2.1 - Regulation of Gene Expression pt.1

Question 1

what is the genome? what is genomics?

Answer 1

-all DNA in a given cell
-always consistent between time points
-the study of the whole genome

Question 2

what is the transcriptome? what is transcriptomics?

Answer 2

-all RNA at a given moment
-not consistent between time points (depends on the cells needs)
-the study of transcription at a given time

Question 3

what is the proteome? what is proteomics?

Answer 3

-all proteins in a given cell
-basis for phenotype
-not consistent between time points (depends on the cells needs)
-the study of the entire complement of proteins

Question 4

how do 2 cell’s transcriptomes and proteomes compare if they have identical genomes?

Answer 4

-they can have different transcriptomes and proteomes
-it is all dependent on external conditions

Question 5

how can growing conditions change the differences in transcriptome and proteome?

Answer 5

-if growing in a closed system, there will be more change as materials will deplete
-if growing in an open system, there will be less change as materials are added

Question 6

how do microbes regulate protein function?

Answer 6

-2 ways
-have mechanisms that either control the amount of an enzyme or protein or control the activity of an enzyme or other protein

Question 7

how do microbes control the amount of an enzyme or protein? what is this considered?

Answer 7

-vary the amount of mRNA made (transcriptional control)
-vary the amount of protein made (translational control)
-control of gene expression

Question 8

how do microbes control the activity of an enzyme or protein?

Answer 8

-post-translational regulatory processes

Question 9

how is controlling the amount of mRNA thats made economical to the cell?

Answer 9

-genes are expressed only when the products are needed and only in the amount thats needed (don’t make more than needed)
-this conserves energy
-prevents interference between expressed products (wont make proteins that do one thing and proteins that do the opposite of that thing simultaneously)

Question 10

how is gene expression regulated?

Answer 10

-majorly by DNA-binding proteins
-processes where the output of genes is changed depending on the state of the cell (if the cell needs certain genes or not)
-responses to changes in physiological conditions (metabolic and environmental)

Question 11

what is central to replication, transcription, translation, and their regulation?

Answer 11

-interactions between proteins and nucleic acids

Question 12

what is a characteristic of protein- nucleic acid interactions?

Answer 12

-they may be specific or non specific
-specific = binding to a specific site on the nucleic acid
- non-specific = binding anywhere along the nucleic acid

Question 13

in what manner do most DNA-binding proteins interact with DNA?

Answer 13

-in a sequence specific manner
-specificity is given by interactions between specific amino acid side chains on proteins and specific chemical groups on the nitrogenous bases and the sugar-phosphate backbone

Question 14

what is the main site of protein binding on DNA and why?

Answer 14

-the major groove
-because it is larger and can fit the size of a protein

Question 15

what is the typical shape/organization of DNA binding proteins?

Answer 15

-homodimeric (2 identical polypeptide subunits)
-each subunit is subdivided into domains (regions with specific structure and function)

Question 16

what are the basic roles of the 2 domains on a homodimeric DNA-binding protein?

Answer 16

-one domain interacts specifically with a region of DNA in the major groove (typically inverted repeats, stem loop structure is not always formed, ex: LacI repressor)
-other domain contains protein-protein contacts to hold the dimer together
-several classes of protein domains which are critical for proper binding to DNA

Question 17

what is the most common domain?

Answer 17

-helix-turn-helix structure
-the turn that links the helices contains 3 amino acids (first is usually glycine)
-1st helix interacts with the DNA specifically (recognition helix)
-2nd helix stabilizes the 1st helix through hydrophobic interactions (stabilizing helix)

Question 18

what proteins are involved with gene expression?

Answer 18

-sigma factors
-transcription factors

Question 19

what are the different sigma factors in E.coli?

Answer 19

-sigma S for proteins needed under starvation conditions
-sigma N involved in nitrogen metabolism
-sigma H and sigma E for heat shock
-sigma F for chemotaxis
-sigma D/ sigma 70 for most constitutive and exponential phase genes
-6 total

Question 20

what does it mean for a genes to be constitutive?

Answer 20

-genes that are expressed all the time (continuous expression)

Question 21

what is the role of transcription factors? what are examples of transcription factors?

Answer 21

-control the rate of gene transcription by binding to specific DNA sequences
-activators and repressors of gene expression

Question 22

how does an activator act to control gene expression?

Answer 22

-positive regulation
-binds to DNA and helps recruit sigma factor/RNA polymerase

Question 23

how does a repressor act to control gene expression?

Answer 23

-negative regulation
-binds to DNA by blocking access of sigma factor/RNA polymerase

Question 24

what genes encode transcription factors? what do the products of these genes affect/regulate

Answer 24

-regulatory genes
-affect expression of one or few genes
-affect expression of many genes (regulon)
-regulate its own expression (autoregulation)

Question 25

what is an operon?

Answer 25

-groups of genes that function in related processes -one transcriptional unit controlled by a single promoter and other cis-acting sequences required for expression and regulation -the single polycistronic message ensures that encoded proteins are translated at the same time -ex: lac operon

Question 26

is a regulatory gene considered part of an operon?

Answer 26

-not unless it is co-transcribed under auto regulation (regulates both itself and the operon)

Question 27

how many genes can an E.coli chromosome encode? how many mRNAs are present during growth (under ideal conditions)?

Answer 27

-it encodes 4000 genes -this is not economical to express all at once -only about 800 mRNAs are present to allow the cell to conserve energy and materials

Question 28

what can cellular proteins be?

Answer 28

-constitutive or inducible/repressible

Question 29

what are constitutive proteins?

Answer 29

-the gene for them is always expressed and the protein is always present -ex: central pathway components and encoded by housekeeping genes (16S rRNA genes)

Question 30

what are inducible/repressible proteins?

Answer 30

-gene expression is regulated in response to change in environmental signals and stresses, growth phase changes, nutrient, and physiological status -ex: antibiotic resistance genes

Question 31

how are biosynthetic enzymes repressed? what is the purpose of doing this?

Answer 31

-when the product is already present in the medium in a sufficient amount, the enzymes that catalyze the synthesis of the product will not be made anymore -ensures that the organism that does not waste energy and nutrients synthesizing unneeded enzymes

Question 32

what is an example of how arginine is repressed?

Answer 32

-arginine is produced only if the amount needed is absent or is not in a sufficient amount -therefore, typically the final product of a particular biosynthetic pathway represses the enzymes of the pathway

Question 33

what is the process of enzyme induction?

Answer 33

-an enzyme is only made when its substrate is present -typically affects degradative enzymes (catabolic)

Question 34

what is an example of how lactose causes enzyme induction?

Answer 34

-enzymes to breakdown lactose are synthesized only in the presence of lactose -ex: B-glactosidase cleaves lactose into glucose and galactose

Question 35

what are effector molecules?

Answer 35

-typically cell metabolites (small molecules of metabolic pathways) like substrates, end products, or intermediates of pathways they regulate -regulate by affecting the activity of transcription factors associated with the pathways -ex: inducers and corepressors

Question 36

what are inducers?

Answer 36

substance that induces enzyme synthesis when it binds to a transcription factor

Question 37

what are corepressors?

Answer 37

-substance that represses enzyme synthesis when it binds to a transcription factor

Question 38

how do effector molecules affect enzyme synthesis?

Answer 38

-through allosteric regulation -the binding of an effector to a domain (enzyme or DNA binding protein) can change the conformation of the target (shape) to its active or inactive form (DIFFERENT FROM A DNA BINDING DOMAIN)

Question 39

where does the activator and inducer complex bind on the DNA? where is this relative to the promoter?

Answer 39

-activator binding site -upstream of the promoter -made to allow RNA polymerase to bind

Question 40

where does the repressor and corepressor complex bind on the DNA? where is this relative to the promoter?

Answer 40

-operator -downstream of the promoter -made to block RNA polymerase from binding

Question 41

what is the underlying mechanism for all regulatory systems employing repressors?

Answer 41

-preventing mRNA synthesis by the activity of specific repressor proteins that are themselves under the control of specific small effector molecules (corepressor)

Question 42

what is the regulation by repressors called?

Answer 42

-negative control

Question 45

how does the mechanism of repression work?

Answer 45

-ex: arginine biosynthetic operon (polycistronic mRNA, 1 promoter, 1 terminator) -arginine synthesis is under negative control (by a helix turn helix homodimeric repressor) -to start out the repressor is inactive and cannot bind to the operator region -arginine (product) acts as a corepressor to shut down biosynthesis (binds to the repressor to change the conformation and allow the repressor to bind to the operator, allosteric regulation)

Question 46

what is de-repression?

Answer 46

-induction can also be controlled by a repressor -the effector molecule is an inducer and the transcription factor is a repressor (binding of the inducer allows for transcription) -the repressor is active in absence of the inducer and blocks transcription by binding to the operator -addition of the inducer causes the repressor to become inactive and it no longer can bind to the operator (transcription is allowed)

Question 47

what is an example of de-repression?

Answer 47

-the lactose operon (polycistronic mRNA, one promoter, one terminator) -the LacI repressor is active in absence of the inducer (allolactose) and therefore blocks transcription by binding to the operator region -when allolactose is added, the LacI repressor inactivates due to allosteric regulation (widens in shape) and unbinds from the operator region

Question 48

what is allolactose?

Answer 48

-isomer of lactose

Question 49

what is the underlying mechanism for all regulatory systems employing activators?

Answer 49

-the activation of mRNA synthesis by the activity of specific activator proteins of which a subset are under control of specific small effector molecules -role of the activator is to start transcription by recruiting RNA polymerase+sigma factor to the promotor region

Question 50

what is the regulation by activators called?

Answer 50

-positive control

Question 51

what is the purpose of the activator binding site being located proximally to the promotor region?

Answer 51

-so it can make contact with the RNA polymerase holoenzyme as it is only loosely bound to the promotor region

Question 52

what happens if the activator binding site is located further upstream (several hundred bps) from the promotor?

Answer 52

-the activator bound DNA must be lopped to make contact with the RNA polymerase in order to activate transcription

Question 53

how does the mechanism of activation work?

Answer 53

-ex: maltose operon (inducible operon with positive transcriptional control) -the synthesis of maltose catabolic enzymes requires the combined function of the maltose activator protein and the inducer which is maltose itself -when maltose is not present, the activator protein is not functional (inactive) -in the presence of maltose, the activator protein becomes functional (active due to the binding of maltose as the inducer) -they will together bind to the activator site and allow the RNA polymerase holoenzyme to begin transcription

Question 54

what is a regulon?

Answer 54

-series of operons or genes that are controlled together by a specific DNA-binding protein that binds only at those genes or operons (regardless of whether it functions as an activator or repressor) -ex: maltose regulatory protein controls maltose genes + operons

Question 55

what genes are contained within the lac operon?

Answer 55

-lacZ -lacY -lacA -all necessary for catabolizing lactose -polycistronic

Question 56

what type of control is de-repression?

Answer 56

-negative control

Question 57

what occurs in the absence of lactose? what type of mechanism is the lac operon under?

Answer 57

-LacI repressor will bind to the operator region (allolactose is the inducer) and prevent RNA polymerase holoenzyme from binding to the promotor (no transcription occurs) -de-repression mechanism

Question 58

is the lacI gene constitutively expressed?

Answer 58

-YES -functional when it is in protein form (naturally in its active form)

Question 59

what is the function of the LacY enzyme?

Answer 59

-Lac permease -transports lactose into the cell

Question 60

what is the function of the LacZ enzyme?

Answer 60

-B-galactosidase -cleaves lactose into glucose and galactose (major reaction) -converts lactose into allolactose (minor reaction)

Question 61

what is the function of the LacA enzyme?

Answer 61

-transacetlyase -modifies lactose and lactose analogs (allolactose) -may prevent toxic buildup in the bacterial cytoplasm

Question 62

what occurs in the presence of lactose?

Answer 62

-lactose is transported into the cytoplasm by LacY (Lac permease) -lactose is converted to allolactose by B-galactosidase (LacZ) -allolactose binds to the LacI repressor and changes its conformation into an inactive form causing it to unbind from the operator -RNA polymerase can now transcribe the lac operon -lactose gets cleaved into glucose and galactose by B-galactosidase (LacZ)

Question 63

how are B-galactosidase (LacZ) and Lac permease (LacY) present before the operon is depressed?

Answer 63

-even when transcription is "turned off" there is still a very low base rate of transcription -this is called basal transcription' -it creates few molecules of these so that gene expression can be induced if lactose were to appear

Question 64

how is basal transcription acheived?

Answer 64

-by a lack of enough repressors

Question 65

what is catabolite repression?

Answer 65

-also known as the glucose effect -a mechanism of global control that controls the use of carbon sources if more than one is present -there is a preferential use of glucose -glucose inhibits the synthesis of catabolic enzymes for other carbon sugars -results in diauxic growth (2 log phases separated by a short lag phase) -called repression but it is actually a process of activation

Question 66

why is there a preferential use of glucose?

Answer 66

-glucose is a better carbon and energy source as it is more efficient (creates more energy with less work) -growth rate is faster with glucose as the carbon source

Question 67

what are the phases of diauxic growth?

Answer 67

-1st log phase where the lac operon is repressed -lag phase where the expression of the lac operon begins -2nd log phase where lactose is catabolized

Question 68

what are additional components involved in the regulation of the lac operon?

Answer 68

-activator binding site for CAP (CAP site) -catabolite activator protein (CAP = cyclic AMP receptor protein) -cyclic AMP (cAMP)

Question 69

where is cAMP derived from?

Answer 69

-a nucleic acid precursor (DATP)

Question 70

what inhibits cAMP synthesis?

Answer 70

-glucose

Question 71

what is the general mechanism of catabolic repression?

Answer 71

-PTS (phosphoenolpyruvate (PEP) phosphotransferase system) -responsible for transporting sugars into the cell -the richness of the carbon source will influence the PEP:pyruvate ratio

Question 72

how does the mechanism of catabolic repression work when there is high glucose?

Answer 72

-there is a low PEP:pyruvate ratio because more phosphates are being donated, therefore creating more pyruvate -PTS is working consistently to move glucose into the cell -cAMP levels are low (adenylate cyclase is not active) because there is a high concentration of unphosphorylated IIA^glc since it is rapidly dephosphorylated (phosphate gets passed to IIB^glc) -when IIA^glc is unphosphorlyated, it binds to LacY to inhibit it

Question 73

how does the mechanism of catabolic repression work when there is low glucose?

Answer 73

-there is a high PEP:pyruvate ratio because the system stall and PEP is not donating its phosphate as much, therefore less of it converts into pyruvate -there is a high concentration of phosphorylated IIA^glc since it receives the phosphate and has no glucose to pass it onto -P-IIA^glc activates adenylate cyclase which produces cAMP -since phosphate stays bound to IIA^glc, LacY is uninhibited

Question 74

what role does cAMP play in the mechanism of catabolic repression?

Answer 74

-cAMP is an inducer molecule that turns on the expression of the lac operon -therefore it acts as a form of positive control

Question 75

how does cAMP function when glucose is absent but lactose is present?

Answer 75

-cAMP levels are high -cAMP binds to CAP -CAP-cAMP binds to the activator binding site (CAP site) -this allows for RNA polymerase holoenzyme to bind and transcription proceeds

Question 76

how does cAMP function when glucose and lactose are both present ?

Answer 76

-cAMP levels are low -cAMP does not bind to CAP -CAP is thus inactive and cannot bind to the activator binding site (CAP site) -RNA polymerase does not bind and transcription does not proceed

Question 77

what is the dual control of the lac operon?

Answer 77

-controlled by both positive and negative control mechanisms -positive = CAP-cAMP binding to the CAP site (glucose must be absent) -negative = allolactose binds to LacI to remove it from the operator (lactose must be present) -both conditions must be met for optimal transcription

Question 78

what genes are contained on the tryptophan (trp) operon?

Answer 78

-trpE -trpD -trpC -trpB -trpA -all needed for synthesis of the amino acid tryptophan -polycistronic mRNA

Question 79

what is the regulatory gene for the trp operon?

Answer 79

-trpR (located upstream of the trp operon) -encodes for TrpR -constitutively expressed but starts inactive (aporepressor) -functions as a repressor when activated by binding of the corepressor (tryptophan itself)

Question 80

why is the LacI repressor not an aporepressor?

Answer 80

-because it starts active and the binding of the inducer deactivates it

Question 81

what happens when levels of tryptophan in the cytoplasm are low?

Answer 81

-the trp operon is transcribed as there is no free tryptophan to bind to the inactive repressor

Question 82

what happens when levels of tryptophan in the cytoplasm are high?

Answer 82

-the trp operon should be shut down -free tryptophan in the cytoplasm binds to TrpR which triggers a conformational change, turning it into an active repressor -active TrpR binds to the operator and blocks RNA polymerase

Question 83

how is the trp operon controlled?

Answer 83

-repressible operon under negative control

Question 84

what is attenuation?

Answer 84

-form of transcriptional control that is exerted after transcription initiation but before the completion of transcription -the leader sequence folds into 2 alternative secondary structures -the first structure allows for continued synthesis of mRNA and the second causes premature termination

Question 85

what determines which secondary structure the leader sequence will fold into?

Answer 85

-depends on either events at the ribosome or on the activity of regulatory proteins (organism dependent) -the level of tryptophanyl-tRNA -transcription and translation is a coupled process which is a requirement for attenuation

Question 86

is attenuation a universal control system?

Answer 86

-NO -attenuation control is absent in eukaryotes because the processes of transcription and translation are spatially separated

Question 87

what is found within the trpR null mutant?

Answer 87

-the levels of enzymes for synthesizing tryptophan are higher in the absence of tryptophan -this is because of attenuation

Question 88

what are the possible hairpin structures that the leader sequence could fold into?

Answer 88

-there are 4 regions in the leader sequence -hairpin structures could form between 1:2, 2:3, 3:4

Question 89

what is the purpose of the 1:2 hairpin structure?

Answer 89

-it pauses RNA polymerase to allow time for the ribosome to load and begin translation on the AUG codon -the ribosome then bumps RNA polymerase off of the pause site (1:2 hairpin)

Question 90

what dictates whether the hairpin 2:3 or 3:4 forms?

Answer 90

-the progress of the ribosome -if it is able to add the 2 tryptophans located in region 1, then it sits on the stop codon and prevents 2:3 from forming -the 3:4 structure will form and terminate transcription -if it is not able to add them, then the ribosome stalls at the trp codons, which allows for 2:3 to form and prevents 3:4 from forming

Question 91

what causes the 3:4 hairpin structure to allow for termination?

Answer 91

-there is a poly-U tail that follows region 4 -this creates unstable base pairing between the DNA and RNA to allow for the RNA to fall off -rho independent transcription termination

Question 92

how do cells regulate cell metabolism?

Answer 92

-in response to many different environmental factors -changes in temp, pH, oxygen, and nutrient availability -changes in the number of other cells present (quorum sensing = cells communicating with each other to see if they are ready) -cells receive signals from the environment and transmit them to the specific target in order to be regulated -some signals are effector molecules (hormones, cell metabolites, nutrients) -majority of signals are detected by a cell surface sensing system that transmits the signal to the rest of the regulatory machinery (signal transduction = movement of the signal through the cell)