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Tutorial II Gene expression: Molecular mechanisms of gene transcription Piergiorgio Percipalle, PhD Program Control of gene transcription and RNA processing mRNA translation and protein synthesis KAROLINSKA INSTITUTET Department of Cell and Molecular Biology Medical Nobel Institute Gene transcription How do cells use information stored In the chromosomes? The central dogma of molecular biology Schematic representation of chromosome 2 from the fruit fly (D. Melanogaster) 1 DNA sequences are transcribed to RNA RNA synthesis Ribonucleic acid (RNA) Similarities between DNA and RNA Transkription Transkription The chemical structure in the RNA molecule A RNA is a linear polymer, just like DNA, consisting of4 Different nucleotides linked to each other through phosphodiester bonds Differences between DNA and RNA B In the RNAmolecule, nucleotides are called ”ribonucleotides” RNA contains the base ”uracil” instead of ”thymine”. Thymine can also base-pair with adenine Uracil base-pairs with adenine DNA have RNA have different ternary structures DNA is a duplex RNA is linear 2 There are several types of RNA Type of RNA mRNAs Function messanger-RNA, protin-coding rRNAs ribosomal-RNAs, ribosome structure tRNAs transfer-RNAs, protein synthesis snRNAs small nuclear RNAs, RNA processing snoRNAs small nuclear RNAs, rRNA processing Non-coding RNAs different cellular processes Transcription is performed by RNA polymerase How is a specific RNA produced form a template DNA? Transcription is a step-wise process Recognition of a specific DNA sequence: RNA synthesis starts Promoter RNA synthesis Recognition of a specific DNA sequence: RNA synthesis ends The transcription cycle Termination Terminator RNA polymerase Initiation Elongation 3 Direction of RNA polymerase Eukaryotes have three different RNA polymerases Type of polymerase Transcription along a chromosome The structure af RNA polymerase II is very conserved Genes which are transcribed RNA polymerase I rRNA genes RNA polymerase II all protein-coding genes RNA polymerase III tRNA genes In eukaryotic cells, RNA polymerase II transcription requires regulatory sequences on the DNA RNA polymerase II requires general transcription factors…….. UAS Transcription 4 RNA polymerase II general transcription factors Factor No of subunits TFIIA 2 TFIIB TFIID 1 TBP TAFs 1 14 TFIIF 3 TFIIE 2 TFIIH 10 Mediator Function Stabilizes TBP and TFIID DNA binding. Blocks transcription inhibitors. Positive and negative gene regulation Binds TBP, Pol II and promoter DNA. Helps fix transcription start site Binds TATA element and deforms promoter DNA. Platform for assemblyof TFIIB, TFIIA and TAFs. Binds INR and DPE promoter elements. Target of regulatory factors. Binds Pol II and is involved in Pol II recruitment to PIC and in open complex formation. Binds promoter near transcription start. May help open or stabilize the transcription bubble in the open complex. Functions in transcription and DNA repair. Kinase and two helicase activities. Essential for open complex formation. Mutations in IIH can cause disease. • Predominant role to enhance the PIC assembly, via pol II From Hahn, Nature Structural & Molecular Biology 2004 Formation of ribonucleoprotein (RNP) particles Chromatin structure Pre-mRNA DNA RNA polymerase II Transport facilitator Transport mediator Splicing factor Splicing repressor Translational regulator Daneholt, Proc. Natl. Acad. Sci. USA 2001 Chromatin structure modifications Chromatin and histone modifications as part of relay mechanisms Percipalle and Östlund Farrants, Curr Op Cell Biol 2006 5 Summary of the transcription initiation process in eukaryotes (Saccharomyces cerevisiae) Eukaryotic gene repressors Repressors compete with activators Crosstalk between transcription and chromatin? RNA processing RNA polymerase II C-terminal domain Pre-mRNA 6 Formation of ribonucleoprotein (RNP) particles In eukaryotes RNA elongation occurs concomitantly with processing RNA capping (5’ end) Pre-mRNA RNA splicing DNA RNA polymerase II RNA polyadenylation (3’ end) Transport facilitator Transport mediator Splicing factor Splicing repressor Translational regulator Daneholt, Proc. Natl. Acad. Sci. USA 2001 RNA-capping RNA splicing 5’ - phopsphate released GMP addition (5’ to 5’) Methylation of guanosine Examples of gene structures In eukaryotic cells, the primary RNA transcript (pre-mRNA), includes both coding and non-coding sequences In pre-mRNA, coding sequences are called Exons whereas non-coding sequences are called Intrones During splicing intronic sequences are removed 7 RNA splicing is mediated by the Spliceosome A B E C F An example of alternative splicing: the α-tropomyosin gene D RNA polyadenylation The Cell Nucleus by Ramon y Cajal (Nobel Prize, Medicine and Physiology) Termination sequences RNA cleavage PAP adds 200 nucleotides at the 3’ end of the RNA transcript The structure of the cell nucleus The nucleolus and its subdomains 8 Mechanisms of rRNA processing Visualization of nuclear structures: Cajal Bodies Human cells Xenopus Oocytes Function of Cajal Bodies Visualization of Speckles CajalBodies/GEMS Bodies/GEMS Cajal Assemblyof of Assembly Transcription-Processing Transcription-Processing Transcription Machinery Machinery Stockpiles of fully mature snRNPs ready to be used in RNARNA-splicing Visualization of chromatin and nuclear structure Speckles Cajal Body Nucleo cytoplasmic transport of mature mRNA molecules Protein synthesis factors Protein Synthesis Cajal Body Speckles 9