Biochem 201 Biological Regulatory Mechanisms Lecture 2 Carol

Biochem 201 Biological Regulatory Mechanisms Lecture #2, Carol Gross; January 21, 2016 Regulation of Transcription in Bacteria General References Chapter 16 of Molecular Biology of the Gene 6 th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M, Losick, R. 547 -587. 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 001. 1 -001. 37 Examples of Control Mechanisms Alternative Sigma Factors Sorenson, MK, Ray, SS, Darst, SA (2004) Crystal structure of the flagellar sigma/anti-sigma complex 28 /Flg. M reveals an intact sigma factor in an inactive conformation. Molecular Cell 14: 127 -138. Gruber, TM, Gross, CA (2003) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev. Microbiol 57: 441 -66 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. 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: 13215 -13220. Jain, D. Nickels, B. E. , Sun, L. , Hochschild, A. , and Darst, S. A. (2004) Structure of a ternary transcription activation complex. Mol Cell 13: 45 -53. Hawley and Mc. Clure (1982) Mechanism of Activation of Transcription from the PRM promoter. JMB 157: 493 -525

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: 973979. Vilar, J. M. G. and Leibler, S. (2003) DNA looping and physical constraints on transcription regulation. J Mol Biol 331: 981 -989. 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 c. I repressor. Genes Dev. 18: 344 -354. 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: 442 -446. [DOI: 10. 1126/science. 1161427] 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): 624 -35. doi: 10. 1016 Proofreading *Zenkin, N, Yuzenkova, y Severinov K Transcript-assisted transcriptional proofreading. Science. 2006 Jul 28; 313(5786): 518 -20 Sydow JF, Cramer P. (2009) RNA polymerase fidelity and transcriptional proofreading. Curr Opin Struct Biol. 2009 Dec; 19(6): 732 -9. Epub 2009 Nov 13. Sydow JF, Brueckner F, Cheung AC, Damsma GE, Dengl S, Lehmann E, Vassylyev D, Cramer P. (2009) Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol Cell. Jun 26; 34(6): 710 -21. Pausing Artsimovitch, I. and Landick, R (2000). Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals. PNAS 97: 7090 -7095

Zhang J, Palangat M, Landick R. Role of the RNA polymerase trigger loop in catalysis and pausing. Nat Struct Mol Biol. 2010 Jan; 17(1): 99 -104. Epub 2009 Dec 6. *Shaevitz, j. Abbondanzieri E, Landick R. and Block S (2003) Backtracking by single RNA polymerase molecules observed at near base pair resolution. Nature 426: 684 -687 Herbert, K. , La Porta, A, Wong B, Mooney, R. Neuman, K. Landick, R. and Block, S. (2006). Sequence-Resolved Detection of Pausing by Single RNA Polymerase Molecules. Cell 125: 1083 -1094 *Weixlbaumer, A, Leon, K, Landick, R and Darst SA (2013) Structural basis of transcriptional pausing in bacteria. Cell. 2013 Jan 31; 152(3): 431 -41. doi: 10. 1016/j. cell. 2012. 020. Regulation through the 2˚ channel Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, Foster JW, Gourse RL. (2004) Dks. A: a critical component of the transcription initiation machinery that potentiates the regulation of r. RNA promoters by pp. Gpp and the initiating NTP. Cell. 6: 311 -22 Measurement of elongation Larson MH, Mooney RA, Peters JM, Windgassen T, Nayak D, Gross CA, Block SM, Greenleaf WJ, Landick R, Weissman JS. Science. 2014: A pause sequence enriched at translation start sites drives transcription dynamics in vivo. May 30; 344(6187): 1042 -7. Shaevitz JW, Abbondanzieri EA, Landick R, Block SM Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature. 2003 Dec 11; 426(6967): 684 -7. Epub 2003 Nov 23.

Important Points 1. Every step in transcription initiation can be regulated to increase or decrease the number of successful initiations per time. 2. In E. coli, transcription initiation is controlled primarily by alternative factors and by a large variety of other sequence-specific DNA-binding proteins. 3. G=RTln. KD. This means that a net increase of 1. 4 kcal/mole (the approximate contribution of an additional hydrogen bond) increases binding affinity by 10 -fold. Many examples of transcription activation in bacteria take advantage of such weak interactions. 4. To activate transcription at a given promoter by increasing KB, the concentration of RNA polymerase in the cell and its affinity for the promoter must be in the range so an increase in KB makes a difference. Likewise, to activate transcription by increasing kf, the rate of isomerization must be slow enough so the increase makes a substantial difference. 5. Network motifs give the regulatory circuit its properties 6. Transcriptional pauses are integral to the transcription process and are extensively utilized for regulatory roles

Transcriptional Control: Bacterial Paradigms 1. Quick review of protein-DNA interactions 2. Overview of bacterial gene regulation 3. Regulating transcription in the cellular milieu 4. An in-depth look at activation and repression 5. Regulatory circuits 6. Elongation control

How proteins recognize DNA

All 4 bp can be distinguished in the major groove

Common families of DNA binding proteins

Overview: Every step of transcription can be regulated NTPs KB R+P RPc initial binding Kf Abortive Initiation RPo “isomerization” Elongating Complex DNA Binding Proteins used to alter promoter properties Negative control: repressors prevent RNAP binding R -35 -10 Positive control: activators facilitate RNAP binding-favorable protein contact Favorable contact A * RNAP holo -35 -10

Gene regulation in E. coli: The Broad Perspective • 3. 6 m. B chromosome • 4400 genes • 300 -350 sequence-specific DNA-binding proteins • 7 factors In E. coli 1 copy/cell ≈ 10 -9 M If KD = 10 -9 M and things are simple: 10 copies/cell 100 copies/cell 90% occupied 99% occupied

Construction of an effective activation system Activating transcription initiation at KB (initial binding) step Positive control: activators ( e. g. CAP); facilitate RNAP binding with favorable protein-protein contact Favorable contact A * RNAP holo -35 -10 ∆ G = RT ln. KD; if * nets 1. 4 kcal/mol, KB goes up 10 -fold

Activating by increasing KB is effective only if initial promoter occupancy is low If favorable contact nets 1. 4 Kcal/mole (KB goes up 10 X) then: a) If initial occupancy of promoter is low RNAP A * RNAP 10% occupied 1% occupied Transcription rate increases 10 -fold b) If initial occupancy of promoter is high RNAP 99% occupied A * RNAP 99. 9% occupied Little or no effect on transcription rate

Strategies to identify point of contact between activator and RNAP 1. Isolate “positive control” (pc) mutations in activator. These mutant proteins bind DNA normally but do not activate transcription M 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 3. Isolate activator-nonresponsive mutations in RNAP S-S-X* RNAP -35 -10 M RNAP -35 -10

Construction of an effective repression system Lac ~ 1980 -35 -10 Lac operator (O 1) Lac 2000 O 3 -90 O 1 -35 -10 O 2 +400 Oehler, 2000 O 2 1/10 affinity of O 1 O 3 1/300 affinity of O 1 What is the function of these weak operators?

The weak operators significantly enhance represssion Oehler, 2000

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

Effects of looping (2 operators) 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. Vilar, J. M. G. and Leibler, S. (2003) J Mol Biol 331: 981 -989 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. 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

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

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

Bistability at the lac operon A P O lac. Z lac. Y Permease lac. A Repressor (imports inducer) Permease-YFP

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

AND NOT logic is used to regulate how E. coli responds to sugar source The CAP activator senses nutritional state c. AMP Inactive CAP high glucose Active CAP—binds DNA Regulates >100 genes positively or negatively A P O lac. Z lac. Y lac. A Repressor Activator CAP-c. AMP Activation of lac requires binding of the activator (high c. AMP; no glucose) AND NOT binding of the repressor (presence of lactose)

Coherent feed-forward loop allows timing of responses Example: response to sugars Transient input CAP-c. AMP Mal. T activator Sustained input

Regulated Elongation Co-evolution of RNAP and Nascent RNA produced functional interplay. RNAP influence on RNA structure: vectorial transcription limits folding landscape RNA influence on RNAP: sequence and structure alter polymerase speed up to 2 orders of magnitude, as well as functionality.

Current view of Pausing (? ) Elemental Pause Elongation Complex

Formation of an RNA hairpin and its effect on RNAP 1. Assemble complex on a synthetic scaffold with a single fluorescent base (*) 2. Add antisense RNA such that RNA stem starts 12 bp from 3’ end of RNA; measure rate of quenching. This mimics a hairpin pause; such hairpins form inside the RNA exit channel * Result: rate of formation slows only 2 -fold 3. Flap tip required to interact with duplex for pause 4. Clamp must be in open position for pause Hein…& Landick Nature Structural & Molecular Biology 21, 794– 802 (2014) doi: 10. 1038/nsmb. 2867

Using a hairpin pause to couple transcription to translation in operons encoding enzymes for biosynthesis of amino acids 1. Pause RNA polymerase at a hairpin pause positioned immediately upstream of a terminator hairpin 2. Pausing of transcription allows initiation of translation; RNA polymerase releases from the pause when the ribosome approaches thus allowing synchronized transcription/translation 3. The ribosome is translating a small ORF with several codons for the amino acid produced by the biosynthetic genes in the operon e. g. hisitidine 4. If translation does not stall ( e. g. Amino acid in excess) the terminator hairpin is formed and very little of the m. RNA for the biosynthetic genes are produced. 5. If translation and the ribosome stall because amino acid is limiting, then the terminator loop does not form and Abundant m. RNA for the biosynthetic genes are formed. 6. This method of regulation is called Attenuation control

Attenuation in biosynthetic operons His codons TAA his. L his. G 1 2 No protein synthesis High His his. L 4 1 2 pause hairpin TAA his. L 3 4 1 Low His 3 2 his. G 3 4 transcription terminator his. G transcription terminator Operon m. RNA level TAA his. L 2 3 1 transcription 4 anti-terminator Low his. G High

Regulated “attenuation” (termination) is widespread Switch between the “antitermination” and “termination” Stem-loop structures can be mediated by: 1. Ribosome pausing ( reflects level of a particular charged t. RNA): regulates expression of amino acid biosynthetic operons in gram - bacteria 2. Uncharged t. RNA: promotes anti-termination stem-loop in amino acyl t. RNA synthetase genes in gm + bacteria 3. Proteins: stabilize either antitermination or termination stem-loop structures 4. Small molecules: aka riboswitches 5. Alternative 2˚ structures can also alter translation, self splicing, degradation

Pauses can also be measured genome wide using NET-seq Matt Larson ( Weissman lab)

RNA polymerase pausing is enriched at translation start site Matthew H. Larson et al. Science 2014; 344: 1042 -1047 Published by AAAS

E. coli Nus. G: A 21 k. D essential elongation factor NTD NGN domain CTD KOW domain Activities: 1. Increases elongation rate 2. suppresses backtracking 3. Required for anti-termination mechanisms 4. Enhances termination mediated by the rho-factor How does one 21 Kd protein mediate all of these activities?

The CTD of Nus. G interacts with other protein partners Nus. G CTD 50 µM 10 Nus. E, a ribosomal protein (S 10) is part of a complex of proteins mediating antitermination/termination depending on its protein partners n. M Rho is an RNA binding hexamer that mediates termination by dissociating RNA from its complex with RNA polymerase and DNA using stepwise physical forces on the RNA derived from alternating protein conformations coupled to ATP hydrolysis Although the CTD mediates the protein interactions involved in termination and antitermination, full length Nus. G is required for both processes, presumably because Nus. G must be tethered to RNA polymerase for these functions

Coupled syntheses. J W Roberts Science 2010; 328: 436 -437 Published by AAAS

Nus. G, the only universal elongation factor, exhibits divergent interactions with other regulators