7.1 Major Modes of Regulation

7.1 Major Modes of Regulation
Gene expression: transcription of gene into mRNA followed by translation of mRNA into protein (Figure 7.1) Most proteins are enzymes that carry out biochemical reactions Constitutive proteins are needed at the same level all the time Microbial genomes encode many proteins that are not needed all the time Regulation helps conserve energy and resources by fine tuning protein levels

Levels of Regulation A Snapshot Figure 7.1 DNA RNA Protein –35 –10 +1
Promoter RBS Structural gene Terminator 5′ 3′ DNA 3′ 5′ Activation Repression Transcription (making RNA) RBS Start codon Stop codon RNA 5′ 3′ 5′-UTR 3′-UTR Translation (making protein) Figure 7.1 Gene expression and regulation of protein activity. Feedback inhibition Protein–protein interactions Mechanisms of controlling enzyme activity Protein Degradation Covalent modifications Figure 7.1

II. DNA-Binding Proteins and Transcriptional Regulation
7.3 Negative Control: Repression and Induction 7.4 Positive Control: Activation 7.5 Global Control and the lac Operon 7.6 Transcriptional Controls in Archaea

7.2 DNA-Binding Proteins mRNA transcripts generally have a short half-life Prevents the production of unneeded proteins Regulation of transcription typically requires proteins that can bind to DNA Small molecules influence the binding of regulatory proteins to DNA Proteins actually regulate transcription

7.2 DNA-Binding Proteins Most DNA-binding proteins interact with DNA in a sequence-specific manner Specificity provided by interactions between amino acid side chains and chemical groups on the bases and sugar–phosphate backbone of DNA Major groove of DNA is the main site of protein binding Inverted repeats frequently are binding site for regulatory proteins

7.2 DNA-Binding Proteins Homodimeric proteins: proteins composed of two identical polypeptides Protein dimers interact with inverted repeats on DNA

Types of DNA Repeats Mirror repeat

7.2 DNA-Binding Proteins Several classes of protein domains are critical for proper binding of proteins to DNA Helix-turn-helix (Figure 7.4)-one class of DNA binding domain First helix is the recognition helix Second helix is the stabilizing helix

A perfect fit for the major groove

7.2 DNA-Binding Proteins Classes of protein domains Zinc finger
Protein structure that binds a zinc ion Eukaryotic regulatory proteins use zinc fingers for DNA binding Leucine zipper Contains regularly spaced leucine residues Function is to hold two recognition helices in the correct orientation

Three important types of DNA binding domains

7.3 Negative Control: Repression and Induction
Defined as control that prevents transcription Several mechanisms in bacteria These systems are greatly influenced by environment in which the organism is growing Through presence or absence of specific small molecules Interactions between small molecules and DNA-binding proteins result in control of transcription or translation

Early on, microbiologists realized that bacteria could respond to environmental signals by starting or stopping to make enzymes: adaptation. Induction-occurs when environmental signal triggers the synthesis of an enzyme Repression-occurs when environmental signal prevents the synthesis of an enzyme

Induction: production of an enzyme in response to a signal (Figure 7.6) Typically affects catabolic enzymes (e.g., lac operon) Enzymes are synthesized only when they are needed No wasted energy

Induction Figure 7.6 Total protein Cell number β-Galacto- sidase
Figure 7.6 Enzyme induction. Lactose added Figure 7.6

Repression: preventing the synthesis of an enzyme in response to a signal (Figure 7.5) Enzymes affected by repression make up a small fraction of total proteins Typically affects anabolic enzymes (e.g., arginine biosynthesis)

Repression Figure 7.5 Cell number Total protein Arginine added
Figure 7.5 Enzyme repression. Arginine biosynthesis enzymes Figure 7.5

Inducer: a substance that induces enzyme synthesis Corepressor: a substance that represses enzyme synthesis (not same as repressor) Effectors: collective term for inducers and repressors Effectors affect transcription indirectly by binding to specific DNA-binding proteins

Paradigm system is the lactose (lac) operon of E. coli. Operon: group of genes related in that they work together Structural genes code for enzymes or soldier proteins Regulatory genes control the structural genes (Figure 7.7) Enzyme induction can also be controlled by a repressor Addition of inducer inactivates a repressor protein (not same as corepressor substance) , and transcription can proceed (Figure 7.8) Repressor's role is to prevent enzyme synthesis, so it is called negative control

Structural genes of lac operon: (contiguous) Lac Z Lac Y Lac A Regulatory genes of the lac operon: (contiguous, overlapping or separate) Lac operator or Lac O Lac promoter or Lac P Lac repressor or Lac I-coded by an independent gene with its own promoter-always active at a low level: constitutive

Transcription blocked Transcription proceeds
lac Promoter lac Operator lacZ lacY lacA RNA polymerase Transcription blocked Repressor lac Promoter lac Operator lacZ lacY lacA RNA polymerase Transcription proceeds Figure 7.8 Enzyme induction in the lactose operon. Repressor Inducer (allolactose) Figure 7.8

Structural genes of arg operon: (contiguous) Arg C Arg B Arg H Regulatory genes of the lac operon: (contiguous, overlapping or separate) Arg operator or Arg O Arg promoter or Arg P Arg repressor or Arg R-coded by an independent gene with its own promoter-always active at a low level

Transcription proceeds Transcription blocked
arg Promoter arg Operator argC argB argH RNA polymerase Transcription proceeds Repressor arg Promoter arg Operator argC argB argH Figure 7.7 Enzyme repression in the arginine operon. RNA polymerase Corepressor (arginine) Transcription blocked Repressor Figure 7.7

Summary: Induction and repression work by way of a protein that changes shape in response to a signal

7.4 Positive Control: Activation
Positive control: regulator protein activates the binding of RNA polymerase to DNA (Figure 7.9) Maltose catabolism in E. coli Maltose activator protein cannot bind to DNA unless it first binds maltose Activator proteins bind specifically to certain DNA sequence Called activator-binding site, not operator

Figure 7.9 Activator- binding site mal Promoter malE malF malG
No transcription RNA polymerase Maltose activator protein Activator- binding site mal Promoter malE malF malG RNA polymerase Figure 7.9 Positive control of enzyme induction in the maltose operon. Transcription proceeds Maltose activator protein Inducer (maltose) Figure 7.9

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 7.11)

Figure 7.11 Activator- binding site Promoter RNA polymerase
Transcription proceeds Activator protein Promoter RNA polymerase Figure 7.11 Activator protein interactions with RNA polymerase. Transcription proceeds Activator protein Activator- binding site Figure 7.11

Genes for maltose are spread out over the chromosome in several operons (Figure 7.12) Each operon has an activator-binding site Multiple operons controlled by the same regulatory protein are called a regulon Regulons also exist for negatively controlled systems

Figure 7.12 M B K A E Y F G Z mal lac oriC malS Mal regulatory
protein Lac regulatory protein T mal Figure 7.12 Maltose regulon of Escherichia coli. Maltose operons make up maltose regulon P Q Lactose operon Direction of transcription Figure 7.12

7.5 Global Control and the lac Operon
Global control systems: regulate expression of many different genes simultaneously Catabolite repression is an example of global control 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 Ensures that the "best" carbon and energy source is used first Diauxic growth: two exponential growth phases

Growth on lactose Glucose exhausted Growth on glucose Figure 7.13
Figure 7.13 Diauxic growth of Escherichia coli on a mixture of glucose and lactose. Figure 7.13

Cyclic AMP and CRP In catabolite repression, transcription is controlled by an activator protein and is a form of positive control (Figure 7.15) Cyclic AMP receptor protein (CRP) is the activator protein Cyclic AMP is a key molecule in many metabolic control systems Derived from a nucleic acid precursor Is a regulatory nucleotide

Figure 7.15 Figure 7.15 Overall regulation of the lac system.
CRP protein cAMP RNA polymerase Binding of CRP recruits RNA polymerase lac Structural genes DNA lacI C P O lacZ lacY lacA Active repressor binds to operator and blocks transcription. Transcription Transcription lacI mRNA mRNA lacZ lacY lacA Figure 7.15 Overall regulation of the lac system. Translation Translation LacI Inducer Active repressor LacZ LacY LacA Lactose catabolism Inactive repressor Figure 7.15

Dozens of catabolic operons are affected by catabolite repression Enzymes for degrading lactose, maltose, and other common carbon sources Flagellar genes are also controlled by catabolite repression No need to swim in search of nutrients

7.6 Transcription Controls in Archaea
Archaea use DNA-binding proteins to control transcription More closely resembles control by Bacteria than 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

Figure 7.16 NrpR NrpR blocks TFB and TBP binding; no transcription.
DNA BRE TATA INIT NrpR binds α-ketoglutarate. α-Ketoglutarate Glutamate ( ) NH3 When NrpR is released, TBP and TFB can bind. NrpR Figure 7.16 Repression of genes for nitrogen metabolism in Archaea. TFB TBP Transcription proceeds. RNA polymerase Figure 7.16

III. Sensing and Signal Transduction
7.7 Two-Component Regulatory Systems 7.8 Regulation of Chemotaxis 7.9 Quorum Sensing 7.10 Other Global Control Networks

7.7 Two-Component Regulatory Systems
Prokaryotes regulate cellular metabolism in response to environmental fluctuations External signal is transmitted directly to the target External signal is detected by sensor and transmitted to regulatory machinery (signal transduction) Most signal transduction systems are two-component regulatory systems

Two-component regulatory systems (Figure 7.17) Made up of two different proteins: Sensor kinase (in cytoplasmic membrane): detects environmental signal and autophosphorylates Response regulator (in cytoplasm): DNA-binding protein that regulates transcription Also has feedback loop Terminates signal

Shows regulator blocking transcription but activation can also occur
Environmental signal Sensor kinase ATP ADP Cytoplasmic membrane His His P P Response regulator Phosphatase activity Figure 7.17 The control of gene expression by a two-component regulatory system. P P RNA polymerase Transcription blocked DNA Promoter Operator Structural genes Shows regulator blocking transcription but activation can also occur

Almost 50 different two-component systems in E. coli Examples include phosphate assimilation, nitrogen metabolism, and osmotic pressure response (Figure 7.18) Some Archaea also have two-component regulatory systems Some signal transduction systems have multiple regulatory elements

7.8 Regulation of Chemotaxis
Modified two-component system used in chemotaxis to Sense temporal changes in attractants or repellents Regulate flagellar rotation Three main steps (Figure 7.19) Response to signal Controlling flagellar rotation Adaptation

Step 1: Response to signal Sensory proteins in cytoplasmic membrane sense attractants and repellents Methyl-accepting chemotaxis proteins (MCPs) Bind attractant or repellent and initiate flagellar rotation Step 2: Controlling flagellar rotation Controlled by CheY protein CheY results in counterclockwise rotation and runs CheY-P results in clockwise rotation and tumbling

Figure 7.19 ATP Repellents bind to MCP and trigger phosphorylation of
CheA-CheW complex. MCP Flagellar motor CheR CheA-CheW phosphorylate CheY and CheB. MCP is both methylated and demethylated. ATP CheW CheA ADP CheY-P binds to flagellar switch. CheB P CheY CheY P Figure 7.19 Interactions of MCPs, Che proteins, and the flagellar motor in bacterial chemotaxis. CheZ CheB CheZ dephosphorylates CheY-P. Cytoplasm Flagellum Figure 7.19

Summary CheY = counterclockwise rotation = run
But CheY-P = clockwise = tumble Repellents increase CheY-P therefore tumbling and direction change Works through two component system using MCP and other Che proteins

Step 3: Adaptation Feedback loop Allows the system to reset itself to continue to sense the presence of a signal Involves modification of MCPs with methyl group Degree of methylation controls sensitivity to attractant and repellent

7.9 Quorum Sensing Prokaryotes can respond to the presence of other cells of the same species Quorum sensing: mechanism by which bacteria assess their population density Ensures that a sufficient number of cells are present before initiating a response that, to be effective, requires a certain cell density (e.g., toxin production in pathogenic bacterium)

7.9 Quorum Sensing 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

Acyl homoserine lactone (AHL)
Activator protein AHL Quorum- specific proteins Other cells of the same species Figure 7.20 Quorum sensing. Chromosome AHL synthase Figure 7.20

7.9 Quorum Sensing Several different classes of autoinducers
Acyl homoserine lactone (AHL) was the first autoinducer to be identified Quorum sensing first discovered as mechanism regulating light production in bacteria including Aliivibrio fischeri (Figure 7.21) Lux operon encodes bioluminescence

Figure 7.21 Bioluminescent bacteria producing the enzyme luciferase.

7.9 Quorum Sensing Examples of quorum sensing
Virulence factors Switching from free-living to growing as a biofilm Quorum sensing is present in some microbial eukaryotes Quorum sensing likely exists in Archaea

7.9 Quorum Sensing-example
Virulence factors 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 7.22b ATP Binding of AIP to ArgC leads to auto-phosphorylation.
Cytoplasmic membrane ArgB ArgC ATP P Cytoplasm Pre-AIP ADP ArgC phosphorylates ArgA. Pre-AIP is converted to AIP by ArgB and exported out of the cell. ArgA ArgA-P activates expression of genes required for pre-AIP and virulence proteins. P Figure 7.22b Quorum sensing regulation of virulence factors. + + Basal transcription Virulence proteins D C B argA Genes encoding virulence Virulence factor production in Staphylococcus Figure 7.22b

Summary Staphylococcus aureus is an opportunitist pathogen
ArgA-P activates arg genes above basal level Also activates virulence factors Exoenzymes to digest tissue Exotoxins as poisons (gastroenteritis) Role in toxic shock syndrome (TSS)

7.9 Quorum Sensing Biofilm formation Pseudomonas aeruginosa
Produces polysaccharides that increase pathogenicity and antibiotic resistance Two quorum-sensing systems Produces AHLs and cyclic di-guanosine monophosphate (c-di-GMP) Leads to exopolysaccharide production and flagella synthesis (Figure 7.23)

Figure 7.23 Production Exopolysaccharide of AHLs and production and
c-di-GMP Exopolysaccharide production and flagella synthesis Increasing cell population Mature biofilm Attachment Figure 7.23 Biofilm formation in Pseudomonas. Figure 7.23

7.10 Other Global Control Networks
Nitrogen utilization: regulation by alternate sigma Under limiting conditions nitrogen uptake and use becomes very important Requires expression of new genes The genes do not have consensus promoter sequence, therefore not recognized by regular RNA pol and sigma factor Nitrogen stress results in expression of alternate sigma: sigma54 from RpoN gene

7.13 Nitrogen Fixation, Nitrogenase, and Heterocyst Formation
Nitrogen fixation is process of reducing N2 to NH3 Only certain prokaryotes can fix nitrogen Reaction is catalyzed by nitrogenase Composed of dinitrogenase and dinitrogenase reductase Sensitive to the presence of oxygen

Highly regulated process because it is such an energy-demanding process Nif regulon coordinates regulation of genes essential to nitrogen fixation (Figure 7.27) Oxygen and ammonia are the two main regulatory effectors Complex regulation uses multiple strategies

Nitrogenase proteins Dinitrogenase reductase FeMo-co synthesis FeMo-co synthesis Dinitro- genase Dinitro- genase reductase processing Homocitrate synthesis FeMo-co synthesis Electron transport β α Mo processing Regulators Pyruvate flavodoxin oxido- reductase FeMo-co insertion into dinitrogenase Metal center biosynthesis Positive Negative Flavodoxin nif DNA Figure 7.27 The nif regulon in Klebsiella pneumoniae, the best-studied nitrogen-fixing bacterium. Q B A L F M Z W V S U X N E Y T K D H J RNA Figure 7.27

Heterocyst-specialized cells for nitrogen fixation in some cyanobacteria (Figure 7.28) Requires metabolic and morphological changes Formation of thickened envelope (3 layers) Inactivation of photosystem II (it releases oxygen) Expression of nitrogenase Patterning of heterocyst differentiation

HetR activates genes necessary for heterocyst formation
Fixed N flow [α-Ketoglutarate] NtcA activates hetR expression Heterocyst HetR activates genes necessary for heterocyst formation Vegetative cells Vegetative cells Fixed C flow A filament of Anabaena Heterocyst—vegetative cell interactions Triggering heterocyst formation Figure 7.28 Regulation of heterocyst formation. Figure 7.28

V. RNA-Based Regulation
Uses non-coding RNAs or non-coding regions of transcripts for regulation 7.14 Regulatory RNAs: Small RNAs (i.e. antisense or cis-RNA and trans-RNA) 7.15 Riboswitches 7.16 Attenuation

7.14 Regulatory RNA: Small RNAs
Small RNAs work by: Block or open a ribosome-binding site (RBS) Increase or decrease degradation of mRNA (i.e. mRNA half-life) May also act at level of transcription

Translation inhibition/stimulation
RNA degradation/protection 1. sRNA 1. sRNA mRNA 3′ 5′ mRNA 3′ 5′ 5′ 3′ 5′ 3′ 5′ 3′ 5′ 3′ RBS RBS RBS RBS Ribonuclease Translation No translation Translation No translation 3′ 2. 2. 5′ 5′ 3′ 5′ 3′ 5′ 3′ 5′ 3′ 5′ 3′ RBS RBS RBS RBS Ribonuclease Figure 7.29 Small RNA mechanisms for modulating the translation of mRNA. No translation Translation No translation Translation Figure 7.29

7.14 Regulatory RNA: Small RNAs
Antisense RNAs first discovered in plasmids, phages, transposons RNA-OUT of Tn10 (IS10) decreases transposase expression Most antisense regulators have not been studied How do we know they exist?

7.14 Regulatory RNA: Small RNAs and Antisense RNA
Second classic example AntiQ RNA from Enterococcus faecalis plasmid pCF10 Small RNA binds to growing mRNA transcript for conjugation genes Alters secondary structure to block further transcription

Types of small RNAs (cont'd) Trans-RNAs are encoded in the intergenic region (not within gene they regulate) Limited complementarity with target Usually require help from a protein for binding

Hfq protein Facilitates RNA binding Also has regulatory functions independent of RNA “Global” regulator

Figure 7.30 Small regulatory RNA mRNA 3′ 5′ Hfq Small regulatory
Figure 7.30 The RNA chaperone Hfq holds RNAs together. Hfq protein Small regulatory RNA recognition sequence Figure 7.30

Useful Reference Annu Rev Genet. Author manuscript; available in PMC 2011 Jan 28. Annu Rev Genet. 2010; 44: 167–188. doi: /annurev-genet PMCID: PMC NIHMSID: NIHMS236247 Bacterial antisense RNAs: How many are there and what are they doing? Maureen Kiley Thomason1,2 and Gisela Storz1 1 Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 2 Department of Biochemistry and Molecular & Cell Biology, Georgetown University Medical Center, Washington, DC 20007 Maureen Kiley Thomason: Gisela Storz: Abstract Antisense RNAs encoded on the DNA strand opposite another gene have the potential to form extensive base pairing interactions with the corresponding sense RNA. Unlike other smaller regulatory RNAs in bacteria, antisense RNAs range in size, from tens to thousands of nucleotides. The numbers of antisense RNAs reported for different bacteria vary extensively but hundreds have been suggested in some species. If all of these reported antisense RNAs are expressed at levels sufficient to regulate the genes encoded opposite them, antisense RNAs could significantly impact gene expression in bacteria. Here we review the evidence for these RNA regulators and describe what is known about the functions and mechanisms of action for some of these RNAs. Important considerations for future research as well as potential applications are also discussed.

7.15 Riboswitches Riboswitches: RNA domains in an mRNA molecule that can bind small molecules to control translation of mRNA (Figure 7.31) Located at 5′ end of mRNA Binding results from folding of RNA into a 3-D structure Similar to a protein recognizing a substrate Found in some bacteria, fungi, and plants

Figure 7.31 Regulation by a riboswitch.

7.15 Riboswitches Riboswitch example: SAM riboswitch or (SAM Box riboswitch) in Bacillus subtilis Regulates expression of genes required for methionine metabolism translation of mRNA No SAM: transcription of mRNA takes place SAM causes shape change that allows formation of a terminator

7.16 Attenuation Transcriptional control that functions by premature termination of mRNA synthesis Trp operon of E. coli Additional control level above and beyond induction or repression Presence of trp down regulates expression of genes for trp biosynthesis Depends on control sequences in 5’UTR or trp leader

Trp mRNA gets translated as it is made
trp structural genes P O L trpE trpD trpC trpB trpA DNA Trp Leader Met-Lys-Ala-lle-Phe-Val-Leu-Lys-Gly-Trp-Trp-Arg-Thr-Ser Trp mRNA gets translated as it is made When there is trp in the cell the trp leader can be translated No trp in the cell translation stalls Figure 7.32 Attenuation and leader peptides in Escherichia coli. Figure 7.32

Figure 7.33 Figure 7.33 Mechanism of attenuation. Excess tryptophan:
transcription terminated Leader sequence DNA Direction of transcription Base pairing RNA polymerase terminates Ribosome 2 3 4 5′ Transcription terminated and tryptophan structural genes not transcribed 1 Trp-rich leader peptide trpE mRNA Direction of translation Leader sequence Limiting tryptophan: transcription proceeds DNA Direction of transcription Figure 7.33 Mechanism of attenuation. Translation stalled RNA polymerase continues 2 3 4 Transcription continues and tryptophan structural genes transcribed Leader peptide 5′ 1 trpE Direction of translation Figure 7.33

VI. Regulation of Enzymes and Other Proteins Post-translational
7.17 Feedback Inhibition 7.18 Post-Translational Regulation

7.17 Feedback Inhibition Feedback inhibition: mechanism for turning off the reactions in a biosynthetic pathway (Figure 7.34a) End product of the pathway binds to the first enzyme in the pathway, thus inhibiting its activity Inhibited enzyme is an allosteric enzyme (Figure 7.34b) Two binding sites: active and allosteric Easily reversible reaction Fast-acting

7.17 Feedback Inhibition Some pathways controlled by feedback inhibition use isoenzymes, different enzymes that catalyze the same reaction but are subject to different regulatory controls Rhodopseudomonas palustris Highly versatile bacterium with multiple life styles Switches between different nitrogenase isoenzymes depending on conditions

7.18 Post-Translational Covalent Modification
Biosynthetic enzymes can also be regulated by covalent modifications Regulation involves a small molecule attached to or removed from the protein (Figure 7.35) Results in conformational change that inhibits activity Common modifiers include adenosine monophosphate (AMP), adenosine diphosphate (ADP), inorganic phosphate (PO42-), and methyl groups (CH3)

7.1 Major Modes of Regulation

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