Why genes are regulated?

1. Why genes are regulated? • Minimize energy consumption--why express a gene you do not need? (economy) • Control growth--many cells in a mature organism do not grow, and expression of genes involved in promoting cell division is tightly regulated. (physiological balance) • Development--inappropriate expression of genes that regulate differentiation may adversely affect development (pathology) • Response to environment (dynamic)

2. How is gene regulation controlled? Transcription- at initiation and at termination. Less happened at elongation. RNA processing- only happened in Eukaryotes via modification, splicing, transport, or stability. not available in Prokaryotes (overcome by transcription is intimately tied up with translation) Translation- its regulation is analogous to those of transcription, happened at initiation and at termination

3. Gene regulation in transcription A principle example by Jacob and Monod (1961) cis-acting element: not convertible, function as a DNA sequence in situ, phyiscal linked trans-acting element:diffusible Gene activity is regulated by the specific interactions of the trans-acting products (usually proteins/RNAs) with the cis-acting sequences (usually sites in DNA). trans-acting element diffuse structural gene regulator gene (cis-acting element: usually upstream of target genes including promoter and terminator.) The outcome of regulation may be positive or negative.

4. Components in regulatory circuits Regulators and mechanisms: Protein regulators:1. allostery - two different sites, one for nucleic acid target, the other for a small molecule 2. multimer (usually has a symmetrical organization) -cooperative binding effect RNA regulators: usually a small RNA molecule 1. changes in 2° structure 2. complementary base pairing Consequence of targeting: a. Formation of the double helical structure may itself be sufficient. b. Duplex formation may be important because it sequesters a region of the target RNA The relationship of regulators in gene regulatory circuits: Coordinate: an operator controls the expression of many genes Network: one regulator is required for the production of another (cascade) Antagonize: a series of regulators each of which antagonizes another Autogenous: a protein regulates expression of the gene that codes for itself

5. A diffusible trans-acting factor bound to cis-acting targeting site(s) is: Negative control Positive control Half of prokaryotic genes Most of eukaryotic genes Half of prokaryotic genes default state of genes is active default state of genes is inactive activators control level Repressors and activators are required very short cis-acting sequences (<10bp) to function. Such as the hexamers of -35 and -10 for the RNA polymerase.

6. Three genes are coordinately regulated. Controlled by the metabolic state of the cell -- carbon source. Negative control lac I gene Positive control CAP-cAMP Lac z Lac y Lac a Bacterial Lactose Operon Lac I P/O By Monod,1940 • Jacob & Monod • Induction of the β- galactosidase gene in response to lactose. • Lactose - Inducer of activity • Synthesis of new protein. • In the absence of lactose, the gene is not expressed.

8. The Lac operon

9. The lac operon- A negative control (Jacob and Monod model of transcriptional regulation of the lac operon by lac repressor ) Polycistron– bacterial structural genes are often organized into clusters, coordinately controlled by means of interactions at a single promoter. Monocistron– only one gene is controlled by an individual promoter. + Operator Structural gene(s) (lacZYA) Operon = T T trans-acting factor Transfer an acetyl group form acetyl-CoA to β-galactosides tetramer of ~500kD 30kD membrane bound protein Transports β-galactoside into the cell tetramer (38kD/each) 10 tetramers in a wild type cell Cleave the β-galactoside into component sugars. Lactose → glucose + galactose (independent transcription unit: monocistron) Cluster is transcribed into a single polycistronic mRNA from a promoter where initiation of transcription is regulated.

10. The Lac operon

11. Metabolic action of LacZ gene product, β-galactosidase

12. LacI repressor form a tetramer bound onto the operator

13. How the lac genes are controlled? Repressor and RNA polymerase bind at sites that overlap around the transcription startpoint of the lac operon. Hence, the transcription of genes are turned off by the Lac repressor binding toOlac. (Plac) Repressor RNA polymerase (~60bp) (Olac) (~26bp) A mutation that inactivates the regulator causes the structural genes to remain in the expressed condition.

14. Lac z Lac z Lac y Lac y Lac a Lac a Repressor and inducer Action • In absence of lactose, repressor binds DNA and blocks expression of LacZYA genes LacI gene product bound • In presence of lactose, repressor releases from DNA and lac zya genes express + lactose LacI released Repressor no longer binds - genes can be active.

15. Induction of lac operon Jacob and Monod model of transcriptional regulation of the lac operon by lac repressor

16. The expression of lac operon: an induction Inductionof mRNA and protein (also happened in yeast) unstable mRNA with ~3min half-life Rapid reverse Rapid induction Protein is more stable Within 2-3 mins, ~5000 enzyme molecules are present and can reach up to 5%-10% of the total soluble protein of the bacterium. (to ensure a minimal amount to start the induction) ~5 molecules

18. IPTG: a gratuitous inducer • Artificial inducer of the beta galactosidase gene. • Not metabolized. vs. Lactose The induction does not depend on the activity of inducer. The system must possess some component, distinct from the target enzyme, that recognizes the appropriate substrate; and its ability to recognize related potential substrates is different from that of the enzyme.

19. Conversion of repressors into an inactive leads to gene expression: an allosteric control LacI repressor possesses dual properties: 1.binds to DNA preventing transcription (Allosteric effect) 2.interacts with small-molecule inducer changing its own conformation 2 Inducer binding changing shape 1 DNA binding preventing Tx polycistron coordinate regulation 1. Sequential expression 2. Relative same amount

20. Mutagenesis is an approach to analyze the operator Un-inducible mutants constitutive mutants cis-acting mutations: map on promoter and operator trans-acting product mutations: lacI locus vs. Oc type: operator loses binding with lacI lacI- type: loss of function of lacI Absence the DNA binding activity Allosteric Recessive mutation: complementary by wild type cis-dominance: 1.mutation(s) at any site that is physically contiguous with the sequences it controls 2. cannot be assigned to a complementation group. Plac mutant--- Promoter loses binding with RNA pol Absence the inducer binding site Consistent with the operator as a typical cis-acting site, whose function depends upon recognition of its DNA sequence by some trans-acting factor. lacIs mutant (locked in to the active form that recognizes the operator and prvents transcription.)

21. lacI-d mutant provides the multimeric property of the LacI protein lacI-d mutant: damages in DNA binding site (as a dominant negative mutant to LacI its own function) negative complementation Tetramer is formed in LacI repressor. Heterotetramer may be formed as interallelic complemenatation. Combination betweenlacI-d and lacI+ leads to occurnegative complementation, suggesting lacI-d is calleddominant negative.

22. WT lacI- complementary WT Non- complementary Oc

23. WT lacIs WT lacI-d

24. Molecular Mechanism of repress working on its operator : palindorme Symmetry in the protein. The operator makes the same pattern of contacts with a repressor monomer. Functional important bases (essential specific contacts) point mutation Contact (modification) Cover by repressor (DNase fingerprint)

25. Structure of LacI repressor Several domains: N-terminal DNA-binding domain (a.a. 1-59) a hinge 2X core domains Fit into the major groove of DNA, make special contacts Conformation change (from core domains) leads tosignal the DNA binding capacity. Headpiece (aa 1-59) independent from core 6X : Cleft between core domains Headpiece changes its orientation Loses contact with DNA inducer bound A half-site of the dyad symmetry sequence can bind an intact repressor monomer. The affinity for DNA is many orders of magnitude higher by intact repressor, that is dimer/tetramer. : contains 2X leucine heptad repeats

26. Higher order of LacI repressor form dimer Inducer-binding cleft Hydrophobic core : form dimer C-terminal helices : form tetramer lacI-d :DNA binding dimer form lacIs inducer binding dimer form lacI- tetramer form

27. Why tetramer? Tetramer can bind two operators simultaneously. O1: in the initial region of the lac operon, strongest affinity for repressor O2: 410bp downstream of startpoint, weaker affinity for repressor O3: 83bp upstream of startpoint, weaker affinity for repressor CAP CAP (catabolite associated protein) O1 O2 X O3 X 2-4X 100X 2-4X Repressor tetramer In fact, the repressor binding onto operator(s) enhances RNA polymerase binding at the promoter. However the bound RNA polymerase is prevented from initiating transcription (stored at closed complex). enable transcription to begin immediately upon induction, instead of waiting for an RNA polymerase to be captured.

29. Repressor is always bound to DNA Proteins that have a high affinity for a specific DNA sequence also have a low affinity for other DNA sequences. Every base pair in the bacterial genome is the start of a low-affinity binding-site for repressor. The large number of low-affinity sites ensures that all repressor protein is bound to DNA. Repressor binds to the operator by moving from a low-affinity site rather than by equilibrating from solution. The operator competes with low-affinity sites to bind repressor In the absence of inducer, the operator has an affinity for repressor that is 107× that of a low affinity site. The level of 10 repressor tetramers per cell ensures that the operator is bound by repressor 96% of the time. Induction reduces the affinity for the operator to 104× that of low-affinity sites, so that only 3% of operators are bound. Induction causes repressor to move from the operator to a low-affinity site by direct displacement. These parameters could be changed by a reduction in the effective concentration of DNA in vivo.

30. A kinetic view of repressors on an operator What affects the repressor binding to the operator: genome size, specificityof the repressor, theamountof the repressor existed/required 1. Repressors have a high affinity for a specific DNA sequence and also have a low affinity for other DNA sequences. 2. Hence, the large number of low-affinity sites ensures that all repressors are bound to DNA. 3. Excessive repressor proteins ensure that the operator is occupied by a repressor at ~96% All of repressors are bound to DNA Re-distribution randomly on the genome Inducer binding leads to lose specificity of bound to operator comparing to other DNA sequences Specificity to the high affinity site Repressor binds to the operator by moving from a low-affinity site rather than by equilibrating from soluation.

31. How inducer binding to free repressor? Free repressor binding to DNA results from the reduction of its affinity. How the repressor tetramer set off from the DNA? 1.Upset equilibrium 2. Directly displacement (affinity change/flow) (unbalance) (involvesconformational change, but not bond breaking) >15 mins Dynamic balance Hence, prefer fast See next slide Not reversible

32. Repression can occur at multiple loci A repressor will act on all loci that have a copy of its target operator sequence

33. A diffusible trans-acting factor bound to cis-acting targeting site(s) is: Negative control Positive control Half of prokaryotic genes Most of eukaryotic genes Half of prokaryotic genes default state of genes is active default state of genes is inactive activators control level Repressors and activators are required very short cis-acting sequences (<10bp) to function. Such as the hexamers of -35 and -10 for the RNA polymerase.

34. Catabolite Repression- positive control • Additional control mechanism prevents Lac operon expression when Glucose is present. • Lactose + Glucose to E. coli-- Lac operon will remain off. • Cells have a glucose sensor.

35. How do bacteria control the carbon sources? Phoenolpyruvate:glycose phosphotransferase = PTS • Glucose repression controls use of carbon sources: • E. coli uses glucose in preference to other carbon sources • Glucose prevents uptake of alternative carbon sources • Exclude expression of the operons coding for the enzymes • that metabolize other carbon sources (such as lac, gal, ara) Phenomenon: IIAglc IIAglc-P IIAglc Mechanism: (crr gene) 1.Inducer exclusion 2. Inhibition of positive regulator, CRP activity (see next)

36. IIAglc Adenylate cyclase cAMP Regulation of CRP activity a positive regulator which may overcome a deficiency in the promoter, e.g. a poor consensus sequence at -35 or -10 CRP activator controls the activity of a large set of operons in E. coli. A dimer of CRP is activated by a single molecules of cyclin AMP -P IIA-P Catabolite activator protein (CAP; also known as cAMP receptor protein, CRP) Glucose in reducing cyclic AMP levels is to deprive the relevant operons of a control factor necessary for their expression

39. Catabolite activator protein (CAP) = cAMP receptor protein, CRP) 22.5KDa protein to form a homodimer. an N-terminal domain required for CAP dimerisation and the binding of cAMP, a C-terminal domain that contains a helix-turn-helix motif required for the binding of DNA. Gene activator: AR1 (activating region 1) region within the C-terminal domain, which interacts with the C-terminal domain of the RNAP alpha subunit (aCTD); AR2 (activating region 2) region within the N-terminal domain, which interacts with the N-terminal domain of RNAP alpha subunit (aNTD); AR3 (activating region 3) region within the N-terminal domain, which interacts with the RNAP sigma70 (s70) subunit. CAP is one of over 300 transcription factors used by Escherichia coli alone. Such as metabolism of sugars and amino acids, transport processes, protein folding, toxin production and pilus synthesis. TGTGA conserved pentamer is essential and an inverted repeat version given the strongest interaction with CRP

40. How CRP activator works to positively control transcription? 1. Increase the rate of initial binding to form a closed comolex 2. CAP + cAMP allow formation of an open promoter comoplex How? CRP:form a dimer (22.5kD/each), each of them has a DNA binding region and a transcription-activating region. Binding ~22bp in a responsive promoter TGTGA conserved pentamer is essential and an inverted repeat version given the strongest interaction with CRP (increase affinity to DAN a lot) CRP binding sites lie in different locations relative to the startpoint in the various operons that it regulates Strong binding Weak binding

41. CRP in regulation of lac operon Only the activating region of the subunit nearer the startpoint is required, presumably because it touches RNA polymerase. >>>> Orientation-independent Dimer promotes the binding affinity of CAP onto DNA

42. The CRP protein can bind at different sites relative to RNA poymerase

43. How CAP work?3 classes of CAP-dependent promoters promoter closed complex formation e.g. lac promoter Class I CAP-dependent promoter activation: CAP dimer interacting with the aCTD of RNAP, which is also comprised of b and s subunits promoter complex to an open complex e.g. galP1 promoter promoter closed complex formation Class II CAP-dependent promoter activation: CAP dimer interacting with the aCTD and aNTD of RNAP 1. involve class I and class II mechanisms of action in an additive manner e.g. malK promoter 2. two CAP dimers could function differently Class III CAP-dependent promoter activation: two CAP dimers interacting with the aCTD of RNAP Journal of Molecular Biology 293, S. Busby and R. Ebright, Transcription Activation by Catabolite Activator Protein (CAP), 199-213 (1999),

44. CRP bends DNA ~90°

48. Diverse control circuits by regulators default state → expressed default state X expressed (need arepressor to switch off) interfering (need an activator to switch on) essential CRP, σ Lac operon presence of inducer via Outcome is expressed 1. Allosteric changes 2. Activation of proteins (e.g. by Oxidation) 3. (de)phosphorylation Trp operon via co-repressor Outcome is not expressed A fail-safe, selective advantage due to increased efficiency (basal level expression)

49. The stringent response produces (p)ppGpp (alarmones) Poor growth conditions cause bacteria to produce the small molecule regulators ppGpp and pppGpp to shut down a wide range of activities associated with inhibition of Tx. 10~20X tRNA+rRNA ↓ 3X mRNA ↓ ~5~10% total RNAs ↓ Protein degradation ↑ Stringent response (Relaxed) L11/S50 via conformation change e.g. EF-Tu EF-G Uncharged tRNA /Ribosome ~20sec Reversed reapidly (Idling reaction) The stringent factor RelA is a (p)ppGpp synthetase that is associated with ~5% of ribosomes. RelA is activated when the A site is occupied by an uncharged tRNA. One (p)ppGpp is produced every time an uncharged tRNA enters the A site. A P

50. (p)ppGpp inhibits transcription of rRNA • Initiation of Tx is specifically inhibited at the promoters of operons coding for rRNA • The elongation phase of Tx of many or most templates is reduced by ppGpp The level of protein synthesis increases in proportion with the growth rate. Ribosome ≡ protein synthesis ≡ cell growth NTP level as 1. an indicator 2. drives the initiation by stabilizing the open complex