Eukaryotic Genetics and Expression PDF

11/28/20 Overview Gene Expression Overview BE301 Eukaryotic Genetics and Expression...

11/28/20 Overview Gene Expression Overview BE301 Eukaryotic Genetics and Expression - Promoters simplified. -cis-acting Vs trans-acting Factors. - Eukaryotes Vs Prokaryotes. Prof. Anne Parle-McDermott Topic 7 Overview: Eukaryotic Gene Expression Eukaryotic Transcription: Polymerases Eukaryotic Transcription: RNApol I, II and III 1 2 References Section 3.4 ‘Genes X’. Lewin. Freeman ‘Biological Science’ Cells of different type ‘Genes X’. Lewin. Chapter 20 ‘Eukaryotic Transcription’. have the same genome- so what makes them ‘ Human Molecular Genetics 4’ Strachan & Read Chapter 11 different? ‘Human Gene Expression’ 3 4 Promoters simplified. RNA polymerases: Prokaryotes Vs Eukaryotes Prokaryotes: The promoter of a gene is the region of DNA that -1 polymerase with several subunits: a, b, b’ and s or controls whether a gene is switched on or off. ‘specificity factors’. The promoter region can be hard to identify but the core - Prokaryotic RNA polymerase can recognise and bind its own promoter is usually upstream from the Transcription start promoter and initiate transcription on its own. site. Eukaryotes: more complex Alternative promoters often produce gene products that differ at the 5’ ends. (back to overlapping genes) -3 polymerases: I, II, III - Eukaryotic polymerases require transcription factors to bind the region surrounding the promoter before they can initiate transcription. 5 6 1  11/28/20 Bacterial Operons: Coordinate control of a number of Genes Euk. Promoters: Control of a Primary Gene Promoter is relatively simple. Promoter is complex: -Core promoter The lac operon includes cis-acting regulator elements -Distal promoter: enhancers can be upstream or and protein-coding structural genes downstream (and a good distance away). 7 8 Regulation of Transcription is Governed by 2 types of Factors: 1. Cis-acting: Encoded within the DNA sequence in the promoter / regulatory region of a given gene. The description cis-acting applies to any sequence of DNA that functions exclusively as a DNA sequence, affecting only the DNA to which it is physically linked. 2. Trans-acting: Gene products (often proteins) that interact with cis-acting sequences to control gene expression. Any gene product that is free to diffuse and find its target is described as trans-acting. 9 10 Potential regulatory points in eukaryotic gene Eukaryotic Transcription expression pathway Chromatin Structure Transcription control Processing & splicing of the pre-mRNA mRNA half-life (stability of mRNA) Translation (mRNA selection) Post-translational modification of the protein product DNA (gene) rearrangements -(immunoglobulin Chromatin must be in an open structure and nucleosomes genes) removed from the promoter region- we will come back to this in later. We will focus for now on what happens once the promoter is ‘chromatin-free’ and available for transcription. © Carol & Mike Werner/Visuals Unlimited 11 12 2  11/28/20 Three Eukaryotic RNA Eukaryotic RNA Polymerases Consist of Many Subunits Polymerases All have ~ 12 subunits and are complexes of ~500kD in size. Some subunits are common to all 3 RNApol. RNA polymerase I – nucleolus located, The 3 RNApol localised at different areas of the nucleus- corresponds with the genes that they transcribe. transcribes rRNA genes, 28S, 18S, 5.8S -RNApol I: Nucleolus. Most prominent activity rRNA. RNA polymerase II – transcribes protein -RNApol II: Nucleoplasm coding genes (mRNAs) & some snRNAs -RNApol III: Nucleoplasm. Minor activity. RNA polymerase III – transcribes small RNAs (tRNAs, 5S rRNA & some snRNAs) 13 14 Transcription Factors (TF)- Important for Efficient Gene Yeast Transcription Initiation in Eukaryotes RNApol II Transcription Factor Definition: Multiple repeats of 7 “Any Protein that is needed for initiation of transcription, amino acids. but is not itself part of RNApol.” Genes X Have DNA binding domains. Are considered trans-acting factors. 2 Broad Categories: (General) Basal TFs: to ensure localisation of RNApol at TSS i.e., in the core promoter. Form part of the Pre- initiation Complex. Basal transcription. Gene specific: Activated transcription. Usually consist of an activation domain and a DNA binding domain. RNA polymerase has >10 subunits RNApol II largest subunit= Carboxy-terminal domain (CTD) Most common DNA binding domains: Zinc Finger, Helix- Turn-Helix, Leucine zippers, High Mobility Group Box motif. 15 16 Revision Gene expression: some similarities between Prokaryotes & Eukaryotes but generally more complex in Euks. RNA polymerases in combination with cis-acting and trans- acting factors control gene expression. Region immediately upstream of TSS is usually the core promoter in Euks. but other regulatory binding sites can be long distances up- or down-stream. Eukaryotics have 3 different types of RNA polymerases and RNApol II is the one that transcribes ‘most’ genes, RNApol I (nucleolus)- mainly rRNA, RNApol III- least active. Transcription factors. 17 3