Lecture 5: Gene Expression & Transcription in Eukaryotes PDF

# Vision Collage of Medicine ## Year 2 ## Level 3 ## Lecture 5 ### Gene expression & Transcription in Eukaryotes #### by #### Dr.Ezat Mersal # Objectives - Gene - Gene Structure - Gene expression - Types of Genes - Post transcriptional modifications: RNA splicing. 3'polyadenylation, 5'c...

# Vision Collage of Medicine ## Year 2 ## Level 3 ## Lecture 5 ### Gene expression & Transcription in Eukaryotes #### by #### Dr.Ezat Mersal # Objectives - Gene - Gene Structure - Gene expression - Types of Genes - Post transcriptional modifications: RNA splicing. 3'polyadenylation, 5'capping # Genes - The DNA that makes up the human genome can be subdivided into information bytes called genes. - The gene is the fundamental unit of inheritance and the ultimate determinant of all phenotypes. - Every gene contains a particular set of instructions that code for a specific protein. - This mean, DNA carries the genetic blueprint that is used to make all the proteins that the cell needs. - The DNA of a normal human cell contains an estimated 30,000 to 120,000 genes, but only a fraction of these are used (or expressed) in any particular cell at any given time. # Gene Structure - Every gene consists of several functional components, each involved in a different facet of the process of gene expression. - Most eukaryotic genes in contrast to typical bacterial genes consists of coding sequences (exons) which interrupted by noncoding DNA (introns). - The average gene 7-10 exons spread over 10-16kb of DNA. - The gene must have Exon; start signals; stop signals; regulatory control elements. - There are two main functional units: the promoter region and the coding region. # Gene Expression - The process by which a gene's information is converted into the structures and functions of a cell by a process of producing a biologically functional molecule of either protein or RNA (end product) is made. - Gene expression is assumed to be controlled at various points in the sequence leading to protein synthesis. # Central Dogma - DNA - RNA - Protein - **Transcription:** DNA → RNA - **Translation:** RNA → protein # Types of Genes - **Constitutive gene (Housekeeping genes)** - Expressed at a reasonably constant rate in every day - Not subjected to regulation - e.g enzymes of glycolysis - **Regulated gene** - Expressed only under certain conditions just for specific time - Expressed in all cells/subset of cells - e.g. expression of insulin gene in pancreas # Flow of Genetic Information in Prokaryotes vs. Eukaryotes - **Prokaryotes** - Transcription - Translation - **Eukaryotes** - Transcription - RNA processing - Translation # Eukaryotic Promoter - A promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5’ region of the sense strand). Promoters can be about 100–1000 base pairs long. - **Conserved eukaryotic promoter elements** - CAAT box - TATA box - GC box - CAP site - **Consensus sequence** - GGCCAATCT - TATAA - GGGCGG - TAC # Eukaryotic gene promoter sequences - **Sequences within the eukaryotic promoter region that are recognized by RNA polymerase II** - CAAT box - GGCCAATCT # Enhancer sequences - Are regulatory DNA sequences that, when bound by specific proteins called transcription factors, enhance the transcription of an associated gene. - An enhancer is a short (50-1500 bp) region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. - Enhancers, are not necessarily close to the genes they enhance. They can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or may be thousands of nucleotides away. # Silencers - Some transcription factors (called repressors) bind to regions called 'silencers' that depress the rate of transcription. # TRANSCRIPTION (SYNTHESIS OF RNA) - Is the process by Which certain area of DNA is copied (transcribed) to mRNA, which carries the information needed for protein synthesis. - It's cope to all the DNA starfs - DNA replication - DNA repair - genetic recombination - RNA synthesis (transcription) # Messenger RNA (mRNA) - Messenger RNA is a type of RNA that is necessary for protein production. - In cells, mRNA uses the information in genes to create a blueprint for making proteins. - The DNA is firstly transcribed into mRNA and subsequently translated into a protein product. # Important Note: - Reverse transcription is the synthesis of DNA from RNA template which catalyzed by reverse transcriptase enzyme. - Retroviruses contain RNA as their genetic material. - The retroviral RNA serves as a template for synthesis of DNA by reverse transcriptase. - The DNA that is generated can be inserted into the genome (chromosomes) of the host cell and be expressed. - Retroviruses Examples: HIV (AIDS), Hepatitis C. # Transcription in Eukaryotes - Transcription takes place in two broad steps: - **Pre-messenger RNA is formed**, with the involvement of RNA polymerase enzymes. The process relies on Watson-Crick base pairing (A pairs with U), and the resultant single strand of RNA is the reverse-complement of the original DNA sequence. - **RNA Polymerase** - Reads DNA - Makes RNA copy of One strand/ One gene # RNA Polymerase - RNA polymerases I, and III transcribe rRNA, and tRNA genes, respectively. - Pol III transcribes a few other RNAs as well. - All 3 are big, multimeric proteins (500-700 kD). - All have 2 large subunits with sequences similar to and in E.coli RNA polymerase (Prokaryotes), so catalytic site may be conserved. - Pol II is the most sensitive to α-amanitin, an octapeptide from Amanita phalloides ("destroying angel mushroom") # Transcription Factors - The three polymerases (I, II and III) interact with their promoters via so-called transcription factors. - Transcription factors recognize and initiate transcription at specific promoter sequences. - Some transcription factors (TFIIIA and TFIIIC for RNA polymerase III) bind to specific recognition sequences within the coding region. # The General Transcription Factors Needed for Transcription Initiation by Eukaryotic RNA Polymerase II - **Name** **Number of Subunits** **Roles in transition initiation** -TFIID 1-11 Recognizes TATA box. Recognizes other DNA sequences near the transcription start point; regulates DNA-binding by TBP. - TFIIB 1 Recognizes BRE element in promoters; accurately positions RNA polymerase at the start site of transcription. - TFIIF 3 Stabilizes RNA polymerase interaction with TBP and TFIIB; helps attract TFIIE and TFIIH. - TFIIE 2 Attracts and regulates TFIIH. - TFIIH 9 Unwinds DNA at the transcription start point, phosphorylates Ser5 of the RNA polymerase CTD; releases RNA polymerase from the promoter. # RNA Polymerase II - Most interesting because it regulates synthesis of mRNA - Pol II consists of 10 different core peptides (RPB1 - RPB10) - RPB1 and RPB2 are homologous to *E. coli* RNA polymerase and - RPB1 has DNA-binding site; RPB2 binds NTP (Nucleotide triphosphate). # CTD is essential and this domain may project away from the globular portion of the enzyme (up to 50 nm!). - Only RNA Pol II whose CTD is NOT phosphorylated can initiate transcription. - TATA box (TATAAA) is a consensus promoter. - 7 general transcription factors TFIID are required. # Unlike DNA replication, in which both strands are copied, only one strand is transcribed. - The strand that contains the gene is called the sense strand, while the complementary strand is the antisense strand. - The mRNA produced in transcription is a copy of the sense strand, but it is the antisense strand that is transcribed. # **Sense** - (5') CGCTATAGCGTTT (3') DNA nontemplate (coding) strand - (3') GCGATATCGCAAA (5') DNA template strand - (5') CGCUAUAGCGUUU (3') RNA transcript # Transcription ends when the RNA polymerase enzyme reaches a triplet of bases (UAA or UAG or UGA), that is read as a "stop" signal - The DNA molecule re-winds to re-form the double helix. # **Note:** Starting triplet base is AUG. # Transcription in Eukaryotes - What are Post-transcriptional modifications or gene processing ? - It is a process by which, primary transcript RNA is converted into mature RNA. - The pre-mRNA molecule undergoes three main modifications: - 5' Capping - 3' polyadenylation - RNA splicing - These modifications are 5' capping, 3' polyadenylation, and RNA splicing, which occur in the cell nucleus before the RNA is translated. # Capping - Capping of the pre-mRNA involves the addition of 7-methylguanosine (m7G) to the 5' end. - To achieve this, the terminal 5' phosphate requires removal, which is done with the aid of a phosphatase enzyme. - The cap protects the 5' end of the primary RNA transcript from attack by ribonuclease. - Ribonuclease (commonly abbreviated RNase) is a type of nuclease that catalyzes the degradation of RNA into smaller components. # Cleavage and polyadenylation - The pre-mRNA processing at the 3' end of the RNA molecule involves cleavage of its 3' end and then the addition of about 250 adenine residues to form a poly(A) tail。 - The poly(A) tail protects the 3'end from ribonuclease digestion. # RNA splicing - The pre-messenger RNA thus formed contains introns which are not required for protein synthesis. - The sequence AAUAAA in hnRNA serves as a signal for cleavage of the hnRNA and addition of the poly(A) tail by poly(A) polymerase. ATP serves as the precursor. - The pre-messenger RNA is chopped up to remove the introns and create messenger RNA (mRNA) in a process called RNA splicing. # The structure of a eukaryotic gene and its products - 5' flanking region - Promoter - Transcribed region - Intron - Exon - Enhancer - CAAT box - GC boxes - TATA box - Protein - Cap site - Start signal - Left splice site - Right splice site - Protein stop signal - Poly (A) addition signal - Polyadenylation site # Synthesis of messenger RNA (mRNA) in eukaryotes. hnRNA, heterogeneous nuclear RNA. - **Nucleus** - 5' cap - Intron - 3' poly (A) - **DNA** - **Nuclear envelope** - **Nuclear pore** # CLINICAL CORRELATES - The most common causes of β-thalassemia are defects in mRNA splicing of the ẞ-globin gene. Mutations that affect the splicing create aberrant transcripts that are degraded before they are translated. If patients inherit a single mutated gene (thalassemia minor), the disease manifests with a mild anemia. However, patients with homozygous mutations (thalassemia major) have severe transfusion-dependent anemia. # Synthesis of ribosomal RNA (rRNA) and assembly of ribosomes - **Nuclear organizer** - 5' - 3' - **Nuclear envelope** - **Nucleolus** - **Nuclear pore** - **Cytoplasm** - **45S rRNA precursor** # Synthesis of transfer RNA (tRNA) - **Nucleus** - Intron - tRNA precursor - tRNA - tRNA - **DNA** - **Nuclear pore** - **Cytoplasm** - Synthesis of transfer RNA (tRNA). D, T, W, and (representing other modified nucleotides) are unusual nucleotides produced by post-transcriptional modifications. # Synthesis of transfer RNA (tRNA) - RNA polymerase III is the enzyme that produces tRNA. The promoter is located within the coding region of the gene. - Primary transcripts for tRNA are cleaved at the 5' and 3' ends. - Some precursors contain introns that are removed. - During processing of tRNA precursors, nucleotides are modified. - Post-transcriptional modification includes the conversion of uridine to pseudouridine (Ψ), ribothymidine (T), and dihydrouridine (D). - Other unusual nucleotides are also produced. - Addition of the sequence CCA to the 3' end is catalyzed by nucleotidyl transferase. # tRNA - **Found in all tRNAs** - **Not found in all tRNAs. Other variable sites are shown in blue as well** - **The modified bases are:** - I = inosine - ml = methylinosine - T = ribothymidine - UH₂ = dihydrouridine - m₂G = dimethylguanosine - ψ = pseudouridine # Reference Books: - *Basic genetics : a human approach / BSCS. Dubuque, IA, Kendall/Hunt Pub. Co., c1999. 147 p. QH431.B305 1999* - *Genes, ethnicity, and ageing. Edited by Lincoln H. Schmitt, Leonard Freedman, Rayma Pervan. Nedlands, Australia, Centre for Human Biology, University of Western Australia ; Singapore, River Edge, NJ, World Scientific, c1995. 100 p.QH455.G45 1995* - *Genetic polymorphisms and susceptibility to disease. Edited by M. S. Miller and M. Т. Cronin. New York, Taylor & Francis, 2000. 266 р.*