Lecture 2 - Eukaryotic Gene Expression - Polymerases and Promoters

Lecture 2 Overview of eukaryotic gene expression. Eukaryotic RNA polymerases and gene promoters 1 8.2 Overview of Eukaryotic Gene Control The primary purpose of gene control in multicellular organisms is the execution of precise developmental an...

Lecture 2 Overview of eukaryotic gene expression. Eukaryotic RNA polymerases and gene promoters 1 8.2 Overview of Eukaryotic Gene Control The primary purpose of gene control in multicellular organisms is the execution of precise developmental and tissue-specific programs so that the proper genes are expressed in the proper cells at the proper times during embryonic development and cellular differentiation. Before coming to class: Watch animation: Eukaryotic Transcriptional Control Pay attention to the multiple factors that regulate the transcription of eukaryotic genes Go back to this animation after each class of our first four classes 3  We will be coming back to this figure many times. By the end of class #4 you Chromatic should understand every condensation detail in this figure. inactivates gene transcription READ THE FIGURE LEGEND Figure 8-3 Differences between prokaryotes and eukaryotes: Transcription in eukaryotes takes place on DNA that is wrapped in chromatin. Chromatin needs to open for a gene to be activated and transcription to proceed. Chromatin- mediated regulation is an entirely new mechanism as compared to prokaryotes. It is often called EPIGENETIC REGULATION of gene expression. 5  You are familiar with this terminology from MCB*1090: Euchromatin and Heterochromatin Heterochromatin: (‘hetero’ = different) - regions of chromosomes that are intensely stained - DNA is more densely packed - rich in repetitive DNA (Transposons, centromeres and telomeres) - not accessible to transcriptional machinery - Inactive genes are found in heterochromatin Euchromatin: (‘eu’ = true) - lightly stained chromosome regions - Active genes are found in euchromatin - accessible to transcriptional machinery 6 Very elaborate transcriptional control Once the gene is in open chromatin, a very elaborate system and a variety of factors regulate the expression of each individual gene: far more complicated as compared to prokaryotes 7  Gene regulation in eukaryotes: The three RNA polymerases We will start with description of the RNA polymerases in eukaryotes In Lectures 2 and 3 we will study the structure of eukaryotic promoters and the factors (called General Transcription Factors) that load the polymerases to the promoters In lecture 3 we will also study various techniques used to analyse promoters and their association with transcription factors. 8  From MBG*2040 you know that Eukaryotes have three RNA polymerases: – RNA polymerase I synthesizes only pre-rRNA. – RNA polymerase II synthesizes mRNAs, some of the small nuclear RNAs that participate in mRNA splicing, and micro- and small interfering RNAs (miRNAs and siRNAs) that regulate the translation and stability of mRNAs. – RNA polymerase III synthesizes tRNAs, 5S rRNA, and several other small stable RNAs. 9  TABLE 8-1: Classes of RNA Transcribed by the Three Eukaryotic Nuclear RNA Polymerases and Their Functions Polymerase RNA Transcribed RNA Function Pre-rRNA (28S, 18S, Ribosome components, protein RNA polymerase I 5.8S rRNAs) synthesis RNA polymerase II mRNA Encodes protein snRNAs RNA splicing Chromatin-mediated repression, siRNAs translation control miRNAs Translation control RNA polymerase III tRNAs Protein synthesis Ribosome component, protein 5S rRNA synthesis snRNA U6 RNA splicing Signal recognition particle for 7S RNA insertion of polypeptides into the You need to memorize endoplasmic reticulum this table !! Other small stable Various functions, unknown for RNAs many Similarities between the RNA polymerases (guide to read the textbook) The crystal structure of the yeast RNA pol II has been resolved at a high resolution (Nobel prize) RNA pol II consists of 12 polypeptides, called RPB1, RPB2…RPB12 All other eukaryotic RNA polymerases share very high level of homology with the yeast RNA pol II You do not need to know details of the structures You need to know that a CLAMP DOMAIN in the polymerase (RPB1) accommodates DNA After positioning over DNA the clamp is closed by a bridge The synthesis of RNA takes place in the catalytic center with the participation of Mg++ The synthesized RNA exits through a ”channel” and is immediately capped by 7m Guanosine. 11 Eukaryotic polymerases are complexes of multiple polypeptides. Note the similarity between the prokaryotic β, β’ subunits and the eukaryotic RPB1, RPB2 Note the structural similarity of the enzymes You do not need to memorize the details about the other subunits or the structures you see Figure 8-7 RNA polymerases are complex molecular machines. Please note: - The clamp and the bridge - The position of DNA in the transcribing polymerase - The channel through which the transcribed RNA is extruded from the complex - The nascent RNA will be processed as you have studied in MBG*2040 Figure 8-9 14  Eukaryotic RNA polymerase II contains a unique CARBOXY-TERMINAL DOMAIN (CTD) of its RPB1 subunit (check for details in the online animation). This is a specialized domain not found in any other polymerase, prokaryotic or eukaryotic. The CTD is involved in multiple regulatory interactions and plays a key role in the initiation, release, elongation and processing of the synthesized mRNAs The CTD in yeast contains of 26 repeats of Tyr-Ser-Pro-Thr-Ser-Pro-Ser – in mammals it contains 52 repeats The Ser residues in the CTD are phosphorylated upon transition from initiation to elongation The CTD is not structured. It is not shown on the previous two or three slides because it can not be analysed by crystallography!! 15  Figure 8-10 CTD of RNA pol II is phosphorylated during in vivo transcription 8.3 RNA Polymerase II Promoters and General Transcription Factors 17  The RNA pol II – transcribed genes are regulated by: o CONSERVED BASAL PROMOTER ELEMENTS, ALSO CALLED CORE PROMOTER SEQUENCES o Promoter-proximal binding sites for transcriptional activators o Distal enhancers or repressors o Chromatin structure We will first get familiar with the CORE PROMOTER SEQUENCES and the proteins that bind to them 18 Positions of Core Promoter Elements Figure 8-11 A+1 = base where transcription starts; Y = Pyrimidine (C or T); N = any base Core promoter sequences in eukaryotic DNA: Transcription starts at a defined point called initiation site Usually A on the coding strand. Four elements direct the positioning of the polymerase at these promoters: TATA box – a tight consensus sequence o prevalent in highly transcribed genes Initiator – less conserved element o some genes contain Initiator but no TATA BRE (TFIIB Recognition Element) and/or o influence the activity of the promoter DPE (Downstream Promoter Element) o influence the activity of the promoter 20 - RNA polymerases must recognize the promoter and correctly INITIATE transcription at a very specific position - They can not do this alone!!! - Several GENERAL POLII TRASCRIPTION FACTORS (GTFs) assemble the so called preinitiation complex over the CORE PROMOTER SEQUENCES - Other factors (DNA helicase) help the polymerase initiate transcription - Other factors (protein kinase) release the polymerase - Other factors help the polymerase elongate - Other factors move nucleosomes out of the way Yankulov_MCB2050_lecture panel 1 21 General Transcription Factors of RNA polymerase II RNA pol I GTFs are labeled as TFI: TFIA, TFIB RNA pol II GTFs are labeled as TFII: TFIIA, TFIIB, TFIID, TFIIE, TFIIH RNA pol III GTFs are labeled as TFIII: TFIIIB, TFIIIS 22  In Lecture 4 we will study how promotor elements and basal transcription factors interact to recruit and load RNA polymerases to promoters In lecture 3 we will focus on the techniques used to study these interaction 23