Lecture 7 - Regulation of Gene Expression PDF

Lecture 7 - Regulation of gene expression Gene Expression ▪ In addition to controlling gene expression in response to environmental or other stimuli, a bacterial cell needs to be able to produce some proteins (e.g. structural proteins, ribosomal proteins) at very high levels, while other proteins...

Lecture 7 - Regulation of gene expression Gene Expression ▪ In addition to controlling gene expression in response to environmental or other stimuli, a bacterial cell needs to be able to produce some proteins (e.g. structural proteins, ribosomal proteins) at very high levels, while other proteins (such as some regulatory proteins) are only produced at a very low level ▪ Although these levels may go up and down in response to environmental changes, or at different stages of growth, the maximum potential expression of genes is fixed at different levels ▪ Fortunately, the mechanisms used for fixed and variable controls are similar Lecture 7 - Regulation of gene expression Gene Expression may 314 ▪ Gene expression can be controlled at any of 5 stages: 1. Chromatin rearrangements: regulates chromatin conformation and DNA's accessibility for transcription 2. Transcriptional control: regulates RNA polymerase binding to a promoter and initiation of transcription Most Prokaryotic gene regulation occurs via transcriptional control 3. Post-Transcriptional control: regulates modifications to RNA after transcription 4. Translation control: regulates initiation and elongation steps of translation 5. Post-translational control: regulates modifications to proteins after translation one step elongation termini Lecture 7 - Regulation of gene expression Gene Expression DNA Protein t ▪ Past focus has been on understanding transcription initiation ▪ There is increasing elucidation of post-transcriptional and translational regulation ▪ Regulation relies on precise protein-DNA and protein-protein contacts Lecture 7 - Regulation of gene expression Gene Regulation 2. Regulated gene 1. Housekeeping gene ▪ Under constitutive expression ▪ ▪ Constantly expressed in approximately all cells Levels of the gene product rise and fall with the needs of the organism ▪ Such genes are inducible - able to be turned on ▪ Such genes are also repressible - able to be turned off T.ES onloff ▪ Structural genes: encoding proteins ▪ Regulatory genes: encoding products that interact with other sequences and affect the transcription and translation of these sequences ▪ Regulatory elements: DNA sequences that are not transcribed but play a role in regulating other nucleotide sequences Lecture 7 - Regulation of gene expression Gene Expression ▪ Positive regulation is that regulation in which the presence of a specific regulatory element increases the expression of genetic information quantitatively ▪ Negative regulation is regulation in which the presence of specific regulatory elements diminishes the expression of genetic information always gene regulator D gene structure Lecture 7 - Regulation of gene expression Positive and Negative regulation of the Lac operon req M F Y ▪ In the Lac operon there are three structural genes: 1. LacY encodes the enzyme permease which is necessary for allowing lactose to enter the cell 2. lacZ encodes the protein β-Galactosidase Ia 3. LacA encodes the protein transacetylase ▪ Both LacZ and LacA play a role in lactose catabolism (the breakdown of lactose) ___ Lecture 7 - Regulation of gene expression Positive and Negative regulation of the Lac operon s ▪ All three of these proteins work together to help the cell bring in and metabolize lactose when it's available in the environment, this breakdown of lactose provides energy for the cell ▪ These genes all share a promoter and an operator ▪ the Regulatory Gene Lac I which encodes an active, repressor when there's no lactose in the environment Lecture 7 - Regulation of gene expression Positive and Negative regulation of the Lac operon RNApoly When there is NO lactose in the environment Lac I Promotor • This repressor can bind to the operator Lac I Promotor • This prevents RNA polymerase from binding to the promoter of the operon • Which therefore limits the expression of the Lac genes indeed5 A Lackoto produceprefer Repressor RNA polymerase NO Expression ▪ This is beneficial to the cell because it wouldn't want to waste energy and resources to make AA the proteins to break down lactose when lactose isn't present in the cell Lecture 7 - Regulation of gene expression Positive and Negative regulation of the Lac operon Lac I ▪ For these Lac operon structural genes to be expressed two things need to happen: First, The repressor needs to be deactivated so it cannot bind to the operator and prevent RNA polymerase from transcribing the lac genes Promotor Repressor lactose/ Allo lactose Lac I off Promotor Repressor In order for this event to occur lactose needs to be present when lactose is present allo lactose can be derived from it For Allo lactose acts as an inducer for this operon by binding to the repressor changing the shape of its DNA binding domain and making it inactive Deactivated In this way, the presence of lactose helps promote the transcription of the genes necessary for lactose catabolism Lac I Promotor on Repressor Lecture 7 - Regulation of gene expression Positive and Negative regulation of the Lac operon Lac I Promotor Second, The binding of the CRP c-AMP complex to the promoter Cyclic Adenosine Mono-Phosphate also called cyclic AMP (cAMP) is a small nucleotide that is synthesized from ATP by the enzyme adenyl cyclase c-AMP receptor protein CRP cyclic AMP receptor protein which is named CRP is a DNAbinding protein that when attached to c-AMP then binds to the Lac operon promoter when the CRP-cAMP complex binds to the Lac operon promoter it can help RNA polymerase interact with the promoter and transcribe the Lac operon genes Cyclic Adenosine Monophosphate c-AMP CRP-cAMP complex Lac I Promotor RNA polymerase Lecture 7 - Regulation of gene expression Glucose Repression What controls whether the CRP-cAMP complex is available to bind to the Lac promoter? ▪ cAMP production is dependent on the presence of glucose Lactose glucose fgr responseenvironment condition ▪ When both lactose and glucose are present in the cell's environment glucose is the preferred molecule to break down the cell can more efficiently use glucose than it can lactose (the cell prefers to use glucose for energy over lactose) ▪ It will repress the Lac Operon even though lactose is present, this allows the cell to focus on the use of glucose for energy Lecture 7 - Regulation of gene expression Glucose Repression When glucose is available even if lactose is present: ➢ Decreased adenyl cyclase activity ➢ Less cAMP production ➢ NO formation of CRP-cAMP complex ➢ NO RNA polymerase interaction with promoter ➢ Lac genes NOT EXPRESSED Lecture 7 - Regulation of gene expression Positive and Negative regulation of the Lac operon Negative regulation Positive regulation Repressor CRP-cAMP complex Repressor When active, prevents RNA polymerase When attached to the promotor, supports binding RNA polymerase binding Lac I Promotor Repressor RNA polymerase NO Expression RNA polymerase Lecture 7 - Regulation of gene expression trp operon ggulator structant ▪ Bacteria such as E. coli need amino acids to survive ▪ Tryptophan is one such amino acid that E. coli can ingest from the environment ▪ E. coli can also synthesize tryptophan using enzymes that are encoded by five genes ▪ These five genes are next to each other in what is called the tryptophan (trp) operon ▪ If tryptophan is present in the environment, then E. coli does not need to synthesize it; the switch controlling the activation of the genes in the trp operon is turned off ▪ However, when tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized Lecture 7 - Regulation of gene expression trp operon when active repressor and Inactive When this repressor is in its inactive form it cannot repressor Inactive Repressor bind to the operator and therefore cannot limit Induce transcription of the structural genes in the trip operon In this situation, RNA polymerase can freely bind to Inactive Repressor the promoter and transcribe the structural genes leading to the production of enzymes that help make tryptophan RNA polymerase In this state, the operon is De-repressed meaning the structural genes are expressed Inactive Repressor Tryptophan Lecture 7 - Regulation of gene expression trp operon When there's an excess of tryptophan in the environment or in the cell it would be a waste of energy and resources for the cell to continue making tryptophan so the transcription of the structural genes should be limited Inactive Repressor Excess of tryptophan in the environment It turns out that tryptophan is a co-repressor of the trip operon it's called a co-repressor because it works with the repressor to prevent transcription of the structural genes It will bind to the inactive repressor changing its shape and making it active in this state the DNA binding region of the trip repressor is able to bind to the operator allowing it to prevent RNA polymerase from transcribing these genes Active Repressor r Active Repressor RNA polymerase NO Expression In this way a high concentration of tryptophan in the environment will limit transcription of these structural genes saving the cell lots of energy in this state the operon is repressed Lecture 7 - Regulation of gene expression trp operon Not enough tryptophan Tryptophan is excess RNA Active Repressor polymerase Inactive Repressor RNA Tryptophan polymerase NO Expression Inactive repressor Active repressor trp genes expressed trp genes NOT expressed present Tryptophan produces Tryptophan NOT produces in med Lecture 7 - Regulation of gene expression Two-component sensor regulator system in bacteria ▪ Types of regulatory systems: 1. Two-component regulator system (TCS) 2. Quorum sensing (QS) 3. Global regulators (GR) Lecture 7 - Regulation of gene expression Two-component sensor regulator system in bacteria ▪ TCS: It’s a signaling system used by bacteria to transfer signals from outside into cytoplasm to do that, sensors are required to detect chemical and/or physical signals found in its environment ▪ A system that allows prokaryotes to regulate cell metabolism in response to environmental fluctuations which leads to stress conditions Lecture 7 - Regulation of gene expression Two-component sensor regulator system in bacteria ORR ▪ TCS consists of a sensor kinase (Histidine kinase HK) that responds to specific signals by modifying the phosphorylated state of a cognate response regulator ▪ The HK senses specific signals, and that leads to activation of the kinase activity and autophosphorylation of a conserved histidine residue ▪ The phosphoryl group is subsequently transferred to a cognate response regulator to activate its activities Lecture 7 - Regulation of gene expression Two-component sensor regulator system in bacteria Why two components? most signal transduction stems contain two parts Examples: Lecture 7 - Regulation of gene expression Two-component sensor regulator system in bacteria Environmental signal Mechanism of TCS: Sensor Kinase Phosphorylate Histidine Kinase ATP → ADP Response Regulatory RNA Polymerase Transcription Protein Lecture 7 - Regulation of gene expression I Gene silencing - RNA interference (RNAi) ▪ RNA silencing is a novel gene regulatory mechanism that limits the transcript level by either suppressing transcription (transcriptional gene silencing [TGS]) or by activating a sequence-specific RNA degradation process (posttranscriptional gene silencing [PTGS]/RNA interference [RNAi]) ▪ MIntron stopgene expression The term RNA interference (RNAi) was coined to describe a cellular mechanism that uses the gene's own DNA sequence to turn it off, a process that researchers call silencing. In a wide variety of organisms, including animals, plants, and fungi, RNAi is triggered by double-stranded RNA (dsRNA) ▪ dsRNA Gene silencing is important for development, stress responses, and suppression of viruses, transposons, and transgenes ▪ Several epigenetic phenomena such as genome imprinting and X chromosome inactivation are caused by transcriptional gene silencing (TGS) 0 Lecture 7 - Regulation of gene expression Gene silencing - RNA interference (RNAi) alwase 1. siRNA dobffrard ➢ Short interfering RNA Types of RNA interference (RNAi): ➢ Exogenous dsRNA Or 2. miRNA ➢ micro RNA ➢ Non-coding RNA that forms hairpins or Lecture 7 - Regulation of gene expression Gene silencing - RNA interference (RNAi) Argonaut and the antisense RNA form RISC an RNAinduced silencing complex as in The double-stranded siRNA is cleaved into smaller fragments by an enzyme called Dicer (ribonuclease enzyme cleaves siRNA) Intron DICER cleaves siRNA in ~ 21bp fragments 20 22 Another protein from the Argonaut family then binds to the siRNA and discards the sense strand together with other proteins Argonaut my Since the antisense strand is complementary to the send strand of the mRNA the RISC complex is able to pair with the mRNA to either inhibit translation or cleaving of the mRNA both resulting in silencing brock Lecture 7 - Regulation of gene expression Gene silencing - RNA interference (RNAi) ▪ To study the function of a gene, loss-of-function mutants are made RNA interference: ▪ In research, double-stranded RNA is often used for silencing or gene knockdown ▪ If we want to find out a gene function, we usually make a mutant variant of this gene and compare the phenotypic differences with RNA interference we just synthesize double-stranded RNA complementary to a gene of interest and introduce this double-stranded RNA into an organism like C. elegans, then see a gene knockdown in this organism without making a mutant