BIOL10221 Molecular Biology Lecture Notes PDF

BIOL10221 “Molecular Biology” Dr David Boam Module 5 - Gene expression in Eukaryotes Lecture 5/1 transcriptional regulation in eukaryotes  Aims of this lecture Aims General description of transcription activation mechanisms Describe...

BIOL10221 “Molecular Biology” Dr David Boam Module 5 - Gene expression in Eukaryotes Lecture 5/1 transcriptional regulation in eukaryotes  Aims of this lecture Aims General description of transcription activation mechanisms Describe and explain the role of chromatin in transcription activation Demonstrate how intermolecular interactions play a key role in gene regulation To describe how extracellular stimuli change gene transcription Lecture 5/1 transcriptional regulation in eukaryotes  Transcriptional regulation in eukaryotes Chromatin structure Transcriptional regulation – What are transcription factors? – Mechanisms of activation Keywords Histone, nucleosome, chromatin, histone acetyltransferase (HAT), adaptor, mediator Lecture 5/1 transcriptional regulation in eukaryotes  Transcriptional regulation in eukaryotes Eukaryotic DNA is highly organised into chromatin……. So how does the transcription apparatus gain access genes to transcribe them? Lecture 5/1 transcriptional regulation in eukaryotes DNase hypersensitivity and gene activation tissue not tissue expressing gene of interest expressing gene of interest X extract DNA, digest with DNAase Implication : nucleosomes are cleared from active chromatin – either sliding or “eviction” Lecture 5/1 transcriptional regulation in eukaryotes  Histone structure non-polar globular domain Histone ++++ +++++++++ fold N-terminal C-terminal variable basic variable basic arm arm  highly conserved in eukaryotes.  high proportion of basic amino acids (lys and arg)  General structure is very similar  Lecture 5/1 transcriptional regulation in eukaryotes Crystal structure of a nucleosome Lecture 5/1 transcriptional regulation in eukaryotes  Post-translational modification of histones Methylation -CH3 groups added Gene activation to lys and his and repression, DNA replication. Acetylation Addition of CH3CO2- nucleosome group to lysines of assembly; gene core histones activation Phosphorylation Addition of PO4- to cell division, lysines of H1 transcription, chromatin structure. Lecture 5/1 transcriptional regulation in eukaryotes  A class II regulatory region enhancer 0 to 10kb promoter TATA (TATAAA)  Multiple binding sites for sequence-specific transcription factors  Position and  Co-operate and interact with orientation polymerase independent Bipartite: Transcription activation domain DNA binding domain Lecture 5/1 transcriptional regulation in eukaryotes  Transcriptional stimulation in eukaryotes general factors/TFIID etc RNA polymerase Histone acetylase (HAT) “Mediator” complex adaptors ATP-dependent chromatin modification complexes Long distance interactions: DNA bending/looping Recruitment of general (basal) factors/polymerase Recruitment/co-recruitment of HATs and chromatin Lecture 5/1 transcriptional modifier complexesregulation in eukaryotes  Signal transduction pathways which result in phosphorylation of transcription factors at plasma Ligand-receptor interaction membrane Generation of 2nd messenger small molecule (e.g. cAMP) Activation of protein kinases Regulation of transcription factor activity Charge, structure, protein-protein interactions DNA binding, activation, nuclear localisation, degradation Lecture 5/1 transcriptional regulation in eukaryotes Activation of transcription by signal transduction Gene activation in response to viral infection ligand binds to receptor, causes activation of transcription factor which binds to enhancer and activates transcription Lecture 5/1 transcriptional regulation in eukaryotes  Activation of transcription by signal transduction CREB – gene activation by cyclic AMP Hormone-receptor ATP cAMP ATP cAMP Protein kinase A Protein kinase A CREB CBP P CBP CREB= Cyclic AMP Regulatory Element Binding factor CREB CBP = CREB-Binding Protein Histone acetylation Lecture 5/1 transcriptional regulation in eukaryotes  Nuclear hormone receptors Large superfamily Estrogens of ligand- Androgens dependent Corticosteroids transcription Retinoids factors/receptors Thyroid hormone Sub-families Vitamin D Homodimers Ecdysone Heterodimers Fatty acids monomers Prostaglandins Lecture 5/1 transcriptional regulation in eukaryotes  Nuclear hormone receptors Hormone dependent gene activation Hydrophobic steroid hormone diffuses through plasma membrane Binds receptor Receptor translocates to nucleus to activate transcription Lecture 5/1 transcriptional regulation in eukaryotes C4 zinc fingers - nuclear hormone receptor family Lecture 5/1 transcriptional regulation in eukaryotes  Keywords/concepts Chromatin structure must be modified for transcription factor DNA-protein interactions access protein-protein Promoters and interactions enhancers bind factors co-activators/co- which repressors – Recruit polymerase – Modify chromatin Diverse signalling pathways control transcription factor/gene activity Lecture 5/1 transcriptional regulation in eukaryotes  Learning outcomes for this lecture Define and describe the functional components of a typical eukaryotic transcription regulatory DNA sequence Understand structure and bipartite nature of sequence-specific transcription factors Understand the role of chromatin and the basal transcription apparatus in activating transcription Understand how signal transduction pathways allow an extracellular stimulus to selectively modulate gene transcription in different model systems – Nuclear hormone receptors – Regulation by protein phosphorylation Lecture 5/1 transcriptional regulation in eukaryotes