Lecture 18 RNA and Transcription PDF

Lecture 18 Information Flow in the Cell: RNA and Transcription This morning, we will cover: The Central Dogma of Molecular Biology The RNA world hypothesis Structure of RNA and properties Types of RNAs Transcription in Prokaryotes Transcription in Eukaryotes Regulation of gene...

Lecture 18 Information Flow in the Cell: RNA and Transcription This morning, we will cover: The Central Dogma of Molecular Biology The RNA world hypothesis Structure of RNA and properties Types of RNAs Transcription in Prokaryotes Transcription in Eukaryotes Regulation of gene expression in Prokaryotes  The Eukaryotic cell: nucleus and cytoplasm Genes code for proteins; and in eukaryotes genes are located in the nucleus nucleus cytoplasm But all proteins are synthesized in the cytoplasm What is the nature of the information that moves from the nucleus to the cytoplasm? Information flow: what is it that moves to the cytoplasm? 5’ ~ 1,000 bp DNA 3’ (Located in the Nucleus) 3’ 5’ What is the coding ? intermediate? (Produced in the Cytoplasm)  Chemical nature of the intermediate that travels from the nucleus to cytoplasm A likely candidate = RNA Circumstantial evidence: 1. DNA is located in the nucleus (in chromosomes) But proteins are made in the cytoplasm 2. RNA is synthesized in the nucleus 3. RNA is transported to the cytoplasm where proteins are synthesized Information flows via mRNA 5’ ~ 1,000 bp DNA 3’ (In Nucleus) 3’ 5’ The intermediate = messenger RNA ? (mRNA) (Made in the cytoplasm)  Overview: Information flow in the cell Francis Crick’s ‘Central dogma’ of molecular biology: ‘DNA makes RNA makes protein’ DNA makes RNA makes protein this probably evolved first Origins of life (Work by William Martin; Nick Lane and others)  Life goes on... Selection for persistence: Things that have the property of tending to persist, persist Things that don’t, don’t Things that out-persist other things come to predominate, especially if that persistence is achieved through replication  The RNA World Hypothesis This hypothesis proposes that RNA, rather than DNA or proteins, was the first molecule capable of both storing genetic information and catalyzing chemical reactions. It suggests that life on Earth may have begun with a world dominated by RNA. The invention of genetic material (Kevin Mitchell; Free Agents, 2023)  RNA proteins DNA RNA RNA mutation mutation X DNA proteins DNA * proteins * * * * Improved/novel protein function Disrupted protein function More robust persistence/reproduction Less robust persistence/reproduction Positive selection Negative selection Structure and properties of RNA  Structure of RNA RNA is a polymer with a sugar-phosphate backbone Bases (4 kinds): Purines: Adenine, Guanine Pyrimidines: Uracil, Cytosine It is usually a single-stranded molecule (unlike DNA) But intramolecular base-pairing (via hydrogen bonds) may give rise to double-stranded regions RNA is a single-stranded 5’ polymer ▪ Sugar-phosphate backbone One end has an exposed 5’phosphate The other end an exposed 3’hydroxyl 3’ RNA is made of nucleotide 5’ monomers Nucleotide = sugar, phosphate, base Phosphate Base Sugar 3’ The key biochemical differences between RNA and DNA 1. The sugar: Ribose in RNA vs 2-deoxyribose in DNA 1’ 1’ 2’ 2’ 2- 2. Base difference: Uracil in RNA vs Thymine in DNA But Uracil (like Thymine) can ‘base pair’ to Adenine Different types of RNA 1. Messenger RNA (mRNA) This is the RNA transcribed from ‘protein-coding’ genes mRNAs encode the information for specific proteins: Eukaryotic polyA tail UTR = Untranslated region Low abundance (< 10% of all types of RNA in a cell) Short half-life (i.e. subject to rapid turnover: minutes to hours) 2. Ribosomal RNA (rRNA) Is a structural & functional component of Ribosomes The most abundant RNA in a cell (>90% of total RNA) rRNA is very stable rRNA is a non-coding RNA Ribosome rRNAs associate with ribosomal proteins in the large and small subunits of the ribosome 3. Transfer RNA (tRNA) Very small - less than 100 bases long A non-coding RNA ‘Clover-leaf’ secondary structure due to internal (intramolecular) base-pairing Plays a key role in Translation (protein synthesis) Very abundant and very stable 4. ‘Non-coding’ regulatory RNAs a. RNAs involved in the regulation of gene expression MicroRNA (miRNA) 21 –26 bases Short interfering RNA (siRNA) Long non-coding RNA (lncRNA): 200 - 1000s bases b. RNAs involved in mRNA processing (splicing) Small nuclear RNA (snRNA): 164 bases Transcription: the synthesis of RNA based on a DNA template  Overview of transcription Transcription via RNA polymerase (RNAP) 1. Transcription in Prokaryotes  Transcription - role of RNA polymerase (RNAP) Prokaryotes contain a single type of RNA polymerase How does RNA polymerase know where to start making mRNA? ………. and where to stop? There must be signals in the DNA sequence itself  DNA sequences that control transcription a. Promoters DNA sequences that guide RNAP to the beginning of a gene to start transcription (at a transcription initiation site) b. Terminators DNA sequences that specify the termination of RNA synthesis, and the release of RNAP from the DNA What does a Prokaryotic promoter look like? How does RNA polymerase (RNAP) recognize one?  Prokaryotic promoters contain 2 highly conserved regions RNAP binds a similar region in the promoters of many different genes Transcription “-35” spacer “-10” TTGACA TATAAT This diagram compares the Promoter sequences from 12 different E. coli genes -10 region  Roles of RNA polymerase (RNAP) Scans the DNA to identify a Promoter Initiates transcription Elongates the mRNA chain Terminates transcription And interacts with Regulatory proteins: Activators and/or Repressors The process of Transcription in Prokaryotes  Transcription occurs in 3 distinct phases: 1. Initiation: assembly of the machinery to carry out transcription 2. Elongation: the process of making an RNA copy of the DNA sequence 3. Termination: stopping transcription in the right place; Release of the RNA transcript, and also RNAP 1. Transcription Initiation RNA polymerase (RNAP) scans the DNA looking for a promoter RNA polymerase (RNAP) Transcription Initiation (contd) The DNA strands are unwound and RNAP initiates synthesis of RNA in the 5’ to 3’ direction Template strand of DNA 2. Elongation RNAP moves downstream unwinding the DNA as it goes, and elongating the RNA transcript Elongation – the Template strand of DNA determines the sequence of the bases in the RNA Incoming RNA nucleotides 5 ’ end of 3. Termination Transcription of a Terminator sequence element in the DNA results in release of the RNA transcript and of RNA polymerase Terminator element RNAP disengages from DNA 2. Transcription in Eukaryotes is much more complicated  Major differences between Eukaryotic and Prokaryotic transcription (1) Transcription occurs exclusively in the nucleus which is surrounded by a double membrane (2) In the nucleus DNA is wrapped around histones to give chromatin (3) Chromatin must be in an ‘open conformation’ to allow RNAP access to the DNA (4) Different kinds of RNA polymerase (RNAP) In Prokaryotes: a single RNAP In Eukaryotes: RNAP I - makes rRNA RNAP II - makes mRNA RNAP III - makes tRNA Plants also have: RNAP IV and V (gene silencing) (5) Gene structure is much more complicated in Eukaryotes Upstream Control Transcription start site elements (Enhancers) Promoter Coding region of gene The coding region of most Eukaryotic genes is interrupted by segments of non-coding DNA called Introns The coding segments are known as Exons In some genes, introns make up most of the gene’s length (6) Promoters often have many Upstream Control Elements, (= Enhancers) that bind Transcription Factors Upstream Control elements (Enhancers) promoter (7) RNA polymerase II, which transcribes protein-coding genes, associates with many different Regulatory proteins: such as Transcription Factors  Transcription in Eukaryotes also occurs in 3 distinct phases 1. Initiation: assembling the machinery to begin transcription 2. Elongation: the process of making an RNA copy of the DNA sequence 3. Termination: stopping transcription in the right place on the DNA Transcription initiation in Eukaryotes highlighting the ‘TATA box’ Transcription found in most promoters initation site RNAP II is recruited and a (TIC) (= pre-mRNA) The production of mRNA in Eukaryotes requires multiple processing steps  mRNA processing steps in Eukaryotes 4. A 5’ CAP and a 3’ polyA tail are added to the pre-mRNA ends 5. The pre-mRNA contains EXONS and INTRONS 6. The INTRONS are spliced out of pre-mRNA in the nucleus by spliceosomes 7. The processed mature mRNA must then be exported from the nucleus to the cytoplasm via nuclear pores  Spliceosomes remove introns from pre-mRNA Exon 1 Intron Exon 2 5’ pre-mRNA Spliceosome Exon 1 Exon 2 Spliced mature mRNA with 5’ ligated exons (giving a continuous coding region) 2. Intron removal via splicing creates a continuous coding segment in the mature mRNA Exon Exon Exon Intron removal via splicing and addition of a polyA tail The ‘mature’ processed mRNA is now ready for export to the cytoplasm SUMMARY: Information flow in the eukaryotic cell: (1) Transcription in the nucleus produces a large precursor mRNA (pre-mRNA) The average length of pre-mRNA in humans is 27,000 nucleotides (2) The pre-mRNA is modified and processed Modifications: (a) Addition of a 5’-cap (b) Addition of a polyA tail at the 3’-end Processing: (c) Removal of Introns via splicing The average length of mature mRNA in humans is 1,200 nucleotides (3) The ‘mature’ mRNA is exported to the cytoplasm The mature processed mRNA is exported to the cytoplasm In the cytoplasm: It associates with Ribosomes And the information in the mRNA is Translated to produce a polypeptide (= protein) Regulating Gene Expression 1. Regulation of gene expression in Prokaryotes: Operons  Organisms respond to changes in their external and internal environments by altering gene expression But gene expression is costly from a metabolic viewpoint So organisms need to be able to regulate how and when their genes are expressed (turned on) in order to: a. Grow and develop b. Compete for limited resources; c. Survive adverse conditions  Operons are the fundamental unit of gene organization and regulation of gene expression in Prokaryotes 1. An operon is a set of genes encoding enzymes each of which is required in the same metabolic pathway 2. The genes are clustered together on the chromosome, and form a single transcription unit 3. Transcription of the operon produces a single mRNA that encodes all of the required enzymes Historically, the first investigations of how gene expression might be regulated were carried out in bacteria Specifically: How E. coli responds when the milk sugar lactose becomes available in its growth environment If lactose is present: a set of genes called the lactose (lac) operon are switched ON These genes encode the enzymes required to metabolize lactose But if lactose is absent: the lac operon is switched OFF  Genetic structure of the lac operon Regulatory gene lacI codes for Repressor Transcription unit: 3 genes encoding the Control region: proteins needed for lactose metabolism Promoter & Operator The proteins required for lactose metabolism The proteins required for lactose metabolism: (i) Lactose permease (encoded by the lacY gene) A membrane transporter: imports lactose into the cell (ii) Beta-galactosidase (encoded by the lacZ gene) Cleaves lactose to give glucose and galactose (iii) Galactoside acetyltransferase (encoded by the lacA gene)  The lactose operon shows inducible gene expression E. coli prefers glucose to all other carbon sources However, if no glucose is available, it can use other sugars such as lactose E. coli responds within 15 minutes, if lactose becomes available It responds by expressing the 3 genes of the lac operon Expression of these genes is said to be induced by lactose  Lactose induces expression of the lac operon A. If lactose is absent: The lac operon is not being actively transcribed (i.e. it is turned OFF) B. If lactose is present: Transcription of the lac operon is induced Within 15 minutes expression of the operon increases by approximately 1000 fold  Key questions 1. Why is the lac operon turned OFF when lactose is absent? 2. How does lactose induce (turn ON) expression of the lac operon’s genes?  The lac operon’s control region Regulatory scenario 1: Lactose is absent Repressor binds to the Operator which prevents RNA polymerase from Repressor attaching to the promoter and transcribing the genes downstream. Expression of the operon is OFF  Regulatory scenario 2: Lactose is present Repressor cannot bind to operator If lactose present: lactose binds to the Repressor, changing its shape, and making it unable to bind to the Operator Summary: 1. The operon contains a Regulatory Gene called lacI that codes for an allosteric regulatory protein: Repressor. 3. Repressor can bind to a DNA sequence called the operator located between the Promoter and the 3 genes lacZ, lacY, lacA. 4. When the Repressor is bound to the operator, RNA pol cannot bind the promoter to initiate transcription of the operon’s genes. 5. If lactose is present, it binds to the Repressor. This changes its shape so that it cannot bind to the operator. RNA polymerase is then able to transcribe the operon.  General principles of gene regulation revealed by the lac operon and other systems The existence of: 1. Regulatory Genes: genes that encode Regulatory Proteins that control the expression of other genes 2. Regulatory Proteins: that may function either as Repressors (or Activators) of gene expression 3. Regulatory sequences in the DNA itself: e.g. Operators (and Enhancers) - to which Regulatory Proteins bind, to influence gene expression 2. Regulation of gene expression in Eukaryotes Thanks for your attention!  How do cells become what they are? A typical mammal is constructed from ~ 2000 different cell types Yet, every cell contains the same genes ~ 25,000 in humans Most genes are not being actively expressed The expression of ‘cell- specific’ sets of genes plays a decisive role in both determining, and maintaining cell and tissue identity, and their biological functions  Regulation of Gene expression In any given cell type ~ 20% of genes are in the ON (active) state that is: being transcribed by RNA polymerase and the resulting mRNA being translated to produce the encoded protein These genes are said to be expressed genes In different cell types, different sets of genes are expressed: e.g. in red blood cells very few genes are expressed; but the gene coding for haemoglobin is expressed at a very high level What aspects of Gene Expression are subject to regulation? Regulation determines: 1. Where a gene should be expressed i.e. in which cells and tissues? In plants, the Rubisco gene is only expressed in green tissues It is not expressed in root tissue. 2. Timing: when, and for how long, a gene should be expressed e.g. some genes are expressed for only a few days during embryo development and are never expressed in the adult 3. How much expression i.e. low or high levels of expression In plants the Rubisco gene codes for the most abundantly expressed protein in leaves. Rubisco protein = 50% of the total leaf protein i.e. the gene is very highly expressed  Regulation of gene expression in Eukaryotes is much more complex Because the process of gene expression itself is more complex: 1. Eukaryotic cells contain a nucleus 2. DNA is not naked, but is packaged with histone proteins to form chromatin 3. The coding region of most eukaryotic genes is interrupted by non-coding segments: Introns There are many opportunities for regulating gene expression in Eukaryotes 1. Modification of chromatin - to make the DNA accessible for transcription 2. Regulation of Transcription 3. Regulation of Splicing - for removal of introns 4. Transport of mRNA to the cytoplasm 5. Factors affecting the half-life of mRNA (mRNA degradation) 6. Regulation of Translation of mRNA b. Regulating gene expression by controlling Transcription  Gene structure is much more complicated in Eukaryotes 1. Promoters often have many Upstream Control Elements, (e.g. Enhancers) that strongly influence promoter function Transcription unit Enhancers Promoter 2. RNA polymerase II, which is recruited to the promoter region may also interact with different kinds of regulatory proteins: such as: General Transcription Factors, and Specific Transcription Factors Model for action of distant Enhancers on gene expression in Eukaryotes Specific Transcription Factors (Activators) Many 1000’s of bases between Distant upstream control element (Enhancer DNA sequences) …../contd a DNA-bending protein brings the Enhancers with their bound Activators close to the gene’s Promoter The bound Activators interact with: (i) Mediator proteins (ii) General transcription factors (iii) RNA polymerase II (RNAPII) and set up a multi-protein Transcription Initiation Complex at the promoter Cell type-specific gene expression  Differential gene expression Consider two different cell types: a liver cell a lens (eye) cell Both cells types contain the same genes But in a liver cell: only the Albumin gene is being expressed While in the lens cell: only the Crystallin gene is being expressed How cell type-specific gene expression works:  General principles of gene regulation The existence of: 1. Regulatory Genes: genes that encode Regulatory Proteins that control the expression of other genes 2. Regulatory Proteins: that may function either as Repressors (or Activators) of gene expression 3. Regulatory sequences in the DNA itself: e.g. Operators (and Enhancers) - to which Regulatory Proteins bind, to influence gene expression