1 Transcription Initiation and its Regulation in BacteriaTranscription, chromatin and Its Regulation (Carol A. Gross; Geeta Narlikar) January 19, 2017 – Transcription Initiation and its Regulation in Bacteria January 23, 2017 – Transcription Initiation and its Regulation in Eukaryotes January 26, 2016 – Chromatin 1 January 30, 2016 – Chromatin 2 February 2, 2016 – Transcription Elongation and its regulation in Bacteria and Eukaryotes February 6, 2016 – In class discussion of problem set Transcription Initiation and its Regulation in Bacteria References 1. General Chapter 12,16 of Molecular Biology of the Gene 6th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M, Losick, R Ptashne, M. and Gann, A. (2002) Genes and Signals. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Luscombe, N.M., Austin, S.E., Berman, H.M., Thornton, J.M. (2000) An overview of the structures of protein-DNA complexes. Genome Biology 1(1): reviews 2. Reviews Murakami KS, Darst SA. (2003) Bacterial RNA polymerases: the wholo story. Curr Opin Struct Biol 13:31-9. Campbell, E, Westblade, L, Darst, S., (2008) Regulation of bacterial RNA polymerase  factor activity: a structural perspective. Current Opinion in Micro. 11: Herbert, KM, Greenleaf, WJ, Block, S. (2008) Single-Molecule studies of RNA polymerase: Motoring Along. Annu Rev Biochem. 77: Werner, Finn and Dina Grohmann (201). Evolution of multisubunit RNA polymerases in the three domains of life. Nature Rev. Microbiology 9: 85-98 Grunberg, S. and Steven Hahn (2013) Structural Insights into transcription initiation by RNA polymerase II. TIBS 38: Transitioning to transcription. Both DNA polymerase--the replication machine and RNA polymerase--the transcription machine are similar: they both bind to DNA and perform templated addition of eith DNA (replicaton) or RNA (transcription). However, the two processes have different goals, Replication: constancy: mulitiplie mechanisms to achieve once and only once replication Transcription: diversity—copy genes as needed in the appropriate amounts multiple mechanisms to achieve diversity of extent of transcription and that impacts the way each process is constructed.

2 3. Studies of Transcription Initiation Roy S, Lim HM, Liu M, Adhya S. (2004) Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step. EMBO J. 23: Sorenson MK, Darst SA. (2006).Disulfide cross-linking indicates that FlgM-bound and free sigma28 adopt similar conformations. Proc Natl Acad Sci U S A. 103: *Kapanidis, AN, Margeat, E, Ho, SO,.Ebright, RH. (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science. 314: Revyakin A, Liu C, Ebright RH, Strick TR (2006) Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science. 314: Murakami KS, Masuda S, Campbell EA, Muzzin O, Darst SA (2002). Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science. 296: 4. A few of the many insights from RNA polymerase structures Cramer, P. (2002) Multisubunit RNA polymerases. Curr Opin Struct Biol 12:89-97. Murakami KS, Darst SA. (2003) Bacterial RNA polymerases: the holo story. Curr Opin Struct Biol 13:31-9. *Cramer, P. (2004) RNA polymerase II structure: from core to functional complexes. Curr Opin Genet Dev 14: Review. Wang, D. Bushnell DA, Westover KD, Kaplan, CD, Kornberg RD. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell Dec 1;127(5): *Cramer, P. (2007). Gene transcription: extending the message. Nature, 448(7150), 5. Discussion Paper **Feklistov A and Darst, SA (2011) Structural basis for Promoter -10 Element recognition by the Bacterial RNA Polymerase s Subunit. Cell 147: 1257 – 1269 Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock and Key to Transcription: –DNA Interaction. Cell: 147:

3 6. Examples of Control Mechanismsa. Alternative Sigma Factors Sorenson, MK, Ray, SS, Darst, SA (2004) Crystal structure of the flagellar sigma/anti-sigma complex 28 /FlgM reveals an intact sigma factor in an inactive conformation. Molecular Cell 14: Gruber, TM, Gross, CA (2003) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev. Microbiol 57:441-66 b.Increasing the Initial Binding of RNA Polymerase Holoenzyme to DNA Lawson CL, Swigon D, Murakami KS, Darst SA, Berman HM, Ebright RH. (2004) Catabolite activator protein: DNA binding and transcription activation. Curr Opin Struct Biol. 14:10-20. c.Increasing the Rate of Isomerization of RNA Polymerase *Dove, S.L., Huang, F.W., and Hochschild, A. (2000) Mechanism for a transcriptional activator that works at the isomerization step. Proc Natl Acad Sci USA 97: Jain, D. Nickels, B.E., Sun, L., Hochschild, A., and Darst, S.A. (2004) Structure of a ternary transcription activation complex. Mol Cell 13: Hawley and McClure (1982) Mechanism of Activation of Transcription from the l PRM promoter. JMB 157: d. DNA looping **Oehler, S., Eismann, E.R., Kramer, H. and Mueller-Hill, B. (1990) The three operators of the lac operon cooperate in repression. EMBO 9: Vilar, J.M.G. and Leibler, S. (2003) DNA looping and physical constraints on transcription regulation. J Mol Biol 331: Dodd, I.B., Shearwin, K.E., Perkins, A.J., Burr, T., Hochschild, A., and Egan, J.B. (2004) Cooperativity in long-range gene regulation by the  cI repressor. Genes Dev. 18: e. The dynamics of lac Repressor binding to its operator Elf, J., Li, G.W., and Xie, X.S. (2007). Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 1191–1194.  Li, G.W., Berg, O.G., and Elf, J. (2009). Effects of macromolecular crowding and DNA looping on gene regulation kinetics. Nat. Phys. 5, 294–297 Li, G.W., and Xie, X.S. (2011). Central dogma at the single-molecule level in living cells. Nature 475, 308–315. Hammar, P., Leroy, P., Mahmutovic, A., Marklund, E.G., Berg, O.G., and Elf, J. (2012). The lac repressor displays facilitated diffusion in living cells. Science 336, 1595–1598 *Choi, PJ, Cai,L, Frieda K and X. Sunney Xie (2008) A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell Science 2008: [DOI: /science ] f. In vivo logic of absolute rates of protein synthesis Li, GW, Burkhardt D, Gross, C and Weissman JS (2014). Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell.157(3): doi:

4 e. The dynamics of lac Repressor binding to its operatorElf, J., Li, G.W., and Xie, X.S. (2007). Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 1191–1194.  Li, G.W., Berg, O.G., and Elf, J. (2009). Effects of macromolecular crowding and DNA looping on gene regulation kinetics. Nat. Phys. 5, 294–297 Li, G.W., and Xie, X.S. (2011). Central dogma at the single-molecule level in living cells. Nature 475, 308–315. Hammar, P., Leroy, P., Mahmutovic, A., Marklund, E.G., Berg, O.G., and Elf, J. (2012). The lac repressor displays facilitated diffusion in living cells. Science 336, 1595–1598 *Choi, PJ, Cai,L, Frieda K and X. Sunney Xie (2008) A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell Science : [DOI: /science ] f. In vivo logic of absolute rates of protein synthesis Li, GW, Burkhardt D, Gross, C and Weissman JS (2014). Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell.157(3): doi:

5 Important concepts Cellular RNA polymerases are conserved across all organisms. These important machines not only produce the transcript but also play regulatory roles. The discrete requirements of initiation and elongation mean that all RNA polymerases have initiation subunits. In bacteria, sigma (s) is the Initiation subunit. 3. The core prokaryotic promoter has two binding sites for s ( -35 and -10 nucleotides from the transcription start site). During initiation the transcription start site is opened. Strand opening event initiates in the -10 region of the promoter. 4. Bacteria contain a single housekeeping s and multiple alternative s s, which generally coordinate responses to stress. 5. Transcription is regulated positively by activators and negatively by repressors. There are many quantitative considerations in designing successful regulatory regimes. In particular, binding sites of RNA polymerase promoters) activators and repressors must be weak to achieve meaningful regulation. Thus, these sites often differ significantly from the “consensus” binding sites that have been determined. 6. Bacterial activators and regulators bind very close to the promoter. Almost all activators directly contact RNA polymerase at either the s or a subunit. 7. Regulatory circuits contain common network motifs. Negative and positive feedback loops are predominant motifs. 8. Regulatory circuits often combine motifs to achieve the desired response to an environmental state.

6 Outline Introduction to Transcription/RNA polymeraseBacterial paradigm for transcription initiation A. Process of Transcription Initiation B. Transition to elongation: Abortive Initiation C. Regulating Transcription initiation Explain why start with bacteria

7 The Transcription cycle: Initiation, Elongation, TerminationBinding: closed complex Promoter melting: open complex Initial transcribing complex Elongation Termination

8 A Schematic view of RNA polymerase transcribing DNARNA polymerase (pale blue) moves stepwise along DNA unwinding the DNA at its active site indicated by the Mg2+ (red), which is required for catalysis. The polymerase adds nucleotides to the RNA chain, using the DNA in the active site as a template. The RNA/DNA hybrid is about 9 nt in length, after which the RNA peels off and exits through the RNA exit channel. NTPs enter through the uptake (secondary) channel. (adapted from MBOC p.304)

9 Structure of RNAP in the three domainsUniversally conserved Archaeal/eukaryotic Archaea Eukarya Bacteria Features of enzyme: crab claw: DNA enters through the big channel in middle; 2 pincers; one is callehe clamp and is moveable; open state accommodates ds DN A closed state ss DNA; closed during transcription; bridge helix; a series of moving parts ects from catalytic center to clamp; implications of closed vs open for initation: Note this important for mechanism of initiation: if closed in PIC, melting must occur outside cleft and ss DNA enter active site; if open melting can occur in cleft Discuss conservation; much greater at structural than sequence level. Extra subunits—away from center; discuss plasticity of enzyme—proliferation of enzymes in eukaryotes, insertions and deletions in proks Explain why discuss basics of system in prokaryotes Transcription Werner and Grohmann (2011), Nature Rev Micro 9:85-98 Extra RNAP subunits provide interaction sites for transcription factors, DNA and RNA, and modulate diverse RNAP activities

10 Cellular RNA polymerases are Important1. Produce all RNAs in the cell at appropriate amount 2. Coordinate transcription in response to environmental/developmental changes 3. Coordinate transcription with downstream events

11 Transcription initiation

12 Steps in Transcription InitiationKB Kf initial binding “isomerization” Abortive Initiation Elongating Complex RPo RPc R+P NTPs Initiation transition Elongation/ termination When we discussed the trx cycle, we talked about how RNAP first binds ds strand dna and then opens it. Here is a closer look at this stage of trx What does it tell you that process can be modeled as an equilibrium event? Which step differs for strong and weak promoters?

13 All cellular RNAPs have initiation subunitsBOARD: WHY DO YOU NEED AN INITATING FORM OF RNAP? Job of RNAP is to make transcripts—has to elongate approximately without regard to sequence; and the enzyme is specialized in this way; starting trx at defined positions requires specific recognition; orthogonal to elongation—whole new apparatus to do this. The initiating enzyme must not only recognize DNA but open strands template strand is available to guide transcription. Contours of that process just being unraveled now—Discussion paper Also participates in 2 other activities: binding the iniitiating nucleotide and the transition when enzyme leaves promoer How does the enzyme leave the promoter, when there are all these DNA – protein interactions to bind it to the promoter? Abortive initiation and its relationship to initiation factors is key

14 The Bacterial paradigm for InitiationCore RNAP + sigma a2bb’ s Holoenzyme a2bb’s Initiation factor BOARD: WHY DO YOU NEED AN INITATING FORM OF RNAP? Job of RNAP is to make transcripts—has to elongate approximately without regard to sequence; and the enzyme is specialized in this way; starting trx at defined positions requires specific recognition; orthogonal to elongation—whole new apparatus to do this. The initiating enzyme must not only recognize DNA but open strands template strand is available to guide transcription. Contours of that process just being unraveled now—Discussion paper Also participates in 2 other activities: binding the iniitiating nucleotide and the transition when enzyme leaves promoer How does the enzyme leave the promoter, when there are all these DNA – protein interactions to bind it to the promoter? Abortive initiation and its relationship to initiation factors is key How was sigma discovered? At the time, there were very few multisubunit enzymes, and Dick burgess worried that some of the subunits werent neessary for the process—so he tried to pruify the enzyme further—and he succeeded!

15 Peak 1 restored activityImproved purification of RNA polymerase leads to the discovery of s Improved fractionation lysate phosphocellulose column Labmate Jeff Roberts reported that the new, improved preparation of RNAP (peak 2) had no activity on l DNA salt 1 2 Activity (*ATP) CT DNA Fraction # OD 280 Peak 1 restored activity  increases rate of initiation s Transcription  DNA Assay: incorporation P ATP using l as template Two lessons: You can purify too much Assay must be an appropriate reflection of the activity you are assaying: why did they use CT DNA originally? Advantages of lambda Why did they use gamma ATP rather than alpha ATP SDS gel analysis Peak Peak 2  '   

16 Recognition of the Prokaryotic promoter-35 logo -10 logo Level of conservation at each position Sequence logos: in binary code—need 2 bits of info ( 2 binary numbers) to count to 4: when have 2 bits of information only 1 nt present at that position; when have 0 bits of information all nt present at = probability . It was already known that 4 universally conserved aromatic residues in sigma contributed to melting Determine a high resolution structure of s2 bound to non-template strand of the -10 element The provocative conclusion they reach is that binding and melting occur concommitantly, thus exposing the template strand at the end of the binding process. Their results suggest that sigma does not recognize any portion of the -10 that ends up melted as duplex. Helix-turn-helix in Domain 4 Recognizes -35 as duplex DNA Is the -10 promoter element recognized as Duplex or SS DNA?

17 s is positioned for DNA recognitionExplain sigma undergoes transition from free to bound

18 Transition to elongation:Abortive initiation

19 Abortive Initiation and Promoter escapeKB Kf initial binding “isomerization” Abortive Initiation Elongating Complex RPo RPc R+P NTPs BOARD During abortive initiation, RNAP synthesizes many short transcripts, but reinitiates rapidly. How can the active site of RNAP move forward along the DNA while maintaining contact with the promoter?

20 Three models for Abortive initiation#1 Predicts movement of both the RNAP leading and trailing edge relative to DNA #2 Predicts expansion and contraction of RNAP One model postulates that the RNAP molecule makes transient downstream excursions on the template, briefly breaking its bonds with the promoter, until the short RNA is released, and then the enzyme diffuses back to the promoter Such a model is not easily reconciled with bulk footprinting data, which suggest that the abortive initiation process results from an inability of RNAP to break its promoter contacts. These observations led Straney & Crothers (to propose that the energy required to break free of the promoter might be somehow stored in a “stressed intermediate” and that abortive initiation was a consequence of this energy not being used productively. One particular instance of this concept, the “inchworming” model, postulates that flexible elements inside RNAP might allow the active center to move forward transiently with respect to the upstream face during synthesis, storing up energy like a stretched spring that retracts upon aborted synthesis. In a third model, the flexible element that stores the energy ultimately used for promoter escape lies not in RNAP but in the single-stranded DNA of the transcription bubble and its interactions with the enzyme. In this scrunching model, RNAP functions more or less as a rigid body. The downstream DNA is pulled progressively into the enzyme with each nucleotide addition cycle, producing a scrunched form within the enzyme footprint .Abortive RNA transcripts lead to the release of the scrunched DNA, which is then extruded out the downstream face of RNAP (1, 56–58), only to be reeled in again upon further RNA synthesis. #3 Predicts expansion and contraction of DNA Science ( : ; ; Slide 38-41

21 Using single molecule FRET to monitor movement of RNAP and DNAFörster (fluorescence) resonance energy transfer (FRET) allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution Experimental set-up for single molecule FRET: Single transcription complexes labeled with a fluorescent donor (D, green) and a fluorescent acceptor (A, red) are illuminated as they diffuse through a femtoliter-scale observation volume (green oval; transit time ~1 ms); observed in confocal microscope Figure 2 Single-molecule fluorescence methods. Fluorescence may be used to track binding and residence times of accessory factors (upper panel) or the position of the RNAP holoenzyme (green) by covalently attaching a fluorescent dye (star) and exciting it with an appropriate wavelength (wavy arrows). Förster (fluorescence) resonance energy transfer (FRET) (lower panel) allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. In the lower left diagram, the donor (yellow star) is excited (blue arrows) and emits light. When the donor fluorophore moves sufficiently close to the acceptor (lower right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes. FRET can follow the distance between two appropriately selected fluorophores by means of the nonradiative coupling of fluorescence energy from one to another, which leads to a change in the emission properties. This technique requires both a donor and an acceptor dye, which are each covalently attached to the molecule(s) of interest in close proximity, typically within 2–10 nm. When the donor fluorophore is exposed to excitation light, it can transfer some of its excited-state energy to the acceptor fluorophore in a process that depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution (26). ALEX: alternating laser excitation of both donor and acceptor; permits focusing analysis on molecules that have both fluors Conclusion: DNA shortens (scrunching!)

22 s is positioned to block elongating transcriptsIn vitro transcription: #1 full-length s; #2: truncated s: no domain 4 or s3-4 in exit tunnel) Murakami, Darst 2002

23 The Bacterial paradigm for RegulatingInitiation

24 Gene regulation in E. coli: The Broad Perspective• 3.6 mB chromosome 4400 genes 7  factors (housekeeping  and alternative s) • sequence-specific DNA-binding proteins Size for a binding site to be unique; how important is it to be unique Talk about alternative sigmas Fraction of genome devoted to DNA binding 5-10% across organisms commentary from Gene Wei Li paper: Increased concentration of a trx factor would drive binding but also have to consider whole picture—how much space on DNA; in a recent paper measuring abundance of ~ 200 trf found ave distance between trx factors only `~36nt; close to optimal distance for rapid binding Do rest later The bacterial chromosome is densely covered with TFs that bind DNA both specifically and nonspecifically (Li et al., 2009). The crowded space on DNA imposes constraints on the abundance of TFs because overcrowding by nonspecifically associated DNA-binding proteins could drastically reduce the overall binding kinetics (Hammar et al., 2012; Li et al., 2009). Thus, although higher concentrations of any given TF would allow it to find its cognate DNA sites more rapidly (von Hippel, 2007), too many TFs in total would mask binding sites. Based on our protein abundance estimates, we found that the average distance between DNA-binding proteins is only36 bp on the E. coli chromosome (assuming that most DNA-binding proteins are associated with DNA nonspecifically and are randomly distributed throughout the genome; which is close to the theoretically optimal density for rapid binding (Li et al., 2009). How cells allocate the limited space on DNA to maximize rapid regulation by each TF remained obscure. Our data indicate that the 200 well-characterized TFs in E. coli show a wide variation in level—more than 60% of the TFs are found to have an upper bound of fewer than 100 monomers per genome equivalent (Figures 6A and 6B). A low copy number for a TF implies a slow association rate to DNA, which could lead to slow transcriptional responses (Winter et al., 1981). For example, single-molecule imaging in vivo previously revealed that it takes 6 min for one Lac repressor to find a single binding site in a cell (Elf et al., 2007). Compared to the celldoubling time, which can be as short as 20 min, the binding kinetics for a low copy number TF would make it difficult to achieve timely regulation. This can be circumvented with the use of TFs that are always bound to their target but whose ability to recruit RNA polymerase depends on the presence of ligands because the kinetics of regulation would be determined by diffusion of the small ligand rather than by diffusion of the bulky and far less abundant protein. We therefore hypothesize that the low copy number TFs have evolved to bind to DNA independent of their activity. In E. coli 1 copy/cell ≈ 10-9 M If KD = 10-9M and things are simple: 10 copies/cell % occupied 100 copies/cell % occupied

25 Overview: Every step of transcription can be regulatedKB Kf initial binding “isomerization” Abortive Initiation Elongating Complex RPo RPc R+P NTPs Negative control: repressors prevent RNAP binding R -35 -10 Hardwired promoter hierarchy: set relative amounts and as hear later req for activators Initial bdg kb: sigmas, repressors activtors Isomerization: c1; also repressors: gal\ abortives: altered start site Positive control: activators facilitate RNAP binding-favorable protein-protein contacts A -35 -10 RNAP holo Favorable contact *

26 Construction of an effective activation systemActivating transcription initiation at KB (initial binding) step Activators ( e.g. CAP); facilitate RNAP binding with favorable protein-protein contact A -35 -10 RNAP holo Favorable contact * H bond yields about 1 kcal/mol (p. 45 MBOC) ∆ G = RT lnKD if * nets 1.4 kcal/mol, KB goes up 10-fold

27 Activating by increasing KB is effective only if initial promoter occupancy is lowIf favorable contact nets 1.4Kcal/mole (KB goes up 10X) then: a) If initial occupancy of promoter is low 1% occupied RNAP 10% occupied A * Transcription rate increases 10-fold Promoter seq hard wired; regulated promoters are far from consensus. Why? Also,very few binding sites for activators and repressors are consensus Lose regulation as go towards consensus—general property of all regulated events RNAP 99% occupied A 99.9% occupied * b) If initial occupancy of promoter is high Little or no effect on transcription rate

28 Strategies to identify point of contact between activator and RNAP1. Isolate “positive control” (pc) mutations in activator. These mutant proteins bind DNA normally but do not activate transcription M 2. “Label transfer” (in vitro) from activator labeled near putative “pc” site to RNAP Activate X*; reduce S-S; X* is transferred to nearest site; determine location by protein cleavage studies; X* transferred to -CTD -35 -10 S-S-X* RNAP Why PC mutants Label transfwer x-link—proximity; depnds on reach of reagent as to how specific it is; introduce cysteine in vicininty –modify with photactivatable X-linker attached with disulfid bonde reduce bond to transfer label to nearest site ( 16A) 3. Isolate activator-non-responsive mutations in RNAP -35 -10 M RNAP

29 Construction of an effective repression system-35 -10 Lac operator (O1) Lac ~ 1980 Lac 2000 -35 -10 -90 O3 O1 O2 +400 Oehler, 2000 O /10 affinity of O1 O /300 affinity of O1 What is the function of these weak operators?

30 A mutant Lac repressor that cannot form Through DNA looping, Lac repressor binding to a “strong” operator (Om) can be helped by binding to a “weak” operator (OA) Om OK Om Oa Better! M A mutant Lac repressor that cannot form tetramers is not helped by a weak site EMBO J (1990) 9: Slide 42.

31 Effects of looping (2 operators)Vilar, J.M.G. and Leibler, S. (2003) J Mol Biol 331: Om (main operator) binds repressor more tightly than Oa (auxiliary operator). Transcription takes place only in the states (i) and (iii), when Om is not occupied. One operator: a single unbinding event is enough for the repressor to completely leave the neighborhood of the main operator. Two operators: repressor can escape the neighborhood of the main operator only if it sequentially unbinds both operators. Insensitivity to fluctuations in repressor level per cell The biological importance of DNA looping: 1. facilitating interaction between different sites; increasing local concentration 2. Allows control of gene regulation on multiple time scales through different kinds of dissociation events. The presence of DNA looping allows the use of rare complete dissociation events to control a bistable genetic switch. DNA LOOPING IS EXTENSIVELY EMPLOYED IN EUKARYOITIC GENE REGULATION TO BRING ENHANCERS IN CONTACT WITH PROMOTER Sunney Xie followed kinetics in living cells; takes on the order of minutes to find site—spends most timesearching ns DNA sites. With multiple operators during partial dissociation from main operator-can initaite 1 round of trx ( ~ molecueles); if totally dissociated must find site again—trx ssevaral hundered until find site. This is important in bistability of the lac switch; no bistability c/o aux operators. Allows control of gene regulation on multiple time scales through different kinds of dissociation events Partial dissociation: can initiate 1 round of transcription (~10-20 molecules) Full dissociation: 6 min to find site again

32 Regulatory Circuits are composed of network motifsMBOC: Slide 43 Regulatory Circuits are composed of network motifs Negative feedback loops: tunes expression to cellular state Blue line: negative feedback Red line: constant rate of A synthesis unaffected by R interations between modeling and expts

33 Positive feed back loopsPositive feedback loops can generate bistability and switch-like responses

34 Bistability at the lac operonScience : Bistability at the lac operon O lacZ lacY lacA P R Repressor Permease (imports inducer) Permease-YFP

35 Combinatorial control of gene expressionAND Logic; e.g. arabinose operon AND NOT Logic, e.g. lac operon

36 The CAP activator senses nutritional stateAND NOT logic is used to regulate how E. coli responds to lactose Inactive CAP Active CAP—binds DNA Regulates >100 genes positively or negatively cAMP high glucose The CAP activator senses nutritional state Talk about actual inducer: we use IPTG but in the cell it’s a byproduct of the 1st enzymatic reaction; b-gal cleaves lactose into glu + gal but also makes allo lactose; b 1-6 instead of 1-4; talk about 2 classes of repressors—rare ones and abundant ones—rare ones are constiutively bound to DNA; then speed of rxn detn by more abundant and small ligands O lacZ lacY lacA P A Repressor Activator CAP-cAMP Activation of lac requires binding of the activator (high cAMP; no glucose) AND NOT binding of the repressor (presence of lactose)

37 Additional slides

38 A. N. Kapanidis et al., Science 314, 1144 -1147 (2006)Initial transcription involves DNA scrunching Lower E* peak is free DNA; higher E* peak is DNA in open complex; distance is shorter because RNAP induces DNA bending Open complex A. N. Kapanidis et al., Science 314, (2006)

39 Initial transcription involves DNA scrunchingOpen complex Abortive initiation complex We postulate that the accumulated DNA-scrunching stress in the stressed intermediate provides the driving force for abortive initiation and also provides the driving force for promoter escape and productive initiation. Thus, we postulate that the accumulated DNA-scrunching stress in the stressed intermediate can be resolved in two ways: either (i) by releasing the RNA product, retaining interactions with promoter DNA, retaining interactions with initiation factors, retaining an unchanged position of the RNAP trailing edge, extruding scrunched DNA, and re-forming RPo (abortive initiation); or (ii) by retaining the RNA product, breaking interactions with promoter DNA, breaking interactions with initiation factors, translocating the RNAP trailing edge, and forming RDe (promoter escape and productive initiation). At a typical promoter, promoter escape occurs only after synthesis of an RNA product ~9 to 11 nt in length (1–11) and thus can be inferred to require scrunching of ~7 to 9 bp (N – 2, where N = ~9 to 11; Fig. 3C). Assuming an energetic cost of base-pair breakage of ~2 kcal/mol per bp (30), it can be inferred that, at a typical promoter, a total of ~14 to 18 kcal/mol of base-pair–breakage energy is accumulated in the stressed intermediate. This free energy is high relative to the free energies for RNAP-promoter interaction [~7 to 9 kcal/mol for sequence-specific component of RNAP-promoter interaction (1)] and RNAP-initiation-factor interaction [~13 kcal/mol for transcription initiation factor {sigma}70 (31)]. We propose that our results demonstrate the existence of an obligatory stressed intermediate, and we propose that the energy accumulated in that obligatory stressed intermediate is the energy that drives the disruption of interactions between RNAP and promoter DNA and between RNAP and initiation factors and, thus, is the energy that drives the transition from initiation to elongation. Higher E* in Abortive initiation complex than open complex results from DNA scrunching

40 Initial transcription involves DNA scrunchingOpen complex Abortive initiation complex The energy accumulated in the DNA scrunched “stressed intermediate could disrupt interactions between RNAP,  and the promoter, thereby driving the transition from initiation to elongation We postulate that the accumulated DNA-scrunching stress in the stressed intermediate provides the driving force for abortive initiation and also provides the driving force for promoter escape and productive initiation. Thus, we postulate that the accumulated DNA-scrunching stress in the stressed intermediate can be resolved in two ways: either (i) by releasing the RNA product, retaining interactions with promoter DNA, retaining interactions with initiation factors, retaining an unchanged position of the RNAP trailing edge, extruding scrunched DNA, and re-forming RPo (abortive initiation); or (ii) by retaining the RNA product, breaking interactions with promoter DNA, breaking interactions with initiation factors, translocating the RNAP trailing edge, and forming RDe (promoter escape and productive initiation). At a typical promoter, promoter escape occurs only after synthesis of an RNA product ~9 to 11 nt in length (1–11) and thus can be inferred to require scrunching of ~7 to 9 bp (N – 2, where N = ~9 to 11; Fig. 3C). Assuming an energetic cost of base-pair breakage of ~2 kcal/mol per bp (30), it can be inferred that, at a typical promoter, a total of ~14 to 18 kcal/mol of base-pair–breakage energy is accumulated in the stressed intermediate. This free energy is high relative to the free energies for RNAP-promoter interaction [~7 to 9 kcal/mol for sequence-specific component of RNAP-promoter interaction (1)] and RNAP-initiation-factor interaction [~13 kcal/mol for transcription initiation factor {sigma}70 (31)]. We propose that our results demonstrate the existence of an obligatory stressed intermediate, and we propose that the energy accumulated in that obligatory stressed intermediate is the energy that drives the disruption of interactions between RNAP and promoter DNA and between RNAP and initiation factors and, thus, is the energy that drives the transition from initiation to elongation.

41 The energy accumulated in the DNA scrunched “stressed intermediate could disrupt interactions between RNAP,  and the promoter, thereby driving the transition from initiation to elongation At a typical promoter, promoter escape occurs only after synthesis of an RNA product ~9 to 11 nt in length (1–11) and thus can be inferred to require scrunching of ~7 to 9 bp (N – 2, where N = ~9 to 11; Fig. 3C). Assuming an energetic cost of base-pair breakage of ~2 kcal/mol per bp (30), it can be inferred that, at a typical promoter, a total of ~14 to 18 kcal/mol of base-pair–breakage energy is accumulated in the stressed intermediate. This free energy is high relative to the free energies for RNAP-promoter interaction [~7 to 9 kcal/mol for sequence-specific component of RNAP-promoter interaction (1)] and RNAP-initiation-factor interaction [~13 kcal/mol for transcription initiation factor {sigma}70 (31)].

42 The weak operators significantly enhance represssionDon’t add to repression by themselves, but enhance the ability of major locus to repress; you will read this paper EMBO J (1990) 9:

43 Coherent feed-forward loop allows timing of responses Example: response to sugars Sustained input Transient input CAP-cAMP MalT activator