Homework answers / question archive / Reminders Watch the video lectures • Add to your notes so that you can understand the material • Replay/re-watch sections as necessary • Take breaks • Change the video speed as necessary; use shortcut keys Read the textbook

Reminders Watch the video lectures • Add to your notes so that you can understand the material • Replay/re-watch sections as necessary • Take breaks • Change the video speed as necessary; use shortcut keys Read the textbook

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Reminders Watch the video lectures • Add to your notes so that you can understand the material • Replay/re-watch sections as necessary • Take breaks • Change the video speed as necessary; use shortcut keys Read the textbook. Check the textbook for answers to your questions before posting on the Discussion Board. BIO 230 Lecture 3 : Prokaryotic Transcriptional Regulation Continued… 1) Recap of prokaryotic gene regulation 2) Bacteriophage Lamba 3) Synthetic Biology 4) Transcription Attenuation Readings (Alberts et al. custom text) Pages 400-405, 413-416, 876-878 2 3 Catabolite Activator Protein Trp Repressor Lac Repressor Recap: Prokaryotic Gene Regulation Example 1: The Tryptophan Operon Tryptophan repressor contains a Helix-Turn-Helix DNA binding motif (most common DNA-binding motif) Helix-Turn-Helix Binds in major groove of DNA double helix Tryptophan Repressor Tryptophan binding induces Conformational change Fits in major groove 4 Recap: Prokaryotic Gene Regulation To summarize: Negative regulation: Competition between RNA polymerase and repressor protein for promoter binding Positive regulation: activator protein recruits RNA polymerase to the promoter to activate transcription 5 Recap: Prokaryotic Gene Regulation Gene regulatory elements are typically close to the transcriptional start site of prokaryotic genes BUT regulatory elements can also be found Far upstream of gene Downstream of gene (eukaryotes) Within gene (introns; eukaryotes) 6 Recap: Prokaryotic Gene Regulation Some regulatory elements are distant from the transcriptional start site and influence transcription - How? DNA looping (Euk. Video) NtrC protein is a transcriptional activator DNA looping allows NtrC to directly interact with RNA polymerase to activate transcription from a distance Bacteriophage Lambda Virus that infects bacterial cells Positive and negative regulatory mechanisms work together to regulate the lifestyles of bacteriophage lamba Two proteins repress each others synthesis Bacteriophage Lambda Bacteriophage lambda can exist as one of two states in bacteria Under favorable bacterial growth conditions When host cell is damaged Two gene regulatory proteins are responsible for initiating this switch Bacteriophage Lambda Two gene regulatory proteins are responsible for initiating the switch between prophage and lytic pathways lambda repressor protein (cI) and Cro protein Repress each other’s synthesis, giving rise to the two states. Bacteriophage Lambda Bacteriophage lamba: a genetic switch State 1: Prophage Lambda repressor occupies the operator. blocks synthesis of Cro activates its own synthesis most bacteriophage DNA not transcribed Bacteriophage Lambda eg. bacteriophage lamba: a genetic switch State 2: Lytic Cro occupies the operator blocks synthesis of repressor allows its own synthesis most bacteriophage DNA is extensively transcribed What triggers switch? DNA is replicated, packaged, new bacteriophage released by host cell lysis Bacteriophage Lambda eg. bacteriophage lamba: a genetic switch What triggers switch between prophage and lytic states? Host response to DNA damage -switch to lytic state inactivates repressor Under good growth conditions repressor protein turns off Cro and activates itself positive feedback loop -maintains prophage state Example of a transcriptional circuit. Different types exist, control various biological processes Transcriptional Circuits Transcriptional Circuits eg. repressor protein eg. Cro / Repressor switch Transcriptional Circuits Transcriptional Circuits Positive Feedback loops can be used to create cell memory Transcriptional Circuits Transcriptional Circuits Feed-forward loops can measure the duration of a signal - both A and B required for transcription of Z Brief input B does not accumulate Z not transcribed Prolonged input B accumulates Z is transcribed Transcriptional Circuits Transcriptional Circuits Combinations of regulatory circuits combine in eukaryotic cells to create exceedingly complex regulatory networks Scientists can construct artificial circuits and examine their behavior in cells synthetic biology Gene circuit of developing sea urchin embryo Synthetic Biology Synthetic Biology eg. creating a simple gene oscillator using a delayed negative feedback circuit – “the repressillator” A: Lac repressor B: Tet repressor (response to antibiotic) C: Lambda repressor Predicted: delayed negative feedback gives rise to oscillations Introduced this circuit into bacterial cells and observed expression of the repressor genes Synthetic Biology Synthetic Biology Synthetic Biology: “the repressillator”, how does it work? 1) A expressed A expression 4) 2) B repressed 3) C expressed 4) C represses A expression Synthetic Biology Synthetic Biology: “the repressillator”, how does it work? 5) A repressed A expression 6) B expressed 7) C repressed 8) Repeat 1-4 Did it work? Synthetic Biology Synthetic Biology eg. creating a simple gene oscillator using a negative feedback circuit Looking at 1 Protein (Fluorescently tagged) Observed Predicted Increasing amplitude due to bacterial growth Transcriptional Circuits Feedback loops also circadian gene regulation ~ 24-hour cycle: eg. Drosophila http://www.hhmi.org/biointeractive/drosophila-molecular-clock-model Delayed Negative Feedback Loop Transcription Attenuation -In both prokaryotes and eukaryotes there can be a premature termination of transcription called Transcription attenuation -RNA adopts a structure that interferes with RNA polymerase -Regulatory proteins can bind to RNA and interfere with attentuation -Prokaryotes, plants and some fungi also use Riboswitches to regulate gene expression Transcription Attenuation Riboswitches Short RNA sequences that change conformation when bound by a small molecule eg. prokaryotic riboswitch that regulates purine biosynthesis Recall that bases making up DNA/RNA include: pyrimidines (C,T,U) purines (A,G) Transcription Attenuation Riboswitches eg. prokaryotic riboswitch that regulates purine biosynthesis Low guanine levels -Transcription of purine biosynthetic genes is on Transcription Attenuation Riboswitches eg. prokaryotic riboswitch that regulates purine biosynthesis High guanine levels -Guanine binds riboswitch -Riboswitch undergoes conformational change -Causes RNA polymerase to terminate transcription -Transcription of purine biosynthetic genes is off Remember to read the textbook. Check the textbook for answers to your questions. After reading the textbook, questions are welcome… please ask on the Discussion Board, and/or after classes. Help one another on the Discussion Board. Reminders Watch the video lectures • Add to your notes so that you can understand the material • Replay/re-watch sections as necessary • Take breaks • Change the video speed as necessary; use shortcut keys Read the textbook. Check the textbook for answers to your questions before posting on the Discussion Board. BIO 230 Lecture 4: Eukaryotic Gene Regulation 1) Eukaryotic transcriptional activation 2) Eukaryotic transcriptional repression Readings (Alberts et al. custom text) Pages 310-314, 187-193, 196-197, 198-201 2 Reminder from a couple of lectures ago… Transcriptional Regulation Gene expression in both prokaryotes and eukaryotes is regulated by: Gene Regulatory Proteins (transcription factors) Which bind specifically to: Regulatory regions of DNA (cis elements) Gene regulatory proteins can turn genes: -ON; Positive regulators; activators -OFF; Negative regulators; (eg. Trp operon) repressors 4 Transcriptional Regulation Recall that DNA is transcribed into RNA by the enzyme RNA polymerase 5 Transcriptional Regulation Cells produce several types of RNA: Different RNAs transcribed by different RNA polymerases in eukaryotes Prokaryotes have a single type of RNA polymerase Transcriptional Regulation Transcription initiation in eukaryotes requires many proteins: general transcription factors Help position RNA polymerase at eukaryotic promoters contain TATA box Required by nearly all promoters used by RNA polymerase II Eukaryotic Gene Regulation Eukaryotic transcription - RNA polymerase II transcribes protein coding genes - Requires five general transcription factors; TFIID, TFIIB, TFIIF, TFIIE, and TFIIH (prokaryotes only need one; σ factor) - Eukaryotic genomes lack operons - Eukaryotic DNA is packaged into chromatin which provides an additional mode of regulation - Eukaryotic transcriptional activation requires many gene regulatory proteins Eukaryotic Gene Regulation Eukaryotic transcription - Mediator acts an intermediate between regulatory proteins and RNA polymerase RNA Polymerase Eukaryotic Gene Regulation -Eukaryotic gene expression controlled by many regulatory proteins (~2000 encoded by the human genome) both activators and repressors -Gene regulatory proteins can act over very large distances, sometimes >10000 base pairs away - One mechanism is DNA looping Eukaryotic Gene Regulation Eukaryotic gene regulatory proteins often function as protein complexes on DNA Coactivators and corepressors assemble on DNA-bound gene regulatory proteins do not directly bind DNA Eukaryotic Gene Regulation Eukaryotic Activator Proteins Modular design: 1) DNA binding domain (DB) - recognizes specific DNA sequence 2) Activation domain (AD) - accelerates rate of transcription Can mix-and-match DBs and ADs Eukaryotic Gene Regulation How do Activator Proteins activate transcription? Attract, position and modify: General transcription factors Mediator RNA polymerase II They can do this either: 1) Directly by acting on these components 2) Indirectly modifying chromatin structure Eukaryotic Gene Regulation 1) Activator proteins can bind directly to transcriptional machinery or mediator and attract them to promoters (like prokaryotic activators) Eukaryotic Gene Regulation 2) Activator proteins can alter chromatin structure Nucleosomes are the basic structure of Eukaryotic chromatin - DNA wound around a histone octamer (H2A, H2B, H3, and H4 x 2) Eukaryotic Gene Regulation Nucleosomes pack as compact chromatin fibers Zigzag model Solenoid Model Transcriptional machinery cannot assemble on promoters tightly packaged in chromatin Activator proteins can alter chromatin structure and increase promoter accessibility How? Eukaryotic Gene Regulation 4 major ways activator proteins can alter chromatin 1. 2. 3. 4. Eukaryotic Gene Regulation Nucleosome structure can be altered by chromatin remodeling complexes in an manner to increase promoter accessibility 1) Nucleosome sliding ATP-dependent Eukaryotic Gene Regulation 2, 3) Nucleosome removal and histone exchange Requires cooperation with histone chaperones Eukaryotic Gene Regulation 4 major ways activator proteins can alter chromatin 1. 2. 3. 4. Signal for chromatin remodeling Eukaryotic Gene Regulation 4) Histone modifying enzymes produce specific patterns of histone modifications histone code phosphorylation Enzyme: kinase acetylation Enzyme: acetyltransferase methylation Enzyme: methyltransferase Addition of phosphate group: Addition of acetyl group: Addition of methyl group: Histone modifications occur on specific amino acids of histone tails Eukaryotic Gene Regulation The histone code: Specific modifications to histone tails by histone modifying enzymes “writers” Histone H3 Eukaryotic Gene Regulation The histone code: Code- “reader” proteins can recognize specific modifications and provide meaning to the code Histone H3 Eukaryotic Gene Regulation Transcriptional regulation using the histone code eg. human interferon gene promoter Step 1: Activator protein binds to chromatin DNA and attracts a histone acetyltransferase (HAT) Step 2: HA acetylates lysine 9 of histone H3 and lysine 8 of histone H4. Eukaryotic Gene Regulation Transcriptional regulation using the histone code eg. human interferon gene promoter Step 3: Activator protein attracts a histone kinase (HK) Step 4: HK phosphorylates serine 10 of histone H3. Can only occur after acetylation of lysine 9 Eukaryotic Gene Regulation Transcriptional regulation using the histone code eg. human interferon gene promoter Step 5: Serine modification signals the acetyltransferase to acetylate lysine 14 of histone H3 Histone code for transcription Initiation is written Eukaryotic Gene Regulation Transcriptional regulation using the histone code eg. human interferon gene promoter Step 6: TFIID and a chromatin remodelling complex bind to acetylated histone tails and initiate transcription Eukaryotic Gene Regulation Transcriptional Repression - Unlike prokaryotes, eukaryotic repressor proteins rarely compete with RNA polymerase for access to DNA - Instead use a variety of mechanisms to inihibit transcription 1) Interfering with activator function Eukaryotic Gene Regulation Transcriptional Repression Interfering with activator function 2) 3) Eukaryotic Gene Regulation Transcriptional Repression by altering chromatin structure 4) Eukaryotic Gene Regulation Transcriptional Repression by altering the histone code 5) 6) Eukaryotic Gene Regulation Guided by gene regulatory proteins histone “reader” and “writer” proteins can establish a repressive form of chromatin histone code can spread This chromatin can be stabilized Eukaryotic Gene Regulation Spreading the histone code along chromatin carried out by Reader-writer complexes Eukaryotic Gene Regulation DNA methylase enzyme is attracted by Reader and methylates nearby cytosines in DNA DNA methyl-binding proteins bind methyl groups and stabilize structure -Methylation and therefore gene expression patterns can be inherited A process called epigenetic inheritance Remember to read the textbook. Check the textbook for answers to your questions. After reading the textbook, questions are welcome… please ask on the Discussion Board, and/or after classes. Help one another on the Discussion Board. Webinar Quiz 1 Started: Sep 21 at 12:37pm Quiz Instructions Questions Question 1 Question 2 Question 3 Question 4 Webinar Questions for Review Webinar 1-2 (Lectures 3 & 4). Time Elapsed: Hide Attempt due: Sep 22 at 12pm O Minutes, 9 Seconds Question 1 1 pts The figure below illustrates a complex transcriptional circuit. Which circuit is NOT indicated in the rectangles (blue, green, and red)? D-E-F input G output XYZ Positive Feedback Loop O Negative Feedback Loop Flip-Flop Device O Feed Forward Loop Next Not saved Submit Quiz Webinar Quiz 1 Started: Sep 21 at 12:37pm Quiz Instructions Question Ques Ques Ques Ques Time Elapsed Attempt due: Sep Webinar Questions for Review Webinar 1-2 (Lectures 3 & 4). es Question 2 1 pts Which of the following is the best example of a coactivator? O CAP Histone acetyl transferase. OTFIID Cro • Previous Next Not saved Submit Quiz Webinar Quiz 1 Started: Sep 21 at 12:37pm Quiz Instructions Webinar Questions for Review Webinar 1-2 (Lectures 3 & 4). Question 3 1 pts For the chimeric protein containing a LexA DNA binding domain and a Gal4 activation domain, which of the following will most likely occur? The chimeric protein will inhibit the transcription of genes normally regulated by LexA. The chimeric protein will inhibit the transcription of genes normally regulated by Gal4. The chimeric protein will activate the transcription of genes normally regulated by Gal4. The chimeric protein will activate the transcription of genes normally regulated by LexA. Previous Next Not saved Submit Quiz Ques Webinar Quiz 1 Started: Sep 21 at 12:37pm Quiz Instructions Webinar Questions for Review Webinar 1-2 (Lectures 3 & 4). Time El Attempt D Question 4 1 pts Which of the following would you expect to see at the interferon gene promoter and accompanying histones if an activator protein was present, but histone kinase was inhibited? Acetylated H3K9 and H4K8 Acetylated H3K9 and H4K8: Phosphorylated H3510 Acetylated H3K9 and H4K8: Phosphorylated H3510: TFIID and chromatin remodelling complex binding No histone acetylation or phosphorylation • Previous Not saved Submit Quiz