Lecture 13 Midterm Review Lecture PDF

Quiz 3: Covers Lectures 9-11 Quiz 3 closes TODAY! It will be available on Brightspace until Wednesday Oct. 16th at 11:59pm. There are 8 multiple choice questions in this quiz. You will have 30 minutes to complete the quizzes once initiated. Reminder: The quizzes are worth 20% of the final course...

Quiz 3: Covers Lectures 9-11 Quiz 3 closes TODAY! It will be available on Brightspace until Wednesday Oct. 16th at 11:59pm. There are 8 multiple choice questions in this quiz. You will have 30 minutes to complete the quizzes once initiated. Reminder: The quizzes are worth 20% of the final course grade. Six online quizzes (multiple choice questions) spread through the term. They will be a mix of problem-solving and conceptual questions. Your top five quizzes will be used to tabulate your total quiz mark.  Midterm: Friday October 18th Midterm covers material in lectures 2-12. In-person exam. Midterm will be written during class time (11:35am-12:55pm) and in this room (TB360), or TB204 if you’re left-handed, or the McIntyre Centre for students with accommodations. Midterm will be a closed book exam. You can use a calculator. Mix of multiple choice and written answer, problem-solving and conceptual questions. Use the quizzes and worksheet questions to study, and I will be posting some additional practice questions *Chapter Lecture Date Topic Readings 1 Sept 4 (Wed) Course Introduction 1 2 Sept 6 (Fri) DNA Structure, Replication and Mutations I 10, 12, 18 3 Sept 11 (Wed) DNA Structure, Replication and Mutations II 19 4 Sept 13 (Fri) Gene Expression: Overview I 13 5 Sept 18 (Wed) Gene Expression: Overview II 13, 14 Midterm 6 Sept 20 (Fri) Gene Expression: Overview III 15, 18 7 Sept 25 (Wed) Eukaryotic Gene Regulation I 11, 17 8 Sept 27 (Fri) Eukaryotic Gene Regulation II 17 9 Oct 2 (Wed) Eukaryotic Gene Regulation III 17 10 Oct 4 (Fri) Eukaryotic Gene Regulation IV 14, 17 11 Oct 9 (Wed) Recombinant DNA Technologies I 19 12 Oct 11 (Fri) Recombinant DNA Technologies II 19 13 Oct 16 (Wed) Review Class 14 Oct 18 (Fri) MIDTERM EXAM (Lectures 2-13) Review Lecture: slides with critical concepts and techniques to know for the midterm. Bring your questions on any muddy points!  Midterm: Format The midterm will be a mix of multiple choice (8), shorter answer (4-6), and longer answer questions (2-3). Answer directly on the exam paper. There are multiple versions of the midterm exam. Equal in difficulty. Remember to bring pencils and eraser or pen. Write legibly! You may use a non-programmable calculator. Put all other items (e.g. bags) under your desk or leave it at the front of the class. Question 1 from Lecture 12 Worksheet 1. A biotechnology company has recently discovered a 13-amino acid polypeptide that stimulates milk production in cows. A sequence of DNA containing the open reading frame of the peptide is shown below with the start (ATG) and stop (TAA) codons underlined (RNA-like strand). There are no introns. 5’-GTACGATGGGTACTTGTGAGAAGAGCACTGGGTTCACTGCGTGTTAATCGAA-3’ 3’-CATGCTACCCATGAACACTCTTCTCGTGACCCAAGTGACGCACAATTAGCTT-5’ (a) Indicate the sequences of the two 20-nucleotide primers that would be used to amplify by PCR the 42-base pair region containing the open reading frame (start to stop codons, inclusively). Be sure to designate the 5’ and 3’ ends of the primers. PRIMER 1: 5’-ATGGGTACTTGTGAGAAGAG-3’ PRIMER 2: 3’-GACCCAAGTGACGCACAATT-5’ PRIMER 2 (written 5’ to 3’, left to right): 5’-TTAACACGCAGTGAACCCAG-3’ Question 1 from Lecture 12 Worksheet 5’-GTACGATGGGTACTTGTGAGAAGAGCACTGGGTTCACTGCGTGTTAATCGAA-3’ 3’-CATGCTACCCATGAACACTCTTCTCGTGACCCAAGTGACGCACAATTAGCTT-5’ (a) Indicate the sequences of the two 20-nucleotide primers that would be used to amplify by PCR the 42-base pair region containing the open reading frame (start to stop codons, inclusively). Be sure to designate the 5’ and 3’ ends of the primers. PRIMER 1: 5’-ATGGGTACTTGTGAGAAGAG-3’ PRIMER 2: 5’-TTAACACGCAGTGAACCCAG-3’ (b) Calculate the Tm of each of your primers using the manual method shown in class. Tm = 2°C * (A + T) + 4°C * (C + G) Primer 1: Tm = 2°C * (11) + 4°C * (9) = 58°C Primer 2: Tm = 2°C * (10) + 4°C * (10) = 60°C Question 1 from Lecture 12 Worksheet (c) What would be the sequences of the primers if you wanted to use HindIII (A↓AGCTT) and EcoRI (G↓AATTC) to directionally sub-clone the amplified product into a plasmid digested with the corresponding enzymes, such that the HindIII site is next to the promoter and the EcoRI site is next to the transcription terminator? Add FULL restriction enzyme sites to the 5’-ends of the primers: 5’-AAGCTTATGGGTACTTGTGAGAAGAG-3’ 5’-GAATTCTTAACACGCAGTGAACCCAG-3’ In this case, HindIII primer should be on primer with the start codon (ATG), since that will be on the same side as the promoter. EcoRI on the primer with the stop codon (TAA – reverse complement -> TTA). Add 3-6 nucleotides on the 5’-ends to improve digestion with enzymes, can be any sequence but best to keep about 40-60% GC content of primers: 5’-XXXAAGCTTATGGGTACTTGTGAGAAGAG-3’ (e.g. XXX = GTC) 5’-XXXGAATTCTTAACACGCAGTGAACCCAG-3’ (e.g. XXX = CAC)  Question 1 from Lecture 12 Worksheet (d) Provide the sequence of the amplified DNA segment after it has been digested with HindIII and EcoRI. Note that the arrows in the restriction sites denoted above indicate the sites of cleavage. Provide both the top and bottom complimentary strands (duplexed together) and designate the 5’ and 3’ ends. After PCR, the amplified product (or amplicon) will look like (red -> add on sequences): 5’-GTCAAGCTTATGGGTACTTGTGAGAAGAGCACTGGGTTCACTGCGTGTTAAGAATTCGTG-3’ 3’-CAGTTCGAATACCCATGAACACTCTTCTCGTGACCCAAGTGACGCACAATTCTTAAGCAC-5’ HindIII (A↓AGCTT) + EcoRI (G↓AATTC) 5’-GTCAAGCTTATGGGTACTTGTGAGAAGAGCACTGGGTTCACTGCGTGTTAAGAATTCGTG-3’ 3’-CAGTTCGAATACCCATGAACACTCTTCTCGTGACCCAAGTGACGCACAATTCTTAAGCAC-5’ 5’-AGCTTATGGGTACTTGTGAGAAGAGCACTGGGTTCACTGCGTGTTAAG -3’ 3’- ATACCCATGAACACTCTTCTCGTGACCCAAGTGACGCACAATTCTTAA-5’ 5’ overhangs for directional cloning into expression plasmid also digested with HindIII and EcoRI  What is a Gene? Gene Functional Context Structural Context (unit of heredity) (sequence of DNA) Gene enhancers transcribed to RNA promoter terminator open reading frame (mRNA) *a gene does not necessarily encode mRNA/protein, it could encode a structural or regulatory RNA (e.g rRNA, tRNA, miRNA) **initial RNA that is produced (e.g. pre-mRNA) usually needs to be processed to be functional ***more than one gene product is possible from a single locus (e.g. alternative splicing)  Molecular Nature of Genetic Material ‘Anlage’ = Genes (factors) Genetic material must have three key properties: 1) Replication: stores genetic information and accurately transmitted from parent to offspring 2) Gene Expression: material controls phenotype of the organism (coded information), & contains complex info 3) Mutable: must be able to change on rare occasion to allow for variation Structure of DNA Double Helix -anti-parallel orientation of complementary strands -strands held together by by interchain H-bonds -complementary base pairing results in specific association of the two chains of the double helix Convention: read chain 5’ to 3’ ACTGA -> 5’-ACTGA-3’ 5’-ACTGA-3’ 3’-TGACT-5’  Nucleic Acid Hybridization Hybridization is based on base-pair complementary and can be: DNA-DNA, RNA-RNA, or DNA-RNA duplexes Need to start with single-stranded nucleic acids (e.g. denatured) Naturally occurring: e.g. RNA folding (e.g. rRNAs and tRNAs), siRNAs, miRNAs and CRISPR-based immunity Many techniques: e.g. PCR, DNA sequencing, in situ hybridization (e.g. FISH or mRNA transcript localization), DNA microarrays (genomics), Southern and Northern blotting, screening genomic and cDNA libraries  DNA Synthesis DNA polymerases replicate DNA in a 5’ to 3’ direction only DNA polymerases only add deoxyribonucleotides to the 3’ end of a growing nucleotide chain, using a single strand of DNA as template, which has been exposed by localized unwinding of the DNA double helix DNA Replication  Polymerase Chain Reaction (PCR) Amplify DNA sequence in vitro: Kary Mullis 1993 Nobel Prize Ingredients: 1. DNA template with at least some sequence known 2. Primers (oligonucleotides) complementary to the sequence at each end of the region to be amplified 3. Four deoxyribonucleotides (dNTPS – A, G, C, and T) 4. DNA Polymerase (thermostable; e.g. Taq Polymerase) 5. Buffer with appropriate salts (e.g. MgCl2) Thermocycler: Step 1. Heat to 92-95ºC to denature DNA strands Step 2. Cool to typically 50-60ºC to allow primers to anneal (primers are in vast excess over template) Step 3. Typically raise to 72ºC to allow thermostable DNA polymerase (e.g. Taq) to replicate DNA Cycle: repeat steps 1-3 many times (30 cycles is typical) PCR: Polymerase Chain Reaction (PCR), Another Look at the 1st Few Cycles:  Primer Design Primers generally have the following properties: Length of 18-24 bases (but can be longer, especially if adding non-template sequence to the 5’-end) 40-60% G/C content Melting temperature (Tm) typically 50-65 (annealing temperature will usually be 3-6 lower) Estimate Tm by using this formula: Tm = 2°C * (A + T) + 4°C * (C + G) *more accurate Tm calculators are online (e.g. NEB Tm calculator) 3’-end is critical for specificity Primer pairs should have a Tm within 5°C of each other Primer pairs should not have complementary regions (avoid ‘primer dimers’) Real-Time Quantitative PCR (qPCR) Fluorescence-based detection of amplification products during the PCR reaction in real time Measures input quantity of a nucleic acid by determining the number of cycles required to reach a set level of product In contrast, traditional PCR is used to amplify DNA using end point analysis to distinguish products Exponential increase in product is limited, eventually reaching a plateau PCR end-point analysis is not appropriate for quantification Real-time qPCR enables quantification during the exponential phase Source: Sean Taylor, BioRad qPCR Fluorescence Detection Methods 5’ nuclease Assays SYBR Green (e.g. TaqMan Probes) qPCR machines  Real-Time Quantitative PCR (qPCR) Ct value - or - The Cq or Ct value of a reaction is determined by the amount of template present at the start of the amplification reaction. High starting template will give low Ct value; small amount of starting template will give a high Ct value DNA-dependent RNA Polymerases (RNAPs) Synthesize RNA from DNA templates (transcription) Overall Rxn: DNA template determines which base is added (complementary base pairing) Synthesis proceeds in a 5’ to 3’ direction (adding to 3’-OH) Unlike DNA synthesis, no primer is needed to initiate RNA synthesis Targeted to specific genomic DNA sequences (genes)  Some Essential Terminology Nontemplate (RNA-like) Template Another Look: Overview of Transcription  Generalized Components of a Gene Encoding a Protein Gene non-template mRNA strand 5’ 3’ promoter terminator 5’ 3’ 3’ open reading frame (mRNA) 5’ template +1 ATG Stop strand 5’-UTR (untranslated region) 3’-UTR … -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 … “upstream” “downstream” *note: enhancers are DNA sequences that are not considered part of the core transcription unit, but these DNA sequences influence the level of transcription and are often very far away from the promoter sequence (we will return to this)  Transcriptional Initiation Varies Between Prokaryotes and Eukaryotes Eukaryotic genes often have enhancers: Can be thousands of base pairs away from the promoter Required for efficient transcription  Multiple Eukaryotic RNA Polymerases Three distinct RNA polymerases in nuclei of eukaryotes: 1. RNAP I: located in nucleolus, synthesizes precursors of most rRNA (except 5S rRNA) 2. RNAP II: located in nucleoplasm, synthesizes mRNA precursors (protein-coding genes), snoRNAs, some miRNAs, and some snRNAs 3. RNAP III: located in nucleoplasm, synthesizes small RNAs such as 5S rRNA, tRNAs, some miRNAs, and some snRNAs + RNAP IV and RNAP V in plants for siRNAs + Mitochondrial RNA Polymerase (monomeric, like T7 RNAP) + Chloroplast RNA Polymerase (plants, prokaryotic-like)  Eukaryotic Promoters Eukaryotic RNAPs do not have single, removable σ subunit Eukaryotic RNAPs cannot accurately initiate transcription on their own Need accessory proteins called General Transcription Factors (GTFs) to recognize promoters and recruit RNAP to the transcription start site (basal transcription apparatus) Eukaryotic promoters are far more complex and diverse than prokaryotic promoters – especially those involving RNAP II, which synthesizes mRNA Three core eukaryotic RNAPs recognize different types of promoters  RNA Polymerase II Promoters Promoters are complex! *not every gene transcribed by RNAP II will have all these core promoter elements -> lots of diversity **regulatory promoter elements are even more diverse, and these affect the rate of transcription initiation (distinct from enhancers which act from far away) Assembly of GTFs and Transcription Initiation TBP binds TATA box * TFIIB determines start site TFIIH induces open complex formation preinititiation for RNA synthesis complex to begin  TATA-binding protein (TBP) TBP recognizes TATA-box and induces large bend in DNA Note: many class II core promoters lack TATA box - often contain Inr element at -6 to +11 TBP is universal eukaryotic transcription factor! TBP required for RNAP I, RNAP II, and RNAP III initiation: :SL1 = TBP + TAFs for RNAP I :TFIID = TBP + TAFs for RNAP II :TFIIIB = TBP + TAFs for RNAP III RNAPII Transcription Initiation: Connections with Enhancers via Mediator  Elongation and Termination of RNAP II Transcription After RNAP II initiates RNA synthesis and produces a short transcript, machinery shifts to elongation mode Phosphorylation of one of the subunits of RNAP II RNAP II clears promoter leaving behind most GTFs (except TFIIF and TFIIH) Sequences signalling transcriptional termination in eukaryotes have not been precisely identified Termination is imprecise (transcript is processed)  RNA Processing Prokaryotes vs. Eukaryotes Prokaryotes: - transcription/translation are coupled - mRNAs often encode more than one protein (polycistronic) - most mRNAs translated without further modification Eukaryotes: - transcription/translation are spatially separated - primary transcripts generally not functional - mRNAs generally encode only one protein (monocistronic), or various related proteins due to alternative RNA splicing - mRNAs undergo extensive modification while still in nucleus  Processing of Eukaryotic mRNAs Produces Mature Transcripts 1. Addition of ‘cap’ to 5’-end of nascent transcript (protects RNA from degradation and required for translation of mRNA) capping enzyme adds “backward G” Transcribed bases Methylated cap – Triphosphate bridge not transcribed  Processing of Eukaryotic mRNAs Produces Mature Transcripts 2. poly(A) tail added to 3’-end (protective function and important for efficient translation of mRNA into protein) 3. RNA splicing – coding information is fragmented into exons (introns removed) 4. Internal modifications, e.g. methylation of some A residues, N6-methyladenosine (m6A) Structure and Expression of a Typical Eukaryotic Gene  Alternative Splicing Produces Different mRNAs from the Same Primary Transcript  Nature of the Genetic Code - read continuously from a fixed starting point in mRNA Genetic Code: - triplet code - degenerate (most amino acids are specified by more than one codon) - non-overlapping - continuous (no punctuation)  Reading Frames *an mRNA has 3 potential reading frames but generally only one used mRNA 5’-… - 3’ N-… …- C *initiating AUG codon (start codon) sets reading frame *DNA duplex has 6 potential reading frames (AUG -> ATG)  Eukaryotic mRNAs - 7methyl-GTP cap is essential for mRNA binding by eukaryotic ribosomes and enhances the stability of mRNAs by preventing degradation by 5’-exonucleases - poly(A) tail enhances both stability and translational efficiency of eukaryotic mRNAS - there is NO Shine-Dalgarno sequence at the 5’-end of eukaryotic mRNAs  Initiation Phase in Eukaryotes Small ribosomal subunit binds to 5' cap, then scans the mRNA for the first AUG codon Initiator tRNA carries Met Initiation factors (not shown) play a transient role CAP-independent translation can occur on some eukaryotic mRNAs, sometimes in response to special conditions -> internal ribosome entry site (IRES), usually in 5’-UTR The poly(A) Tail of Eukaryotic mRNA Plays a Role in the Initiation of Translation CAP-independent translation can occur on some eukaryotic mRNAs, sometimes in response to special conditions -> internal ribosome entry site (IRES), usually in 5’-UTR  Mutations Genes normally transmitted unchanged from generation to generation due to high fidelity of DNA replication (human replication has error rate of about 3 bps during copying of 6 billion bps of genome!) and DNA repair mechanisms Mutations: Usually neutral, sometimes bad, but occasionally offer a selective advantage (typically referring to heritable mutation) Types of Mutations: 1. Point mutations (base substitutions) :transitions are purine to purine (A to G or G to A) -or- pyrimidine to pyrimidine (C to T or T to C) :transversions are purine to pyrimidine (A to C, A to T, G to C, or G to T) -or- pyrimidine to purine (C to A, C to G, T to A, or T to G) 2. Deletions or Insertions: usually 1 bp, but can be much bigger 3. Inversions: 1800 rotations of a segment of DNA (small or big segments) 4. Epigenetic: DNA sequence itself is unchanged (e.g. DNA methylation or histone modifications, and these can be heritable) Base Substitutions, Insertions and Deletions Types of Mutations in the Coding Sequence of Genes AUG AUG AUG AUG AUG  Mutations in the Coding Sequence of a Gene can Alter the Gene Product Silent mutations do not alter the amino acid sequence Degenerate genetic code – most amino acids have >1 codon Missense mutations replace one amino acid with another Conservative – chemical properties of mutant amino acid are similar to the original amino acid For example aspartic acid [(−)charged] → glutamic acid [(−)charged] Nonconservative – chemical properties of mutant amino acid are different from original amino acid For example aspartic acid [(−)charged] → alanine (uncharged)  Mutations in the Coding Sequence of a Gene can Alter the Gene Product Nonsense mutations change codon that encodes an amino acid to a stop codon (UGA, UAG, or UAA) Result in production of a truncated protein Frameshift mutations result from insertion or deletion of nucleotides with the coding region No frameshift if multiples of three are inserted or deleted  Point Mutations in Non-Coding Regions Can Affect Gene Expression Mutations in Regulatory and Other Noncoding Sequences: - 5’- and 3’-splice sites, and also branch site - binding sites for RNA Polymerase (and associated factors) - binding sites for regulatory transcription factors - polyadenylation signal - ribosome binding sites - other sites that regulate translation - sites that influence RNA stability Overview of Eukaryotic Gene Regulation  Protein-DNA Interactions To access genetic information, proteins must be capable of interacting with DNA Replication and transcription require large numbers of proteins to interact with each other, as well as with DNA Two Types of Interaction: 1) Non-specific: interactions mainly between functional groups on protein and phospho-ribose backbone of DNA e.g.) histones, some proteins involved in replication 2) Specific: recognize specific sequences of nucleotides (base contacts) as well as non-specific portions of DNA e.g.) transcription factors (general and specific types)  Mapping Protein-DNA complexes DNA ‘footprinting’ DNaseI - - = DNA only control = increasing amounts of protein, *NOTE: only one strand labelled, e.g. purified transcription factor create a ladder of DNA fragments Electrophoretic Mobility Shift Assay (EMSA) Electrophoretic Mobility Shift Assay (EMSA) Electrophoretic Mobility Shift Assay (EMSA) Chromatin: Chromosomal DNA and Proteins  Chromosomal Packaging and Function Heterochromatin – highly condensed, usually inactive transcriptionally Darkly stained regions of chromosomes Constitutive – condensed in all cells (for example most of the Y chromosome) Facultative – condensed in only some cells and relaxed in other cells (for example position effect variegation, X chromosome in female mammals) Euchromatin – relaxed, usually active transcriptionally Lightly stained regions of chromosomes DNase I Hypersensitive Sites Within Chromatin (‘in vivo’ hypersensitivity) DNase I hypersensitive sites: more open chromatin configuration site, upstream of the transcription start site Source: Wang et al., 2012, PLoS One 7(8): e42414 Relaxation (opening) of the chromatin structure allows transcription factors access to binding sites on the DNA  The Nucleosome Core is an Octamer of Two Each of Histones H2A, H2B, H3, and H4 ~160 bp of DNA wrap twice around core of eight histones (octamer) The positive charges (Arg and Lys) at the N-termini of H2A, H2B, H3 and H4 attract the negative charges of the phosphates of DNA 40 bp of linker DNA connects adjacent nucleosomes Histone H1 associates with linker DNA as it enters and leaves the nucleosome core  Chromatin Remodeling and Histone Modifications Activation of eukaryotic transcription is dependent on two things: 1. Relief of repression imposed by chromatin structure (nucleosomes) 2. Interaction of RNAPs with the promoter and transcription regulatory proteins Chromatin Remodeling Complexes  The Four Core Histone Tails Can Be Modified With Chemical Groups Histone tails extend out from nucleosome, are platforms for modification Enzymes can add chemical groups (acetyl groups, methyl groups, phosphate groups, ubiquitin, etc.) Modified tails can alter nucleosomes and bind chromatin modifier proteins Modifications in N-terminal tails of histones: Lys, Arg, Ser, Thr and His residues Histone Tail Modifications Alter Chromatin Structure - Acetylation Acetylation Histone acetyltransferases add acetyl groups to histone tails Prevents close packing of nucleosomes Favors expression of genes in euchromatin The process is reversed by histone deacetylases Histone Tail Modifications Alter Chromatin Structure - Methylation Methylation Histone methyltransferases add methyl groups to histone tails Effect depends on specific amino acid modified (activation or repression) Adding methyl group to H3 lysine 9 favors heterochromatin formation The process is reversed by histone demethylases  Histone Writers, Erasers and Readers Histone ‘Writers’: acetyltransferases, methyltransferes, kinases, ubiquitinases Histone ‘Erasers’: deacetylases, demethylases, phosphatases, de-ubiquitinases Histone ‘Readers’: post-translational modifications of histone N-terminal tails are recognized by proteins (‘readers’) that exert function on gene expression (e.g. bromodomain and chromodomain proteins) Source: Strahl Lab, Univ. of North Carolina Chapel Hill  Histone Writers, Erasers and Readers Histone ‘Writers’: acetyltransferases, methyltransferes, kinases, ubiquitinases Histone ‘Erasers’: deacetylases, demethylases, phosphatases, de-ubiquitinases Histone ‘Readers’: post-translational modifications of histone N-terminal tails are recognized by proteins (‘readers’) that exert function on gene expression (e.g. bromodomain and chromodomain proteins) Source: Strahl Lab, Univ. of North Carolina Chapel Hill Histone Modifications Affect Transcription Histone acetyl transferases (HATs) acetylate histone tails; many transcription factor co-activators are HATs Histone acetylation opens the chromatin – favours gene expression Histone methyltransferases (HMTases) can activate or repress transcription; some HMTases are coactivators and others are corepressors Histone acetylation and methylation are dynamic – modifications can be taken off rapidly by histone deacetylases or histone demethylases  DNA Methylation Another change in chromatin structure associated with transcription is methylation of cytosine residues, which occurs most commonly when adjacent to guanine nucleotides (‘CpG’ methylation). This is distinct from histone methylation! Heavily methylated DNA is associated with repression of transcription in eukaryotes, whereas transcriptionally active DNA is usually unmethylated So called ‘CpG’ islands are found near transcription start sites (promoters) -> methyl groups are removed before initiation of transcription An association exists between DNA methylation and histone deacetylation, both of which repress transcription  Overview of ‘Epigenetic’ Regulation of Gene Expression Source: Wikipedia *Some of these changes in chromatin state, which are not changes in DNA sequence itself, can be passed down during cell division and even sometimes passed to future generations (epigenetics)  Chromatin Immunoprecipitation (X-ChIP) Assay X-ChIP = crosslinked ChIP *know the order of these steps and the reason for each step  Major Cis-Acting Regulatory Elements Core Promoter – DNA sequence that is usually directly adjacent to the gene. Bound by General Transcription Factors (GTFs) Often have a TATA box: TATA A Allow basal level of transcription (unregulated) Regulatory Promoter – Other more gene-specific transcription factors bind nearby at the regulatory promoter  Promoters: Core + Regulatory Promoters contain ‘consensus’ sequences that are mixed and matched in different combinations and in difference promoters. Different transcription factors bind to each consensus sequence, so each promoter responds to a unique combination of transcription factors.  Major Cis-Acting Regulatory Elements Enhancers – DNA sequence that can be far away from gene Augment or repress the basal level of transcription May be located either 5’ or 3’ to the transcription start site Still function when moved to different positions or orientations relative to promoter Reporter Genes Identify Enhancers in Eukaryotes Enhancers can be identified by: Constructing a recombinant DNA molecule that has a putative enhancer sequence fused to a promoter + reporter gene such as the green fluorescent protein (GFP), luciferase (LUC) or β- glucuronidase (GUS) Generating a transgenic organism or cell line that has the recombinant DNA in its genome or transiently Or LUC or GUS reporter genes, for example present. Deletion Constructs to Identify Important Enhancer cis-Acting DNA Elements Ozeki et al., 2001, Biochem J. Foot A +28, therefore includes core promoter Further Finer-Scale Mutations to Identify Important DNA regulatory sequences (cis elements) Ozeki et al., 2001, Biochem J. A + B, enhancer core promoter (-95 to +28)  Proteins Act in Trans to Control Transcription Initiation Transcription factors Sequence specific DNA binding proteins Bind to promoters and enhancers Recruit other proteins to influence transcription Three types: basal factors, activators, repressors Cryo-EM Structure Mediator of RNAP II/Mediator: Human Mediator: Mediator is a complex of more than 20 proteins Mediator doesn’t bind DNA directly – bridges RNA pol II at the promoter and activator or repressor proteins at the enhancer Binds the unphosphorylated form of Pol II but not the phosphorylated form. Phosphorylation of the CTD causes dissociation of Pol II from the mediator to enable initiation of transcription (switch to elongation mode).  Transcription Factors Bind to Sites on DNA and Regulate Transcription Coactivators: mediate interactions between DNA-binding transcription activators and Mediator. Coactivators do not bind DNA directly themselves. Some Coactivators also have histone acetyltransferase activity to locally further open the chromatin for GTFs / RNAP.  Mechanisms of Activator Effects on Transcription Activators bound at enhancers are responsible for much of the variation in levels of transcription of different genes Stimulate recruitment of Recruit coactivators to basal factors and RNA open chromatin structure pol II to promoters  Domains Within Activators Activator proteins have at least two functional domains (modular!) DNA binding domain—binds to specific enhancer Activation domain—binds to other proteins (basal factors or coactivators or mediator) Dimerization domain – some activators also have a domain that allows them to interact with other proteins Repressor Proteins Suppress Transcription Initiation by Recruiting Corepressors Corepressors have two alternate functions: Prevent RNA pol II complex from binding the promoter Modify histones to close chromatin structure Corepressors: Do not bind DNA directly themselves. Often recruit histone deacetyltransferases to locally close chromatin.  Repressor Proteins Can Act Through Competition With an Activator Protein An indirect repressor interferes with the function of an activator Competition due to overlapping binding sites Repressor binds to activation domain (quenching) Binding to activator and keeping it in cytoplasm Binding to activator and preventing homodimerization Cell-Type Specific Transcription is Achieved by Changes in Transcription Factors The function of trans acting proteins changes by: Allosteric interactions (e.g. steroid hormone receptor binds to enhancer only when bound to steroid hormone) Modification of transcription factors (e.g. phosphorylation) Transcription factor cascades Complex Regulatory Regions Enable Fine- Tuning of Gene Expression In humans, ~2000 genes encode transcriptional regulatory proteins Each regulatory protein can act on many genes Each enhancer has binding sites with varying affinities for activators and repressors  How Does an Enhancer Know Which Genes to Regulate? Insulators are sequences located between an enhancer and a promoter. They block access to the promoter.  Example: Galactose Metabolism in Yeast through GAL4 Transcription Factor GAL4 binds to the UASG site and controls the transcription of genes involved in galactose metabolism. *NOTE: ‘Upstream Activation Sequence’ (UAS) in yeast is a type of enhancer (acting at a distance)  Small RNAs Regulate mRNA Stability and Translation Specialized RNAs that prevent expression of specific genes through complementary base pairing; 21-30 nt long micro-RNAs (miRNAs) small interfering RNAs (siRNAs) Piwi-interacting RNAs (piRNAs) These small RNAs are found in eukaryotes and are responsible for a variety of different functions, including the regulation of gene expression, defense against viruses, suppression of transposons, and modification of chromatin structure.  RNA Interference (RNAi) RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic (transposons) and exogenous pathogenic nucleic acids (viruses), and regulates the expression of protein-coding genes. Differences between siRNAs and miRNAs Feature siRNA miRNA Origin mRNA, transposon, or RNA transcribed from distinct virus gene Cleavage of RNA duplex or single- Single-stranded RNA that forms stranded RNA that forms short hairpins of double- long hairpins stranded RNA Size 21–25 nucleotides 21–25 nucleotides Main modes RNA cleavage followed by RNA cleavage followed by of action degradation of mRNA; alter degradation of mRNA; inhibition chromatin structure of translation Target Genes from which they Genes other than those from were transcribed which they were transcribed  Mechanism of siRNA Production and Function Sources of double-stranded RNA (dsRNAs) that are precursors of siRNAs: Transcription of inverted repeats into an RNA molecule that then base pairs with itself to form dsRNA Simultaneous transcription of two different RNA molecules that are complementary to each other (form dsRNA) Infection by virus that makes dsRNA dsRNAs are processed by Dicer to produce short ~22 bp dsRNAs (usually multiple distinct small dsRNAs produced from original longer dsRNA): siRNAs form RNA-protein complex with Argonaute proteins (one of the strands) Interfere with gene expression or may destroy viral mRNAs (RISC -> Slicer) siRNAs are very useful experimental tools to selectively knock down expression of target genes –> RNAi-based reverse genetics  Mechanism of siRNA Production and Function Just one of the RNA strands (ssRNA) is used to form RISC includes *Usually siRNAs Slicer have exact complementarity RISC = RNA- to target RNA induced silencing complex (after cleavage, mRNA is further degraded)  Primary Transcripts Containing microRNAs (miRNAs) Most miRNAs are transcribed by RNA polymerase II, but some are transcribed by RNA polymerase III The primary transcripts have double-stranded stem loops pri-miRNA from primary transcript: inverted repeats miRNAs can be processed from introns of protein-coding transcripts:  miRNA Processing Drosha excises stem-loop from primary miRNA (pri- miRNA) to generate pre-miRNA Dicer processes pre-miRNA to a mature duplex miRNA One strand is incorporated into miRNA-induced silencing complex (RISC)  Two Ways That miRNAs Can Down- Regulate Expression of Target Genes When complementarity is When complementarity is perfect (often in plants): imperfect (usual in animals): Target mRNA is degraded Translation of mRNA target is repressed Usually the binding sites of miRNAs are in the 3’-UTR of the target mRNA  microRNAs 1000s of novel miRNAs in plants, worms, flies, and mammals! miRNAs play important roles in many diseases and disorders, e.g. abnormal expression of miRNAs play a role in many cancers (miRNAs used for cancer prognosis and treatment) Each miRNA can potentially base pair with sequences on hundreds of different target mRNAs (competition and co-regulation)  RNA Interference (RNAi) Screens RNA interference (RNAi) is an endogenous cellular process by which mRNAs are targeted for degradation by double stranded RNA (via siRNAs or miRNAs) You can artificially generate dsRNAs for a gene of interest that you want to silence and then test for a phenotype (usually knock-down the mRNA levels) Amenable to high-throughput, large-scale screening of many genes in a functional genomics screen (rather than just focusing on one gene) Mechanisms Regulating mRNA Translation Control of translation often occurs at initiation Small subunit of ribosome recognizes a complex structure built around the 5’ cap of the mRNA eIFG protein in this complex binds to poly-A binding protein (PABP) at the poly-A tail to circularize mRNA Regulating mRNA Translation in Response to Nutrients 4E-BP1 binds to initiation factor eIF4E, blocks initiation Presence of nutrients and growth factors in the environment leads to phosphorylation of 4E-BP1  Translational Control Through poly-A Tail Length Longer poly-A tails bind PABP more efficiently; translation initiation complex forms more efficiently Artificial Transformation of Bacteria (e.g. E. coli) E. coli: ‘workhorse’ of modern recombinant DNA technology does not naturally take up free DNA, but can be made to artificially 1. Expose bacteria to a salt solution (e.g. CaCl2) and apply heat shock in the presence of foreign DNA 2. Expose bacteria to electric current in presence of foreign DNA Plasmids: Selection of E. coli Transformants by Using an Antibiotic Resistance Gene Khan Academy NOTE: the vast majority of E. coli cells will NOT have taken up the plasmid, antibiotic selection is key to obtain only bacteria (colonies) that have taken up the plasmid. Only the transformants will grow on the selection media. Restriction Enzymes (Endonucleases) -restriction enzymes are endonucleases that recognize and cleave specific DNA sequences, many types -restriction enzymes are produced by bacteria for protection against viruses (digest viral DNA) -recognition site typically 4-8bp in length (Type II) -recognition site typically ‘palindromic’ (dyad symmetry) ‘sticky’ or ‘cohesive’ ends, either with 5’- overhangs or 3’- overhangs depending on the restriction enzyme  Restriction Mapping DNA can also be circular prior to digestion (e.g. plasmid)  DNA Methylation and Restriction Enzymes DNA methylation can affect the ability of a restriction enzyme to digest DNA! Dam and Dcm methylation in prokaryotes (usually use dam- dcm- E. coli) Mammalian CpG methylation  Cloning Fragments of DNA Genomes of animals, plants, and microorganisms are too large to analyze all at once Molecular cloning is a means to purify a specific DNA fragment away from all other fragments and make many identical copies of the fragment Two basic steps: Insert DNA fragments into cloning vectors to specialized chromosome- like carriers that ensure transport, replication, and purification of DNA inserts Transport recombinant DNA into living cells to be copied Group of replicated DNA molecules = DNA clone  Cloning Vectors: Plasmids Features: - have an origin of replication so that DNA can be replicated in host cell - handled easily (e.g. small) - unique restriction sites for cloning of DNA fragments - selectable markers for determining whether the clone has been transferred into cells and verify foreign DNA has been inserted into vector pUC19 Plasmid: A Typical Cloning Vector These restriction sites are part of the ‘multiple cloning site (MCS)’ or polylinker. These are all unique cutting sites in this plasmid. The MCS is within the partial lacZ+ gene to allow for selection of plasmids containing inserts (inserts disrupt the lacZ+ gene function)  Expression Vector Often in gene cloning, the goal is not just to replicate the gene but also produce the protein it encodes, ensuring transcription and translation. The coding sequence inserted can be eukaryotic in origin, so long as the sequence lacks introns. The genetic code is the same between humans and E. coli. For example, functional human insulin can be expressed and purified from E. coli using an expression vector.  Creating Recombinant DNA Molecules With Plasmid Vectors Typically dephosphorylate (5’-ends) the linearized plasmid to reduce self ligation  Host Cells Take Up and Amplify Recombinant DNA Transformation – the process by which a cell or organism takes up foreign DNA In E. coli, only 0.1% of cells will be transformed with plasmid Only cells with plasmid will grow on media with ampicillin Each cell with plasmid will produce a colony on agar plate, the millions of identical plasmids in colony are a DNA clone Construction of a Recombinant DNA Molecule Or an individual DNA fragment (EcoRI digested in this example) that comes from another plasmid, a PCR product, chemically synthesized DNA, etc.  Identifying Bacterial Cells Containing Plasmids with Desired DNA Inserts Example: blue / white colony screening  Identifying Bacterial Cells Containing Plasmids with Desired DNA Inserts Colony PCR is a convenient high-throughput method for determining the presence or absence of insert DNA in plasmid constructs. Individual transformants can either be lysed in water with a short heating step or added directly to the PCR reaction and lysed during the initial heating step. This initial heating step causes the release of the plasmid DNA from the cell, so it can serve as template for the amplification reaction. This direct colony PCR method bypasses both plasmid DNA isolation and restriction digest, providing a fast, easy, and inexpensive solution for screening cloned constructs.  Identifying Bacterial Cells Containing Plasmids with Desired DNA Inserts Colony PCR Primer Design, Three Scenarios: Usually followed by diagnostic restriction enzyme digests and DNA sequencing (Sanger method)  Directional Cloning By using two different restrictions enzymes, the digested insert will ligate into the recipient plasmid in only one direction based on the compatible overhangs (unlike if you digested with a single restriction enzyme, which ligates into the singly digested plasmid in either orientation). Directional Cloning: ‘Subcloning’ Example https://www.addgene.org/protocols/subcloning/ Directional Cloning: A Detailed Look  Cloning with PCR-Amplified Inserts (usually the best way to go!) NOTE: Many restriction enzymes do not cut DNA efficiently at the end of a linear piece. Should add 3-6 bases upstream of your restriction site to improve cutting efficiency. https://www.addgene.org/protocols/pcr-cloning/ Cloning with PCR-Amplified Inserts (usually the best way to go!) https://www.addgene.org/protocols/pcr-cloning/  Verification of Recombinant DNA Construct More typically a ligation reaction involving digested insert and plasmid 1. Verify insert is present in plasmid using colony PCR 2. Grow culture from single colonies, purify plasmid DNA, and do diagnostic restriction digests 3. Sequence the insert (e.g. to verify no PCR-induced mutations)