding of additional required components 4. phosphorylation of the CTD of Pol II leads to transcription initiation  Insulators Insulators = DNA sequences that form boundaries between genes or groups of genes in eukaryotes. They prevent inappropriate cross-signaling between regulatory elements of different genes. DNA sequence (cis-acting) Protein (trans-acting) CTCF= CTC-binding factor 11 Zn fingers  Regulation of Transcription of GAL Genes in Yeast GAL genes have similar promoters Allows coordinated gene expression and regulatory sequences mediated by a common set of proteins Regulation of Transcription of GAL Genes in Yeast Regulation of Transcription of GAL Genes in Yeast Gal4p recruits additional factors: – SAGA complex = involved in histone acetylation and chromatin remodeling – SWI/SNF complex = involved in chromatin remodeling – Mediator Discovering and analyzing regulatory DNA sequences: reporter gene assays The modular nature of gene regulatory proteins: deletion analysis  Nuclear receptor proteins respond to effector molecules that diffuse through the membrane Patterns of gene cellular expression are constantly changing in response to developmental needs and environmental cues Signal cascades are often the result of changing conditions, with transcriptional changes as one of the outcomes Nuclear receptor proteins respond specifically to effectors (such as sex hormones). Nuclear receptors have a DNA binding domain and a ligand binding domain Binding a ligand (like a hormone) induces a conformational change in the nuclear receptor, allowing it to recruit different co-repressors or co-activators (a) More complex networks can be involved. A nuclear receptor outside the nucleus may only be able to enter the nucleus Figure 09-35 Craig et al. Molecular Biology when bound to ligand (b)  Protein-Protein Interactions in Eukaryotic Regulatory Proteins: combinatorial control Eukaryotes have a larger number of genes than activators – yeast have 300 transcription factors for 1000s of genes Most genes are subject to regulation by multiple transcription factors: successful gene activation occurs only when all the factors are bound at their individual sites Combinatorial control = the use of specific combinations of a limited number of regulatory proteins to exert fine control over gene expression. Protein-Protein Interactions in Eukaryotic Regulatory Proteins: combinatorial control  Development depends on asymmetric cell divisions and cell-cell signaling Symmetric division yields identical daughter cells that may have different fates if exposed to different external signals. Asymmetric cell division yields two different types of daughter cells with different fates. It initiates in the form of gradients of mRNA and proteins: a) local translation or b) active transport of molecules at one part of the cell. After gradient formation the mitotic spindle must be aligned properly to the axis of the gradient Combinatorial control of gene expression in D. Melanogaster Cascades of regulatory proteins in development https://www.macmillanhighered.com/BrainHoney/Resource/6716/digital_first_content/trunk/test/hillis2e/asset/img_ch14/c14_fig13.html  Combinatorial control of gene expression in D. Melanogaster Nurse cells deposit some maternal mRNAs in the egg asymmetrically: Bicoid: anterior determinant (transcriptional activator AND a translational repressor) Nanos: posterior determinant (translational repressor) Histone-RFP Bicoid-GFP  Combinatorial control of gene expression in D. Melanogaster Some maternal mRNAs are deposited uniformly throughout the egg cytoplasm: Pumilio, Hunchback and Caudal. Gradients of Bicoid and Nanos proteins lead to accumulation of Hunchback protein in the egg’s anterior region and Caudal protein in its posterior. Coordination of transcription and translation regulatory programs Combinatorial control of gene expression in D. Melanogaster: eve Eve expression The eve gene (pair-rule gene) produces a transcription factor, called even-skipped Even-skipped is expressed in a series of seven stripes along the length of the embryo Eve is essential for embryonic development Combinatorial control of gene expression in D. Melanogaster: eve Biette, Kelly Marie. 2019. Enhancer Interactions in Developmental Gene Regulation. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Combinatorial control of gene expression in D. Melanogaster: eve Literature Nelson & Cox. Lehninger Principles of Biochemistry, 8th edition. W.H. Freeman, chapter 28 Cox, Doudna & O’Donnell. Molecular Biology. Principles and Practice. 2nd edition. Macmillan Education, chapters 19, 21 and 22.