λ s Cro Molecule CI 2 Bound to O R 3 Turns Off P RM Basal Versus Activated Rate CI 2 Bound to O R 2 Turns On P RM P R Active When O R Sites Are Empty

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1 Phage λ ECE/CS/BioEn 6760 Modeling and Analysis of Biological etworks Chris J. Myers Lecture 4: Phage λ: A Simple Genetic Circuit In 1953, Lwoff et al. discovered that a strain of E. Coli when exposed to UV light lyse spewing forth λ viruses. Some of the newly infected E. Coli would soon lyse while others grow and divide normally until exposed to UV light. In other words, some cells follow a lysis pathway while other followed a lysogeny pathway. The decision between the lysis and the lysogeny developmental pathway is made by a fairly simple genetic circuit. Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 1 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 2 / 64 Phage λ Phage λ Developmental Pathways (Courtesy of Maria Schnos and Ross Inman, Institute for Molecular Virology, University of Wisconsin, Madison) (Courtesy of Alberts et al. (2002)) Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 3 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 4 / 64 The O R Operator CI, the λ Repressor M CI CI 2 C C C M Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 5 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 6 / 64

2 λ s Cro Molecule CI 2 Bound to Turns Off Cro 2 Cro M M Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 7 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 8 / 64 CI 2 Bound to Turns On M Basal Versus Activated Rate M RAP othing in biology is clear-cut. Without CI 2 bound to, RAP can still bind to M and initiate transcription of CI at a reduced basal rate. With CI 2 bound to, transcription occurs at enhanced activated rate. Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 9 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 10 / 64 CI 2 Bound to Turns Off M Active When O R Sites Are Empty M RAP M RAP Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 11 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 12 / 64

3 Cro 2 Bound to Turns off M Cro 2 Bound to or Turns off M RAP M Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 13 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 14 / 64 Low Concentrations of CI 2 Cooperativity Aids CI 2 Binding to M M Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 15 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 16 / 64 High Concentrations of CI 2 Low Concentrations of Cro 2 M M Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 17 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 18 / 64

4 Moderate Concentrations of Cro 2 High Concentrations of Cro 2 M M OR M Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 19 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 20 / 64 Cooperativity of CI 2 Binding Cooperativity of CI 2 Binding M M Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 21 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 22 / 64 Cooperativity of CI 2 Binding Cooperativity of CI 2 Binding M RAP M Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 23 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 24 / 64

5 Effect of Cooperativity Configurations of O R 1 % Repression (no coop) Log[CI Total Concentration] At low to moderate concentrations of CI and Cro, there are three common configurations: o molecules bound to O R, Cro produced at full rate and CI produced at low basal rate. CI 2 bound to and, Cro production repressed, and CI activated. Cro 2 bound to, CI cannot be produced, Cro is produced. Feedback of the products as transcription factors coupled with affinities makes O R behave as a bistable switch. In lysis state, Cro produced locking out production of CI. In lysogeny state, CI produced locking out production of Cro. Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 25 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 26 / 64 Immunity Induction In lysogeny state, cell is immune to further infection. genes on DA inserted by further infections are shut off by CI 2 molecules from first infection. Once cell commits to lysogeny, it becomes very stable and does not easily change over to the lysis pathway. UV Light RecA M Activated RecA Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 27 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 28 / 64 Recognition of the Operators ear Symmetry in the Operator Sequences CI 2 and Cro 2 bind to operator sites that are 17 base pairs long. How do these proteins locate these sequences from amongst the millions within the bacteria? Observing from midpo, a strand on one side is nearly symmetric with complimentary strand on other side. Op Operator sequences Operator half sequences T A T C A C C G c C A G A G G T A T A T C A C C G c A T A G T G G C g G T C T C C A T T A C C T C T G T A A C A C C G t G C G T G T T G T A A C A C C G t A T T G T G G C a C G C A C A A C C A A C A C G C T A T C A C C G c A A G G G A T A T A T C A C C G c A T A G T G G C g T T C C C T A T T A T C C C T T O L 1 T A T C A C C G c C A G T G G T A T A T C A C C G c A T A G T G G C g G T C A C C A T T A C C A C T G O L 2 T A T C T C T G g C G G T G T T G T A T C T C T G A T A G A G A C c G C C A C A A C C A A C A C C G c O L 3 T A T C A C C G c A G A T G G T T T A T C A C C G c A T A G T G G C g T C T A C C A A A A C C A T C T Con. T 9 A 12 T 6 C 12 A 9 C 11 C 7 G 9 C 5 C 2 C 3 T 2 T 1 T 4 T 2 T 1 A 1 A 3 C 1 G 1 C 1 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 29 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 30 / 64

6 Consensus Sequence Amino Acid-Base Pair Interactions The consensus sequence is as follows: Many entries are highly preserved. TATCACCGcCGGTGATA ATAGTGGCgGCCACTAT Differences et that cause the differences in affinity for CI 2 and Cro 2 for the different operators. otice that the first half of the operator sites and agree perfectly with the consensus sequence while second half has several differences. gln gly gly leu asn ser val CI 2 ala phe T A C C T C T G A T G G A G A C C gln ala asn ala his ser C A A C A C G C G T T G T G C G T ile Cro 2 lys ile T A T C C C T T A T A G G G A A C Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 31 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 32 / 64 λ Promoters The λ Genome Consensus T T G A C A - 17bp - T A T A A T λm T A G A T A - 17bp - T A G A T T λ T T G A C T - 17bp - G A T A A T (Courtesy of Richard Wheeler) Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 33 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 34 / 64 Patterns of Gene Expression Very Early Events Very Early Inactive CII Early Active CII Very Early Late Lysis Late Lysogeny Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 35 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 36 / 64

7 The Action of The Action of RAP RAP UT L II UT L II Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 37 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 38 / 64 The Action of The Action of RAP UT L II UT L II Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 39 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 40 / 64 The Action of The Action of RAP RAP UT L II UT L II Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 41 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 42 / 64

8 Early Events Retroregulation of Int Early sib Rase3 II Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 43 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 44 / 64 Retroregulation of Int Retroregulation of Int II II Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 45 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 46 / 64 Lysis/Lysogeny Decision Lysis/Lysogeny Decision (cont) Key to lysis/lysogeny decision is the protein CII. CII activates E which jump-starts production of CI. After jump-start, positive feedback in M increases CI and locks out Cro production resulting in lysogeny decision. Activity of CII is determined by environmental factors. Bacterial proteases attack and destroy CII. Growth in a rich medium activates these proteases whereas starvation has the opposite effect. Thus, λ tends to lysogenize starved cells. CIII protein protects CII from degradation promoting lysogeny. Production of CII and CIII enhanced by anti-terminator,. Higher multiplicity of infection means more, I, and II gene copies leading to higher probability of lysogeny. High protease levels and low gene counts CII produced slowly and degrades rapidly little CI is synthesized Q and Cro are synthesized lysis decision Low protease levels and high gene counts more, CII, and CIII are produced CI and Int are made from E and P I Int egrates phage chromosome and CI turns off all genes except CI lysogeny decision Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 47 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 48 / 64

9 Late Lytic Events Late Lysogenic Events Late Lysis Late Lysogeny Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 49 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 50 / 64 Integration and Induction Chemical Reaction etwork Model attp attb Variety of ways to model a system using chemical reactions. This model includes: Five genes:,,, I, and II. Four promoters: M,, E, and. Model somewhat simplified (see appendix at end of Chapter 2). II sib Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 51 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 52 / 64 Phage λ Decision Circuit Model of the Promoter E CI CI 2 Cro 2 Cro M T R1 50% PRE CII E + RAP E + CII KPRE2 KPRE3 E RAP E CII UT R O E 1 O E 2 I E + CII+RAP E RAP KPRE4 kpreb E CII RAP E RAP+nCI CI 2 O L 1 Cro 2 O L 2 UT L n 80% deg II CIII E CII RAP kpre E CII RAP+nCI Constant Value Constant Value K PRE M 1 k PREb sec 1 K PRE M 1 k PRE sec 1 K PRE M 1 n 10 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 53 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 54 / 64

10 Model of the O R Operator Simplified Model of the O R Operator (1 Site Occupied) Modeling method just described requires (n 1) chemical reactions where n is number of potential configurations of transcription factors and RAP bound to the operator and promoter sites. Also requires m reactions for configurations leading to transcription. O R operator has 40 possible configurations with 13 leading to transcription. has four states (empty, CI 2, Cro 2, and RAP). and also can bind all transcription factors but bind to RAP joly (i.e., 10 possibilities). O R + CI 2 O R + Cro 2 O R + RAP O R + RAP RAP 2 RAP KOR2 O R CI 2 KOR5 O R Cro 2 KOR8 KOR9 kprmb kpr RAP 2 RAP RAP+nCI 2 RAP+nCro Constant Value Constant Value K OR M 1 k PRMb sec 1 K OR M 1 k PRM sec 1 K OR M 1 k PR sec 1 K OR M 1 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 55 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 56 / 64 Simplified Model of the O R Operator (2 Sites Occupied) Simplified Model of the O R Operator (3 Sites Occupied) O R + 2CI 2 O R + CI 2 + Cro 2 O R + 2RAP O R + Cro 2 + RAP O R 2RAP O R 2RAP O R Cro 2 RAP KOR10 O R 2CI 2 KOR17 O R CI 2 Cro 2 KOR16 KOR26 kprmb kpr kpr O R 2RAP O R Cro 2 RAP O R 2RAP+nCI O R 2RAP+nCro O R Cro 2 RAP+nCro Constant Value Constant Value K OR M 1 k PRMb sec 1 K OR M 1 k PRM sec 1 K OR M 1 k PR sec 1 K OR M 1 O R + 2CI 2 + Cro 2 O R + RAP+2CI 2 O R RAP 2CI 2 KOR31 O R 2CI 2 Cro 2 KOR37 O R RAP 2CI 2 kprm O R RAP 2CI 2 + nci Constant Value Constant Value K OR M 1 k PRMb sec 1 K OR M 1 k PRM sec 1 k PR sec 1 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 57 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 58 / 64 CI and Cro Dimerazation and Degradation Production from promoter and Degradation + Cro 2 KPL2 Cro 2 + CI 2 KPL4 CI 2 + 2Cro 2 KPL7 2Cro 2 2CI 2Cro CI Cro K2 CI 2 K5 Cro 2 k1 () k4 () Constant k 1 K 2 k 4 K 5 Value sec 1 0.1M sec M 1 + CI 2 + Cro 2 + 2CI 2 + RAP RAP KPL8 CI 2 Cro 2 KPL10 2CI 2 KPL6 kpl k7 () RAP RAP+n Constant Value Constant Value K PL M 1 K PL M 1 K PL M 1 K PL M 1 K PL M 1 k PL sec 1 K PL M 1 k sec 1 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 59 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 60 / 64

11 CIII and CII Production Model for CII and CIII Degradation UT L + RAP+UT L RAP+UT L UT R + 2 RAP+UT R KUT 0.2 kpl kpl KUT 0.5 kpr UT L RAP+UT L + nciii RAP+UT L +nciii UT R 2 RAP+UT R + ncii CII+P1 CII P1 CIII+P1 CIII P1 K8 k10 K11 k13 CII P1 P1 CIII P1 P1 Constant K 8 k 10 K 11 k 13 P1 Value 1.0 M sec M sec 1 35nM 2 RAP+UT R kpr 2 RAP+UT R +ncii ote that K UT is 0.2 M 1. Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 61 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 62 / 64 Why Study Phage λ? Sources Bacteria and their phages multiply quickly making it easier to analyze gene regulation with them than higher organisms. Phage λ has been the subject of study for over 50 years now. It is one of, if not the best, understood genetic circuit. Excellent illustration of a circuit that analyzes its environment makes decision between two competing pathways. Similarities with bacteria that must respond to stress and circuits involved in development and cell differentiation. Genes from phage λ are used in synthetic biology where DA is produced to perform particular functions. Phage λ is an excellent testbed for trying new ideas. Virtually every modeling method has been applied to phage λ. This course also uses it as a running example throughout. Excellent history in paper by Gottesman and Weisberg (2004) Mark Ptashne - A Genetic Switch Wikipedia entry for phage λ Model inspired by Arkin et al. s phage λ model Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 63 / 64 Chris J. Myers (Lecture 4: Phage λ) ECE/CS/BioEn 6760: Modeling Bio etworks 64 / 64