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15. Eukaryotic Transcription.pdf

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Biology (Sci)
BIOL 200
Richard Roy

Naveen Sooknanan McGill Fall 2011 Eukaryotic Transcription: Although the main process of eukaryotic transcription is very similar to that of prokaryotes, the actual factors involved make the mechanism much more complex  Initiation of translation is particularly complex in eukaryotes  Requirement of an RNA polymerase, nucleotides, complement bonding, all still applies to eukaryotes Eukaryotic transcription is limited to two or three organelles  Transcription in the mitochondria and plastids (in plants and algae) have a process mechanically similar to prokaryotes  Transcription in the nucleus is by far the most important and complex There are three eukaryotic RNA polymerases, each differing in gene products and in their sensitivity to the mushroom poison mannitin  RNA polymerase I produces mainly rRNA precursors such as 28S, 5.8S and 18S o Pol I is not sensitive to mannitin  RNA polymerase II is the one we will focus most on, and is responsible for all protein coding genes (mRNA) as well as factors involved in splicing, and other non-coding functional RNA (which we don’t understand) o Pol II is highly sensitive to mannitin o Pol II is the only ATP dependant RNA polymerase  RNA polymerase III is responsible for the transcription of tRNAs, a splicing factor called U6, small stable RNAs and rRNAs produced outside the nucleus o Pol III is mildly sensitive to mannitin All three polymerases are multi-subunit complexes consisting of more than 10 subunits. They are all very similar to prokaryotic RNA polymerase, showing little evolutionary change.  These RNA polymerase molecules have no idea what needs to be transcribed and what doesn’t, so they need the help of some transcription factors which will direct them to the correct genes  Pol II is the only RNA polymerase which has a carboxyterminal domain (CTD) o The CTD is a repeat of 7 amino acids ~52 times in humans and 26 times in yeast o It is very useful in downstream reactions, as we will see later on  These subunits are all very crucial in the functionality of the RNA polymerase. Without one, the organism would die Upstream of the protein coding region of DNA is a region rich in As and Ts. This region is known as the TATA box and is very highly conserved.  The TATA box, 8 nucleotides long, is normally located between -35 and -25 on the gene and acts a promoter to mark the beginning of the transcription process  Some eukaryotes, which are not well understood, are mediated by an initiator sequence which is not well defined 1Naveen Sooknanan McGill Fall 2011 The TATA box binding protein (TBP) does exactly what its name says; binds to the TATA box  TBP binds to the minor groove of the DNA using its conserved CTD and causes a radical bending of the DNA structure where the TBP sits down  This protein has been purified and structurally analyzed, and, strangely enough, eukaryotes without a TAT box still need the TBP in order to initiate transcription The binding of TBP to the TATA box then calls in an armada ot transcription factors (TFs) as well as Pol II to initiate transcription and get it ready for the elongation phase  Once the TBP binds, TFIIB recognizes the bent structure and binds to the TBP forming a complex  Pol II, which is always associated with TFIIF then joins the complex  TFIIE then joins this complex  The complex is then recognized by TFIIH which is a very important enzyme in this process o TFIIH has 2 helicase functions as well as one PNK function o Once TFIIH binds, the pre-initiation complex is formed  TFIIH, through its protein kinase activity, phosphorylates the CTD of Pol II which activates it o The CTD becomes hyperphosphorylated, meaning multiple phosphates are added on  Through the helicase activity of TFIIH, the DNA is unwound to form a transcription bubble  Through the phosphorylation of Pol II, all the general TFs leave except the TBP  Once the CTD of Pol II is hydrolyzed, system switched from initiation to elongation TFIIH is an energy dependant enzyme which uses ATP both in its PNK activity as well as its helicase activity (melting the base pairs)  One of the helicase activities of DNA helicase is a proofreading ability. This is useful for picking up mutations in the DNA as it scans along o One important mutation is the formation of thymine-thymine dimers which can happen in the presence of UV light  Patients with a cancer predisposing disease called Xeroderma pigmentosum cannot enter sunlight because their nucleotide excision repair (NER) system cannot properly repair these thymine-thymine dimers and transcription of all proteins becomes erroneous o These patients are much more likely to develop skin cancer 2Naveen Sooknanan McGill Fall 2011 This dual function of TFIIH leads to a coupling of DNA repair with transcription. This has been proved because genes being transcribed are able to be repaired very quickly because of the proximity of THIIH to the DNA  This allows for efficient transcription RNA polymerase I works a little bit differently, but still requires TFs to help in the process. The most important thing to remember is that the process is not directly ATP dependant, unlike Pol II.  A class I gene contains a core element located ~ -40 - +5 and is essential for binding of Pol I  Also present is an upstream element at ~ -155 - -60 and is crucial for the activation of class I transcription  A complex called the upstream activating factor (UAF) binds to the UE which causes the binding of a trimeric core factor (CF) to the CE o The CF contains TBP  Pol I then associates to the complex and transcription can begin  These factors are different from the TFs used by Pol II, and are also different from the ones used by Pol III Pol III promoters are really different because they are actually located within the transcribed unit, unlike Pol I and Pol II. Like Pol I, however, there is no ATP requirement for class III transcription  In tRNA genes, promoter regions called the A box and B box are protein binding factors which also play a key role in the structure of the final tRNA molecule o TFIIIB and C are required for tRNA transcription  In 5S rRNA, a C box has the same role o In 5S rRNA, TFIIIC complexes with TFIIIA and then joins TFIIIB to initiate transcription It is important to know that while these steps all happen sequentially both on paper and in labs, in vivo transcription may happen all at once through the formation of a holoenzyme prior to transcription. General transcription factors are a broad range of macromolecules which can interact with specific DNA sequence motifs in order to regulate transcription levels  The regulation can be temporal (when the gene is active) or spatial (where the gene is active)  TBP and the TATA box are an example of a transcription factor binding to a specific DNA sequence motif  Normally, the transcription factors contain an α helical domain called a recognition helix which is able to interact, usually, with the major groove of the DNA o TBP is an exception  Transcriptional activators are TFs which are able to sit down on certain DNA sequences and “turn on” certain genes 3control demonto two proteins Supershih T DNA-protein Bare DNA o MINE O FT 1 6 7 8 9 1011 12 13 1415 16 18 20 22 control demonto two proteins Supershih T DNA-protein Bare DNA o MINE O FT 1 6 7 8 9 1011 12 13 1415 16 18 20 22Naveen Sooknanan McGill Fall 2011 o I.e. these genes will not be transcribed in the absence of these TFs Through analysis of linker scanning mutations, it is possible to determine the binding sites of TFs on a desired control region  The control region is placed into a vector upstream of a reporter gene o This reporter gene could be GFP, the lac z gene, thymidine kinase, luciferase, chloramphenicol acetyltrasnferase (CAF)… anything that will report to the scientist when transcription takes place (by light, fluorescence, etc.)  A full control region will obviously activate transcription at a maximal rate, so endon
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