Lecture 3 - Week 3 Transcription -updated (1) PDF

From DNA to RNA – the first step of gene expression BI1CMP1 Dr Susanna Cogo [email protected] Intended Learning Outcomes (ILOs) By the end of this lecture, you will be able to: Describe the structure of eukaryotic genes List and compare the main RNA classes and RNA pol...

From DNA to RNA – the first step of gene expression BI1CMP1 Dr Susanna Cogo [email protected] Intended Learning Outcomes (ILOs) By the end of this lecture, you will be able to: Describe the structure of eukaryotic genes List and compare the main RNA classes and RNA polymerases Outline the stages of eukaryotic transcription Explain the function of promoters and enhancers/silencers Define RNA processing and the concept of alternative splicing The chicken or the egg? DNA stores and transmit the genetic information Proteins are required to catalyse chemical reactions that are essential for life But who came first??? 1981: Thomas Cech discovers that RNA can have catalytic function Catalytic RNAs are called ribozymes and have multiple roles, including cutting or binding sequences, replicate RNA molecules, facilitate the creation of bonds in proteins With evolution, the role of RNA has been relegated mostly to information storage and transfer However, many different RNA classes are known which play diverse roles https://www.nature.com/articles/d41586-024-03212-9 The flow of information DNA synthesis (DNA replication) RNA synthesis (transcription) Protein synthesis (translation) Do all cells need all the information at all times? The genome is organised in genes and intergenic regions. Genes contain most of the information controlling hereditary features (i.e. physical and biological traits) and are transcribed (RNA) and/or translated (proteins). In eukaryotes, only 1-1.5% of the genome is represented by genes, while the remainder are intergenic regions. The set (thousands) of genes expressed in a particular cell at a specific time determines what that cell can do. The structure of eukaryotic genes A gene is a sequence of DNA that functions as a unit to give rise to either an RNA or a protein. Each gene has regulatory and coding regions. Regulatory regions are nucleotide sequences which can increase or decrease gene expression – they can be located very far away from the regulated gene!!! The promoter is a sequence of DNA where proteins bind to initiate transcription – usually it is located next to the transcription start site. The termination region indicates the end of transcription. Watch this video on gene structure: https://www.youtube.com/watch?v=h5HI2OqOJA0 The structure of eukaryotic genes Coding regions also contain introns and exons (RNA coding region). Introns are noncoding sections of an RNA transcript, or the DNA encoding it, that are spliced out before the RNA molecule is translated into a protein. The sections of DNA (or RNA) that code for functional products are called exons. Introns are not part of the final mRNA transcript, and are removed through a process called splicing. The longest human gene is DMD, coding for dystrophin (79 exons, 2300 kilobases – kb). The structure of eukaryotic genes Transcription of a gene to RNA and processing to generate mRNA (messenger RNA) happen in the nucleus. The process of producing RNA and proteins from a gene is called gene expression. Initial model 1 gene = 1 enzyme Modified to 1 gene = 1 polypeptide However, some genes encode structural RNAs, tRNA, miRNAs, rRNA, etc, not proteins. One gene – one protein (?) The molecular basis of phenotypes was discovered before it was known that DNA is the genetic material. Studies of many organisms showed that major phenotypic differences were due to differences in specific proteins. Only 1 in every 10,000-20,000 raccoons is born with albinism. Albinism is an inherited genetic condition characterized by the inability to produce melanin, the pigment in hair, skin and eyes. Archibald Garrod (1908) George Beadle & Edward Tatum (1940s) The basis of transcription Gene expression in eukaryotes is a multistage and compartmentalised process Transcription is the process by which the information in one strand of DNA is copied into a new molecule of RNA. All three major classes of RNA (mRNA, tRNA, rRNA) are synthesized by transcription of the appropriate genes and are involved in protein synthesis. Three main RNA classes are involved in protein synthesis The toolkit of transcription To initiate transcription, these elements are required:  a starting DNA template  a transcription unit (promoter, RNA coding sequence, terminator)  the machinery for transcription  rNTPs Transcription starts in a bubble Happens in 5’ -> 3’ direction !!! Genes are transcribed from one strand – the template strand !!! Information on which strand to be transcribed is in the promoter Transcription is 5′ to 3′ on a template that is 3′ to 5′ or coding strand RNA polymerase – An enzyme that synthesizes RNA using a DNA template The transcription “Christmas tree” The transcription unit The promoter, RNA coding region and terminator represent a transcription unit Upstream Downstream The first nucleotide transcribed is numbered +1 If the DNA strand 5ʹ–GTACCGTC–3ʹ was used as a template, what would be the sequence of the transcribed RNA? A: 5’–GUACCGUC–3’ B: 5’–GACGGTAC–3’ C: 5’–CAUGGCAG–3’ D: 5’–GACGGUAC–3’ E: 5’–GUCGGUAC–3’ Transcription has three sequential stages 1. Initiation: the transcription machinery recognises and binds to the promoter to start synthesis from DNA (no primer needed) 2. Elongation: the RNA polymerase moves along the template during elongation and synthesises RNA, adding nucleotides at the 3’ 3. Termination: the enzyme dissociates at the end of the transcription unit and releases RNA Key players in transcription RNA polymerase Chromatin structure needs to be modified by regulatory proteins binding near the promoter before transcription to make the DNA accessible One enzyme! (plus accessory proteins) RNA polymerase (multisubunit, multifunctional) Other factors Transcription factors Mediator complex Cis DNA elements: promoter and regulatory DNA sequences The information about how frequency and level of transcription is in the promoter Regulation of transcription initiation (i) Eukaryotic RNA polymerase cannot recognize the DNA sequence on its own, but needs many accessory proteins Regulatory proteins bind to DNA to modify chromatin structure RNA pol II + general transcription factors + mediator = basal apparatus https://www.youtube.com/watch?v=9x6p-r7NNKg Different types of RNA polymerases exist in eukaryotes 3 main RNA polymerases exist in eukaryotes: RNA polymerase I, II, and III Different polymerases bind to different promoters and transcribe different genes RNA polymerase II transcribes genes encoding proteins A lot of accessory proteins are required to bind the promoter regions and recruit the polymerase RNA polymerase II promoters RNA pol II promoters are composed of a core promoter and a regulatory promoter; A sequence in the core promoter, named TATA box, is recognized by TFIID – a transcription factor of the general apparatus that can bind to the TATA box via the TATA binding protein (TBP) -> binding leads to DNA bending and unwinding; More TFs and RNA pol II (basal transcription apparatus) bind to the core promoter and help positioning RNA pol II on the start site. -75 Regulatory promoter upstream CCAA T box https://www.youtube.com/ watch?v=9x6p-r7NNKg Regulation of transcription initiation (ii) The regulatory promoter also binds transcription factors (TFs) and regulates the speed of the process (via mediator); TFs can also bind enhancers or silencers to regulate transcription: these can be located very far from the DNA sequence to be transcribed (usually upstream); The entire transcription machinery is made up of RNA pol II + up to 50 other proteins; DNA and RNA pol II change conformation to expose the single strand DNA template, and a short stretch of DNA (11-15nt) is unwound. RNA pol II promoters From structure to function The open complex forms when the DNA is positioned in the active site of the RNA pol and the strands are separated https://www.youtube.com/watch? v=GdKfadJGId4 Abortive initiation It is likely to be a rate-limiting control on gene activation After synthesis of an approximately 9-12nt long strand, elongation can begin When the transcript is approximately 30nt long, RNA pol II leaves promoter region to make space for a new round Elongation RNA pol II moves downstream to continue transcription TFs are left behind to bind a new molecule of RNA pol Nobel Prize in Chemistry 2006 – Roger Kornberg: Structure and function of RNA polymerase Elongation DNA moves through a channel in RNA polymerase and makes a sharp turn at the active site The first 8 rNTPs remain bound to DNA (hybrid molecule) The DNA-RNA hybrid encounters a structural obstacle as it progresses through the polymerase The strands are separated and the newly forming RNA molecule a) continues growing in 5’ to 3’ direction and b) runs through another cleft in the RNA pol Changes in the conformations of certain flexible modules within the enzyme control the entry of nucleotides to the active site DNA is forced to make a turn at the active site by a protein wall. Termination No specific termination sequences for RNA pol II RNA pol II keeps synthetising RNA even 100-1000nts after the end of the coding sequence Pre-mRNA is cleaved while RNA pol II is still transcribing The “extra” RNA stretch is degraded in 5’ too 3’ direction by an enzyme called Rat1 Transcription terminates when Rat1 reaches the polymerase What is the enzymatic activity of Rat1? Could you spot any analogy/difference with DNA replication? Colinear or not colinear? In 1958, Francis Crick suggested that the number of nucleotides in a gene are proportional to the number of aminoacids in a protein (colinearity) 1977: evidence of non-colinearity The intervening sequences: introns Introns are non-coding sequences absent from the final transcript (mRNA) Mostly present in eukaryotes Intron size and number correlates with organismal complexity Protein coding region 5’ UTR 3’ UTR The structure of mRNA Addition of the 5’ cap: Addition of a guanine nucleotide + methylation of the nitrogenous base (7-methylguanosine) via 5’-5’ binding (Sometimes) methylation of the sugar in the 1st and 2nd nucleotide (in position 2, 2’ methyl groups) The cap is added immediately after the initiation of transcription only in RNA pol II transcripts The cap increases mRNA stability, regulates intron removal and is important for translation The structure of mRNA Addition of the poly(A) tail: 50-250 adenine nucleotides are added at the 3’ via polyadenylation The extra material at the end of the coding sequence needs to be cleaved before the poly(A) tail is added Cleavage is mediated by consensus sequences both upstream and downstream of the cleavage site The poly(A) consensus sequence (AAUAAA) is located 11-30nt upstream of the cleavage site A sequence rich in U (and G) nt is downstream The poly(A) tail contributes to mRNA stability, thus regulating gene expression, and helps attachment to ribosomes for translation From pre-mRNA to mRNA Before being transported to the cytoplasm for translation, Gene expression in eukaryotes is a multistage process and introns need to be removed in a process called splicing compartmentalised There is a time and space separation between transcription and translation (in eukaryotes) Consensus sequences are required to indicate where splicing occurs:  5’ splice site (GU at the beginning of an intron)  3’ splice site (AG at the end)  Branch point: A nucleotide located 18-40 nt upstream of the 3’ splice site Splicing is mediated by the spliceosome (5 RNA + 300 proteins) Splicing is a 2-step process 1. The 5’ splice site is cut first and G attaches to A in the branch point forming a lariat 2. The 3’ splice site is cut and the two exon extremities are covalently attached. The intron is released as a lariat and degraded. mRNA is ready to be transported in the cytoplasm. https:// www.youtube.com/ watch? v=cU60bryCRHM One gene – many proteins Alternative splicing: the same DNA molecule can be spliced in different ways to generate different mRNA products (hence, proteins) One gene – many proteins Brain Thyroid RNA transport from nucleus to cytoplasm Nuclear export of mRNA Nuclear export of mRNA https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC8583845/ #:~:text=The%20passage %20of%20mRNAs %20to,spanning%20the %20double%20nuclear %20envelope. https://doi.org/10.1016/j.semcdb.2014.04.027 The Royal Disease An intronic mutation in factor IX leading to incorrect splicing can cause haemophilia https://www.science.org/doi/10.1126/science.1180660? adobe_mc=MCMID %3D51237819785402504810749757823928688151%7C MCORGID %3D242B6472541199F70A4C98A6%2540AdobeOrg %7CTS%3D1728986521 https://www.youtube.com/watch?v=xMNm7XY_m_s