Gene Regulation MCB 126 PDF

MCB 126 | Microbial Genetics Gene Regulation Prof. Cornista | December 15, 2021 TRANS NO. 6 ________________________________________________________________________________...

MCB 126 | Microbial Genetics Gene Regulation Prof. Cornista | December 15, 2021 TRANS NO. 6 ________________________________________________________________________________ OUTLINE OVERVIEW OF GENE REGULATION I. Major Modes of Regulation II. DNA-Binding Proteins III. Negative Control: Repression and Induction IV. Positive Control: Activation V. Global Control and the lac Operon VI. Transcriptional Control in Archaea SENSING AND SIGNAL TRANSDUCTION I. Two Component Regulatory Systems II. Quorum Sensing Figure 7.1 III. Other Global Control Networks DNA sequence, promoter region, ribosome binding site, structural gene that code for specific proteins and terminator OVERVIEW OF GENE REGULATION AUG in the beginning, UGA at end DNA transcribed to mRNA then translated to I. MAJOR MODES OF GENE REGULATION protein Gene expression: transcription of gene into mRNA Several mechanisms of protein activity followed by translation of mRNA into protein (Figure ○ Protein-protein interactions 7.1) ○ Covalent modification ○ DNA → mRNA → protein (central dogma of ○ Degradation molecular biology) ○ Feedback inhibition Most proteins are enzymes that carry out Protein enzyme regulated - not expressed at certain biochemical reactions point when enzyme not needed ○ For DNA replication (DNA pol I, III, gyrase, ligase, ○ Untranscribed 5’ and 3’ end primase, and several other proteins involved) ○ 5’-UTR and 3’-UTR ○ For transcription, RNA polymerase Constitutive proteins are needed at the same level Two major levels of regulation in the cell: ○ One controls the activity of preexisting enzymes all the time Post-translational regulation ○ During time of gene expression there are specific Very rapid process (seconds) proteins always expressed and there are other ○ One controls the amount of an enzyme proteins not expressed and should be regulated Regulates level of transcription Microbial genomes encode many proteins that are Before enzyme is produced not needed all the time Regulates translation ○ Expression of protein should be regulated Post-transcriptional level. Regulation helps conserve energy and resources Slower process (minutes) ○ Protein expression requires energy expenditure of Monitoring gene expression cell through several chemical reactions (turned off, ○ Reporter genes encode for an easy-to-detect if not needed) product ○ Only turned on when needed for biochemical Tagged in laboratory reactions of cell Can be fused to other genes Can be fused to regulatory elements Example: green fluorescent protein (GFP) Used to know if particular gene coded produces enzyme and protein is expressed ________________________________________________________________________________ | BIOMAN 2023 1 of 9 | Gene Regulation ________________________________________________________________________________ II. DNA-BINDING PROTEINS mRNA transcripts generally have a short half-life ○ Prevents the production of unneeded proteins (mechanism of controlling) Reason why it is degraded ○ mRNA transcript - processed mRNA Regulation of transcription typically requires proteins that can bind to DNA ○ Repressor proteins produced by regulatory genes Small molecules influence the binding of regulatory proteins to DNA ○ Proteins actually regulate transcription ○ Binds near promoter to block RNA pol from Figure 7.3 transcribing whole gene in the operator Operator region Dimeric protein normally binds to domain fitting Most DNA-binding proteins interact with DNA in a major groove of DNA sequence sequence-specific manner ○ If regulatory protein is dimer ○ Promoter has sequences found upstream (-10 and ○ DNA binding domain fits in major grooves and -35 sequences, TATA box, Pribnow region) along sugar-phosphate backbone Near promoter region ○ Happen over inverted repeats in DNA sequence Specificity provided by interactions between amino ○ Binding specific to inverted repeats acid side chains and chemical groups on the bases Protein-protein contacts hold protein dimer together and sugar-phosphate backbone of DNA Several classes of protein domains are critical for ○ When repressor protein binds to DNA, it binds on proper binding of proteins to DNA specific sites near promoter region and try to block ○ Helix-turn-helix (Figure 7.4) - for recognition of RNA pol from transcribing gene protein Major groove of DNA is the main site of protein First helix is the recognition helix binding Second helix is the stabilizing helix ○ Grooves of DNA play vital role in binding of Many different DNA-binding proteins from proteins Bacteria contain helix-turn-helix Inverted repeats frequently are binding site for lac and trp repressors of E. coli (lac regulatory proteins and trp operon) ○ Random sequences that are inverted in other side Protein recognizes inverted repeats → ○ Inverted repeats in promoter region and end of Second helix in DNA molecule stabilizes region binding of protein to DNA Homodimeric proteins: proteins composed of two Binding of repressor protein on identical polypeptides helix-to-helix turn ○ Two specific proteins linked together ○ Plays vital role ○ Has binding site within particular DNA sequence ○ There are also single proteins Protein dimers interact with inverted repeats on DNA ○ Each of the polypeptides binds to one inverted repeat (Figure 7.3) Figure 7.4 Part of docking of enzyme to DNA/substrate can be predicted through bioinformatics Majority of DNA binding mechanisms are well studied ________________________________________________________________________________ | BIOMAN 2023 2 of 9 | Gene Regulation ________________________________________________________________________________ Classes of protein domains ○ Zinc finger ○ Transcriptional level Protein structure that binds a zinc ion ○ Repression: preventing the synthesis of an Eukaryotic regulatory proteins use zinc enzyme in response to a signal (Figure 7.5) fingers for DNA binding Enzymes affected by repression make up a Certain DNA have specific binding small fraction of total proteins sites/cofactors Typically affects anabolic enzymes (e.g., arginine biosynthesis) Protein domains bind to specific metal binding sites of gene Zinc finger for regulatory protein ○ Leucine zipper Contains regularly spaced leucine residues Function is to hold two recognition helices in the correct orientation For prokaryotes Binding differs between prokaryotic and eukaryotic proteins Multiple outcomes after DNA binding are possible 1. DNA-binding protein may catalyze a specific reaction on the DNA molecule (i.e., transcription by RNA polymerase) Bind to catalyze production of protein or bind to inhibit transcription (not always Figure 7.5 regulatory) As cell number increases, total protein also Only regulatory if negative increases Some DNA are there to signal RNA pol to ○ Logarithmic increase transcribed whole gene When arginine if added/increased/synthesized into the medium, biosynthesis of arginine at certain 2. The binding event can block transcription point should be regulated (negative regulation) Induction: production of an enzyme in response to 3. The binding event can activate transcription a signal (Figure 7.6) (positive regulation) ○ Typically affects catabolic (breakdown) enzymes (e.g.. lac operon) III. NEGATIVE CONTROL: REPRESSION AND ○ Enzymes are synthesized only when they are needed INDUCTION No wasted energy Several mechanisms for controlling gene expression in bacteria ○ Well known system is of E. coli ○ These systems are greatly influenced by environment in which the organism is growing Presence or absence of certain substrates in culture Ex. media with glucose and lactose (bacteria first utilizes glucose, lac operon - lactose) ○ Presence or absence of specific small molecules Ex. Arginine, maltose, lactose ○ Interactions between small molecules and DNA-binding proteins result in control of transcription or translation Negative control: a regulatory mechanism that stops transcription Figure 7.6 ○ because there is already sufficient amount of enzyme Lac operon (catabolic) particularly if process is anabolic in nature Galactosidase only produced when there is lactose (synthesis) Product itself helps regulate and signal ○ No lactose → no galactosidase promoter to stop because a lot of arginine already present in cell ________________________________________________________________________________ | BIOMAN 2023 3 of 9 | Gene Regulation ________________________________________________________________________________ Inducer: substance that induces enzyme synthesis Corepressor: substance that represses enzyme synthesis Effectors: collective term for inducers and repressors ○ Allosteric and operator Effectors affect transcription indirectly by binding to specific DNA-binding proteins ○ Repressor molecules bind to an allosteric repressor protein Specific binding sites of protein Fit mechanism - it fits into particular site ○ Allosteric repressor becomes active and binds to Figure 7.8 region of DNA near promoter called the operator Operator binds to repressor at very specific Lac operon sites ○ lacZ, lacY and lacA involved in production of Operon: cluster of genes arranged in a linear beta-galactosidase (breakdown lactose) fashion whose expression is under control of a ○ No lactose → repressor protein binds to operator single operator → RNA pol does not transcribe genes to produce ○ Operator is located downstream of the promoter beta-galactosidase ○ Transcription is physically blocked when repressor ○ Lactose (inducer) in excess → lactose binds to binds to operator (Figure 7.7) repressor at specific site and remove repressor Enzyme induction can also be controlled by a protein from operator → RNA pol transcribes gene → beta galactosidase procured → breakdown or repressor lactose ○ Repressor is produced by regulator ○ Inducer is allolactose ○ Addition of inducer inactivates repressor, and transcription can proceed (Figure 7.8) Repressor's role is inhibitory, so it is called negative IV. POSITIVE CONTROL: ACTIVATION control Positive control: regulator protein activates the binding of RNA polymerase to DNA (Figure 7.9) ○ Regulator protein binds to operator in negative control ○ Regulator protein binds to RNA pol in positive control Activates RNA pol by binding to it → transcription occurs Maltose catabolism in E. coli ○ Maltose activator protein cannot bind to DNA unless it first binds maltose ○ Activator protein binds to maltose → maltose activator protein binds to DNA sequence (activator binding site which is not an operator) → Signals RNA pol to start transcription process Activator proteins bind specifically to certain DNA sequence Figure 7.7. ○ Called activator-binding site, not operator Arg (arginine) operon - negative regulation ○ RNA pol binds to promoter ○ 3 genes involved (argC, argB, argH) in production of arginine ○ Arginine in excess → binds to repressor (signals repressor to bind to operator) → repressor binds to operator → RNA pol blocked → process of transcription blocked ○ Products acts as corepressor and binds to repressor to signal stop of arginine production and transcription ________________________________________________________________________________ | BIOMAN 2023 4 of 9 | Gene Regulation ________________________________________________________________________________ B- activator binding site far from promoter (bends so that it is able to bind with RNA pol to signal transcription) Location of activator binding site may vary Genes for maltose are spread out over the chromosome in several operons (Figure 7.12) ○ Because it might be needed to produce them simultaneously ○ Each operon has an activator-binding site Necessary so that RNA pol can recognize gene ○ Multiple operons controlled by the same regulatory protein are called a regulon. If operon located in chromosome has several operons, they are controlled by the same regulatory proteins Regulons also exist for negatively controlled Figure 7.9 systems ○ malE, malF, malG - genes for transcription of maltose ○ Maltose activator binds to activator binding site with help of inducer (maltose) ○ Maltose binds to activator protein → Activator protein signal RNA pol for transcription to proceed (switching on) ○ No maltose → nothing binds to activator protein → activator protein will not signal RNA pol to bind to promoter → No transcription Promoters of positively controlled operons only weakly bind RNA polymerase Activator protein helps RNA polymerase recognize promoter ○ May cause a change in DNA structure. ○ May interact directly with RNA polymerase Activator-binding site may be close to the promoter or be several hundred base pairs away (Figure Figure 7.12 7.11) If genes are regulated at the same time, it falls in ○ Activator proteins helps RNA pol to recognize promoter regulon ○ Without activator protein, DNA will not be Mechanism of regulation is same/similar transcribed ○ Activator binding site always upstream V. GLOBAL CONTROL AND THE LAC OPERON Global control systems: regulate expression of many different genes simultaneously ○ Common in catabolite repression Catabolite repression is an example of global control ○ Repressing breakdown of particular organism ○ Synthesis of unrelated catabolic enzymes is repressed if glucose is present in growth medium (Figure 7.13) ○ lac operon is under control of catabolite repression Breakdown of lactose ○ Ensures that the "best" carbon and energy source is used first If there are two substrates, bacteria will first utilize best carbon source Ex. agar with glucose and lactose → Figure 7.11 glucose first utilized before lactose as source of carbon (lac operon turned off) Same with human body (carbohydrates A - Activator binding site near promoter utilized first before protein) ________________________________________________________________________________ | BIOMAN 2023 5 of 9 | Gene Regulation ________________________________________________________________________________ Diauxic growth: two exponential growth phases present in the same culture of bacteria (catabolite repression mechanism) Figure 7.13 Figure 7.15 Organism will grow and utilize glucose (lactose not yet utilized) ○ Inducer binds to repressor → prevents repressor Glucose exhausted → shift to utilization of lactose from binding into operator → transcription ○ Active repressor binds to operator → blocks as source of energy transcription (lactose absent) Beta-galactosidase - responsible for catabolism of ○ Binding site of CRP recruits RNA Pol (Note: Sir lactose mentioned active repressor proteins na nag-recruit pero ○ Glucose present → level of enzyme small CRP nasa diagram) ○ Glucose exhaustion → enzymes increase, lac Dozens of catabolic operons are affected by operon turned on and transcribed catabolite repression Cyclic AMP and CRP ○ Enzymes for degrading lactose, maltose, and ○ In catabolite repression, transcription is controlled other common carbon sources by an activator protein and is a form of positive Flagellar genes are also controlled by catabolite control (Figure 7.15) repression ○ Cyclic AMP receptor protein (CRP): is the ○ No need to swim in search of nutrients activator protein in global regulatory system ○ Flagellar genes inhibited/repressed at certain ○ Cyclic AMP: is a key molecule in many metabolic points control systems Catabolite repression process common in bacterial Derived from a nucleic acid precursor Is a regulatory nucleotide cell ○ So that if protein is not needed by cell, they are repressed/controlled ○ There are several mechanisms in controlling production of protein VI. TRANSCRIPTIONAL CONTROL IN ARCHAEA Different terms are used but same mechanism Archaea use DNA-binding proteins to control transcription ○ More closely resembles control by Bacteria than Eukarya ○ DNA sequence of Archaea more similar to Eukarya Repressor proteins in Archaea ○ NrpR is an example of an archaeal repressor protein from Methanococcus maripaludis (Figure 7.16) Represses genes involved in nitrogen metabolism ________________________________________________________________________________ | BIOMAN 2023 6 of 9 | Gene Regulation ________________________________________________________________________________ ○ Prerequisite to several other microbial mechanisms (Ex. bioluminescence) ○ If quorum sensing is inhibited → prevention of production of toxins in pathogenic bacteria (density dependent) Each species of bacterium produces a specific autoinducer molecule (Figure 7.20) ○ Diffuses freely across the cell envelope ○ Reaches high concentrations inside cell only if many cells are near ○ Binds to specific activator protein and triggers transcription of specific genes ○ Signals cells that there are other cell near each other Quorum sensing is unique to bacteria ○ Has implication on mechanisms performed by groups of bacterial populations (ex. Toxin production) ○ Figure 7.16 ○ Also have specific receptor proteins ○ Repress genes involved in nitrogen metabolism ○ NrpR released → TBB and TFP bind to RNA pol → signals RNA pol for transcription to take place ○ NrpR blocks TFB and TBP from binding → no transcription SENSING AND SIGNAL TRANSDUCTION I. TWO COMPONENT REGULATORY SYSTEMS Prokaryotes regulate cellular metabolism in response to environmental fluctuations Figure 7.20 ○ External signal is transmitted directly to the target Quorum sensing protein found in chromosome of ○ External signal is detected by sensor and bacteria transmitted to regulatory machinery (signal Acyl homoserine lactone (AHL) - signalling molecule transduction) Most signal transduction systems able to penetrate into cell are two-component regulatory ○ Released by other bacteria to tell that there are systems other microbes near ○ Goes in cell → binds to activator protein → signal genes involved in quorum sensing to be expressed II. QUORUM SENSING (AHL synthase) → produce and release AHL to Prokaryotes can respond to the presence of other signal other bacteria within the same locality cells of the same species ○ Activates cell to cell communication present in Quorum sensing: mechanism by which bacteria bacterial population assess their population density Inhibit signal molecules to prevent quorum sensing ○ When organisms clump to each other at (gene regulation) certain population as a protective mechanism ○ Toxin production can also be prevented → lead to bioluminescence ○ Inhibit signal molecules or activator proteins ○ There are several other factors which can target ○ A type of cell-to-cell-communication quorum sensing (ex.secondary metabolites) ○ Ensures that a sufficient number of cells are Several different classes of autoinducers present before initiating a response that, to be effective, requires a certain cell density (e.g., toxin ○ Acyl homoserine lactone (AHL) was the first production in pathogenic bacterium) autoinducer to be identified ○ To reach certain population density (clumping) Quorum sensing first discovered as mechanism ○ They communicate with each other regulating light production in bacteria including ○ Very important mechanism well studied in bacteria Aliivibrio fischeri (Figure 7.21) ○ Lux operon encodes bioluminescence ________________________________________________________________________________ | BIOMAN 2023 7 of 9 | Gene Regulation ________________________________________________________________________________ ○ Lux gene involved for bioluminescence can be inactivated Insertional inactivation (insert gene between lux operon) Once inactivated, quorum sensing also inactivated and organism no longer able to fluoresce If organism spreads out in plate, they are communicating with each other through quorum sensing Figure 7.22a Staphylococcus aureus ○ Secretes small peptides that damage host cells or alter host's immune system ○ Under control of autoinducing peptide (AIP) Activates several proteins that lead to production of virulence proteins (Figure 7.22b) Figure 7.21 Plate with Vibrio species that luminesce Streak in nutrient agar ○ Plate glows in dark room as long as high population ○ Common in Vibrio species Examples of quorum sensing ○ Virulence factors ○ Switching from free-living to growing as a biofilm (controlled by quorum sensing) If quorum sensing controlled, biofilm is also controlled Ex. bacteria grows on surface of catheter and forms biofilm, pathogens form on surfaces a biofilm Quorum sensing is present in some microbial eukaryotes Quorum sensing likely exists in Archaea Virulence factors ○ Escherichia coli 057:H7 Shiga toxin-producing strain Figure 7.22b Produced through quorum sensing Produces AHL AI-3 Quorum sensing plays a vital role in several Epinephrine plus norepinephrine plus Al-3 microorganisms bind to sensor molecules in plasma ○ Good control mechanism in preventing growth and membrane proliferation of group of microorganism by targeting Activates motility, enterotoxin its pathways or operon that controls mechanism of production, and production of quorum sensing virulence proteins (Figure 7.22a) Biofilm formation ○ Growth of microorganisms on surfaces ○ Pseudomonas aeruginosa Produces polysaccharides that increase pathogenicity and antibiotic resistance when in biofilm (clump in numbers to evade antibiotic) More pathogenic when biofilm Common and causes diseases Can cause pneumonia Extremely pathogenic Inhibit quorum sensing → no biofilm → reduce pathogenicity Two quorum-sensing systems ________________________________________________________________________________ | BIOMAN 2023 8 of 9 | Gene Regulation ________________________________________________________________________________ Produces AHLs and cyclic di-guanosine monophosphate (c-di-GMP) Leads to exopolysaccharide production and flagella synthesis (Figure 7.23) Can thrive on specific surfaces (Ex. tubes, catheter) Figure 7.23 Increase in cell population because of signal molecules → attachment (formation of flagella for them to attach to each other) → thick biofilm Monitored through fluorescence or tagged with dye Once in biofilm, they evade antibiotic ○ Strength in numbers ○ Less likely to be affected by antibiotics III. OTHER GLOBAL CONTROL NETWORKS Several other global control systems ○ Aerobic and anaerobic respiration ○ Catabolite repression ○ Nitrogen utilization ○ Oxidative stress ○ SOS response ○ Heat shock response ABDURAHMAN ALQUERO ________________________________________________________________________________ | BIOMAN 2023 9 of 9