transcription_n_regulation_2022s (2).pptx

RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn Class MED Year 1 Title Transcription and regulation of gene expression Lecturer Salim Fredericks Learning outcomes • Describe the basic structure and types of RNA • Describe the process of transcription in eukaryotes: I...

RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn Class MED Year 1 Title Transcription and regulation of gene expression Lecturer Salim Fredericks Learning outcomes • Describe the basic structure and types of RNA • Describe the process of transcription in eukaryotes: Initiation, elongation & termination • Describe gene structure in eukaryotes • Describe post-transcriptional RNA processing (splicing, capping, polyadenylation, alternative/differential splicing, RNA editing) • Describe the differences between constitutive and inducible gene expression and the role of transcription factors in this process • Define the structure and mechanism of action of transcription factors • Outline the role of non-coding DNA and microRNA in regulating gene expression Regulation of gene expression The expression of gene into a protein (gene expression) involves both transcription (FUN11) and translation (FUN12) Regulation occurs at each step along this process. 1. Chromatin structure 2. Transcription initiation 3. mRNA stability 4. Transcript processing © 2013 Nature Education All rights reserved Gene expression means synthesizing its corresponding protein via the processes of transcription and translation • DNA is considered the “blueprint” of the cell – CONTAINS ALL THE GENETIC MATERIAL • Messenger RNA (mRNA) is the “photocopy” of a portion of the cells genetic material. When the cell needs to produce a certain protein, it activates the region of the genome encoding for that protein and produces multiple copies of that piece of DNA in the form of mRNA. The multiple copies of mRNA are then used to translate the genetic code into protein through the action of the cell’s protein manufacturing machinery, the ribosomes. mRNA expands the quantity of a given protein that can be made and it provides an important control point for regulating when What is RNA? RNA = Ribonucleic acid (similar to DNA but with key differences) Major functions within the cell 1. Important for the copying of genetic information from DNA (primarily a storage form of the genome) 2. Contributes to the formation of ribosomes (ribosomes are particles important for the synthesis of new proteins) Differences between RNA and DNADNA RNA Sugar Nitrogeno us Bases Structure Location Deoxyribo se Adenine (A) Thymine (T) Guanine (G) Cytosine (C) Doublestranded helical Nucleus Ribose Adenine (A) Uracil (U) Guanine (G) Cytosine (C) Singlestranded. 2°/3° structure Nucleus & RNA DNA vs Different types of RNA TYPE OF RNA mRNAs rRNAs tRNAs snRNAs ncRNAs FUNCTION messenger RNAs, code for proteins ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA Non-coding RNAs function in diverse cellular processes, including regulation of gene expression, Xchromosome inactivation, protein 1. RNA species involved in transcrip tion & translati on A species involved in transcription (A) Folded RNA showing only conventional base-pair interactions (B) structure with both conventional (red) and nonconventional (green) base-pair interactions (C) structure of an ›actual a portion of a group 1 mcb.berkeley.edu courses RNA, › mcb110 › Albe A species involved in transcription Synthesis and Location Function Messe • Produced by Nucleus & nger transcription of protein- cytoplasm RNA coding genes mRNA • Translated • Contains instructions for protein synthesis mRNA is a copy of the DNA that encodesStructure a protein of protein coding genes Distinct ‘start’ & ‘stop’ signals are encoded within the DNA sequence of a gene Transcription - Begins at the Transcription start site (TSS) at the end of the promoter region Promoters • Promoters are regions of DNA upstream of the TSS which lead to the initiation of transcription • Each gene has a unique promoter • 2 different types – basal promoter element and enhancer element Transcriptional Start Site (TSS) Enhancer Basal promoter 1. Basal Promoter – bound by a key transcriptional enzyme RNA polymerase II and basal transcription factors (required for RNA pol II binding) 2. Enhancer elements – recognised by proteins that will aid transcription – transcription factors 1. Basal Promoter elements • Essential for transcription of all genes • Functions to recruit in RNA pol II • Binds basal/general transcription factors first • Once these are bound RNA pol II will bind • Two different types of basal elements TATA box – most common – 20-30 base pairs from transcriptional start site – Can bind basal TFs and RNA pol II on own – strong element CCAT box – less common and not as strong – 50-130 base pairs away from start site 2. 1 2 3 1 Transcription Transcription is typically catalysed by RNA Polymerase II [RNA Pol II] RNA Pol II transcribes just one of the two DNA strands of the gene, reading its template 3’ to 5’ and making an RNA copy 5’ to 3’ Incorporates ribonucleotide triphosphates (NTPs - A, G, C & U) when creating the messenger RNA (mRNA) copy of the DNA. Three stages in the transcription process: Initiation RNA Pol II binds to DNA and unwinds a 17-18 bp segment of the promoter (the ‘Open Complex’) Elongation RNA Pol II moves along the template synthesising RNA until it reaches the terminator region During the elongation phase, an area of DNA under the RNA Pol II remains unwound This ‘Transcription Bubble’ moves along the Termination 5’ (ii) Most eukaryotic mRNA precursors have the motif ---AAUAAA---within the transcribed sequence = Polyadenylation signal sequence (iii) Recruits endonuclease – enzyme 5’ New mRNA Molecule Released for Processi ng(ii AA UA A (i) Transcription continues beyond the proteincoding region: 3'UTR (UnTranslated Region) ) Signals RNA Pol II to disengage (iii ) 3’UT R Release & degrade d (i) RNA Pol II Transcription continues past 3’UTR 2 Post-transcriptional mRNA processing The RNA molecule synthesised by RNA pol II is called the primary (1°) transcript The collection of these precursor molecules is known as heterogenous nuclear RNA (hnRNA) Extensive modification: (a) 5’ Capping (b) 3’ poly(A) tail 5’ capping of mRNA precursors 5’ Capping is the addition of a 7-methylguanosine residue Unusual 5’ to 5’ triphosphate linkage to the 5’ end of the mRNA Catalysed by guanylyltransferase (capping enzyme) which is then methylated by a ethyltransferase enzyme st nd The 1 and 2 Function: Protection of mRNA from degradation by nucleotides are also exonucleases Promote nuclear export methylated Aids recognition by translational machinery © 2019 New England Biolabs (UK) Ltd. All Rights Reserved. (b) 3’ poly(A) tail addition to °1 transcript After the Polyadenylation signal sequence has been recognised (----- AAUAAA----) & Endonuclease recruited & Cleave mRNA Then: -Poly(A) Polymerase then adds ~ 40-250 adenine residues to the cleaved 3’ end AAAAAAAAAAAAAAAAAAA An~40-250 New mRNA molecule Post-transcriptional regulation mRNA processing • • • Gene transcribed to produce mRNA with both introns and exons Exons only encode the protein coding region of a gene mRNA must be processed before translated Main areas of post-transcriptional regulation: 1. mRNA stability 2. Differential mRNA splicing mRNA stability mRNAs from different genes have their longevity encoded within them 3’ UTR sequence determines the stability of mRNA Destablising sequences in 3’ UTR Target for endonuclease 5’ CAP protects against degradation Exonucleases will degrade from polyA tail mRNA stability • Poly A tail confers mRNA stability • String of Adenosines at end of mRNA • Bound by poly-A binding protein (PABP) stabilises • Binds Approx 30 residues • Tail gradually shortened over lifetime of mRNA • PABP no longer bind ie < 30bp then degraded Different types of RNA TYPE OF RNA FUNCTION mRNA messenger RNAs, code for proteins s rRNAs ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis tRNAs transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids snRNAs small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA ncRN As Non-coding RNAs function in diverse cellular processes, including regulation of gene expression, X- 1. RNA species involved in protein synthesis 2. RNA species involved transcript processin g 3 Splicing of mRNA precursors Exon 1 Exon 2 Exon 3 3’ 5’ Intron 1 Intron 2 The non-coding intronic sequences are not present in mature RNA; they are spliced (cut) out and adjacent exons are joined together. Introns removed while RNA is being synthesised after the cap added but before transcript transported into the cytoplasm rons vary in size from 50 to ≥ 10,000 nucleotides Splicing carried out by molecular machine known as ‘Spliceosome’ Large complex consisting of RNA and Protein Small nuclear RNAs (snRNA) combine with a number of proteins to form: Small Nuclear RiboNucleoProteins (snRNPs) -Facilitate splicing by recognising and interacting with specific sequences at each end of the intron, cutting and rejoining the uclear RiboNucleoProteins (snRNPs) acts as transcrip Several snRNPs assemble to form a SPLICEOSOME. A specific adenine nucleotide (see A in diagram) attacks the 5’ intron, breaking the RNA. The 5’ end of the intron becomes attached to the A nucleotide, forming a loop of RNA. The free 3’ end of one exon attacks the 5’ end of the other The 3’ and 5’ ends of adjacent exons bond covalently, releasing the intron (which will then degrade). Differential splicing or exon shuffling Cell type A Protein A Calcitonin processing • Calcitonin (thyroid) – hormone • cGRP (Calcitonin generelated peptide) (brain) - neurotransmitter Cell type B Protein B Post-transcriptional regulation: mRNA processing-level Alternative splicing of tropomyosin Why is gene regulation so important? • Every cell type same DNA content – but differentiate to different cells liver embryo muscle bone • Need to turn genes on and off to generate specificity Tissue specificity Stem Cell Differentiation to different cell types and transcription factors involved Transcription can be activated in a constitutive or inducible manner • Constitutive gene expression – Constitutive transcription factor – Always on – Level of gene expression determined by how many binding sites and how much of TF around • Inducible gene expression – Inducible transcription factors – Turned on when necessary – Determine tissue specificity Types of genes A. Constitutive genes B. Inducible genes A. Constitutive genes Some genes are essential and necessary for life, and therefore are continuously expressed, such as those enzymes involved in metabolism/DNA repair or those regulating transcription and translation. These genes are called constitutive genes. They are always being made – B. Inducible genes any genes/proteins only synthesised when required - Inducib etermines development and tissue specificity ows cells to respond to environment 1. Extracellular cues: Hormones, Cytokines, Cell-cell interaction 2. Signal transduced into cell and drives transcription New Proteins 3. Nucleus: Transcription of new genes Inducible gene expression is controlled by transcription factor proteins Steroid hormones Steroid receptor transcript ion factor (TF) cytos ol nucle us Example: Hormones will bind to Steroid receptor TF’s, these form a homodimer that recognises and binds specific Inducible transcription factors • Are present or turned on only when needed • Allow the cells and tissues to respond to environmental cues Beta cells of pancreas – Fasting state ATTC GGCT AATCC CCGG ATTC GGCT AATCC CCGG Insulin Beta cells of pancreas – Fed state ATTC GGCT AATCC CCGG ATTC GGCT AATCC CCGG Insulin Transcription factor activity • • • • Amount and activity of transcription factors regulates the rate of transcription of a given gene Bind specific DNA sequence - response element Interact with RNA pol II and promote transcription Can bring in chromatin modifiers to aid unwinding Can also bring in coactivators SP1 binding sites SP1 binding sites Number of binding sites for that transcription factor in TATA GENE I TATA rate of transcription a promoter determines GENE I SP1 binding sites SP1 binding sites TATA TATA GENE II GENE II Inducible transcription factors allow only the genes that are required to be switched on. - Allows rapid and dynamic response to stimulus Different types of RNA TYPE OF RNA FUNCTION mRNAs messenger RNAs, code for proteins rRNAs ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis tRNAs transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids snRNAs small nuclear RNAs, function in a variety of nuclear processes, including the splicing of premRNA ncRNA Non-coding RNAs function in diverse s cellular processes, including regulation of gene expression, Xchromosome inactivation, protein transport 1. RNA species involved in protein synthesis 2. RNA species involved transcript processin g 3. RNA species involved in regulation of Regulatory non-coding DNA (formerly known as ‘Junk’ DNA) In 2012 ENCODE: Encyclopaedia of DNA Elements project was completed and the findings completely changed how we view non-coding DNA/RNA http://www.nature.com/nature/journal/v489/n7414/full/489052a.ht ml • Only 1% of human genome makes protein • 20% is the elements required for gene expression – introns, promoters, enhancers etc microRNA microRNA - miRNA • Small strands of RNA that regulate gene expression (20-25 nucleotides) • Transcribed from the DNA but non-coding (i.e: they don’t make any proteins) • Partially complementary to a number of mRNAs (protein encoding ones) • Complementarity the basis of miRNA mechanism of action – they seek and bind specific mRNA sequences • Primary function of miRNA is to down regulate gene expression How does miRNA block gene expression? • Promotes RNA degradation – binds complementary sequences in 3’UTR and induces degradation • Binds mRNA and blocks translation Adapted from: Front. Genet., 13 January 2015 | http://dx.doi.org/10.3389/fgene.2014.00472