BIO230 Lecture 3: Prokaryotic Transcriptional Regulation Continued (Fall 2024) PDF

BIO230 Section 1: Regulation of Genome Expression Lecture 3: Prokaryotic Transcriptional Regulation Continued BIO230H1F: From Genes to Organisms Prof. Kenneth W. Yip, Ph.D. Assistant Professor, Teaching Stream Cell & Systems Biology,...

BIO230 Section 1: Regulation of Genome Expression Lecture 3: Prokaryotic Transcriptional Regulation Continued BIO230H1F: From Genes to Organisms Prof. Kenneth W. Yip, Ph.D. Assistant Professor, Teaching Stream Cell & Systems Biology, University of Toronto Fall 2024 Hematopoiesis BIO230 Section 1 Lecture 3: Prok Transcriptional Regulation Continued 1. Recap of prokaryotic gene regulation 2. A genetic switch example from bacteriophage lambda 3. Transcriptional circuits 4. Synthetic biology Pg. 431-434 Differentiated Cells Maintain Their Identity 5. Transcription attenuation Transcription Circuits Allow the Cell to Carry Out Logic Operations Pg. 445-446 Post-Transcriptional Controls Transcription Attenuation Causes the Premature Readings (Alberts et al.): Termination of Some RNA Molecules All listed on Quercus Riboswitches Probably Represent Ancient Forms of Gene Control Pg. 937-938 Circadian Clocks Use Negative Feedback Loops to Control Gene Expression BIO230 Lecture 1-3 2 Recap: Prokaryotic Gene Regulation BIO230 Lecture 1-3 3 Recap: The Tryptophan Operon Tryptophan repressor contains a helix-turn-helix DNA binding motif (most common DNA-binding motif) Helix-Turn-Helix Tryptophan Repressor Binds in the major groove Tryptophan binding induces of the DNA double helix Conformational change Protein fits into the major groove BIO230 Lecture 1-3 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 BIO230 Lecture 1-3 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) BIO230 Lecture 1-3 6 Recap: Prokaryotic Gene Regulation Some regulatory elements are distant from the transcriptional start site and influence transcription NtrC protein is a transcriptional activator DNA looping allows NtrC to directly interact with RNA polymerase to activate transcription from a distance BIO230 Lecture 1-3 7 A Genetic Switch Example from Bacteriophage Lambda Virus that infects bacterial cells Positive and negative regulatory mechanisms work together to regulate the lifestyles of bacteriophage lambda BIO230 Lecture 1-3 8 A Genetic Switch Example from Bacteriophage Lambda Bacteriophage lambda can exist as one of two states in bacteria Two gene regulatory proteins are responsible for initiating this switch and actually repress each other’s synthesis… BIO230 Lecture 1-3 9 A Genetic Switch Example from Bacteriophage Lambda Two gene regulatory proteins are responsible for initiating the switch between prophage and lytic pathways Lambda repressor protein (cI) and Cro protein They repress each other’s synthesis, giving rise to the two states BIO230 Lecture 1-3 10 A Genetic Switch Example from Bacteriophage Lambda State 1: Prophage Lambda repressor occupies the operator: blocks synthesis of Cro activates its own synthesis most bacteriophage DNA not transcribed BIO230 Lecture 1-3 11 A Genetic Switch Example from Bacteriophage Lambda State 2: Lytic Cro occupies the operator: blocks synthesis of lambda repressor allows its own synthesis most bacteriophage DNA is extensively transcribed DNA is replicated, packaged, new bacteriophage released by host cell lysis BIO230 Lecture 1-3 12 A Genetic Switch Example from Bacteriophage Lambda What triggers the switch between prophage and lytic states? It’s host response to DNA damage! switch to lytic state inactivates repressor Under good growth conditions, lambda repressor protein turns off Cro and activates itself in a positive feedback loop maintains prophage state The prophage-lytic control is an example of a transcriptional circuit Different types exist, control various biological processes BIO230 Lecture 1-3 13 Transcriptional Circuits BIO230 Lecture 1-3 14 Transcriptional Circuits Positive feedback loops can be used to create cell memory BIO230 Lecture 1-3 15 Transcriptional Circuits Feed-forward loops can measure the duration of a signal both A and B required for transcription of Z Brief input Prolonged B does not input B accumulate accumulates Z not Z is transcribed transcribed BIO230 Lecture 1-3 16 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. This is called synthetic biology BIO230 Lecture 1-3 17 An Example of Synthetic Biology: The Repressilator The Repressilator: scientists created a simple gene oscillator using a delayed negative feedback circuit 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 BIO230 Lecture 1-3 18 An Example of Synthetic Biology: The Repressilator BIO230 Lecture 1-3 19 An Example of Synthetic Biology: The Repressilator How does it work? 1) A expressed A expression 2) B repressed 3) C expressed 4) C represses A expression BIO230 Lecture 1-3 20 An Example of Synthetic Biology: The Repressilator How does it work? 5) A repressed A expression 6) B expressed 7) C repressed 8) Repeat 1-4 BIO230 Lecture 1-3 21 An Example of Synthetic Biology: The Repressilator Did it work? Looking at 1 Protein (Fluorescently tagged) Predicted Observed BIO230 Lecture 1-3 22 Transcriptional Circuits Feedback loops also circadian gene regulation ~ 24-hour cycle: eg. Drosophila http://www.hhmi.org/biointeractive/drosophila-molecular-clock-model BIO230 Lecture 1-3 23 Transcriptional 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 attenuation Prokaryotes, plants and some fungi also use riboswitches to regulate gene expression BIO230 Lecture 1-3 24 Transcriptional 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) BIO230 Lecture 1-3 25 Transcriptional Attenuation Riboswitches eg. prokaryotic riboswitch that regulates purine biosynthesis Low guanine levels -Transcription of purine biosynthetic genes is on BIO230 Lecture 1-3 26 Transcriptional 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 BIO230 Lecture 1-3 27 Review Question The figure below illustrates a complex transcriptional circuit. Which circuit is not indicated in the rectangles? A. Positive Feedback Loop B. Negative Feedback Loop C. Flip-Flop Device D. Feed Forward Loop BIO230 Lecture 1-3 28 Review the textbook and add to your notes. If that was too quick for you, or if you have additional questions, please review the textbook, review the recording (if available), post on the Discussion Board, stay for the Q&A sessions, and try ChatBIO230. This is your responsibility.