7.1 The lac Operon 7-1

Transcription

1 7.1 The lac Operon The lac operon was the first operon discovered It contains 3 genes coding for E. coli proteins that permit the bacteria to use the sugar lactose Galactoside permease (lacy) which transports lactose into the cells - β-galactosidase (lacz) cuts the lactose into galactose and glucose Galactoside transacetylase (laca) whose function is unclear 7-1

2 Genes of the lac Operon The genes are grouped together: lacz = β-galactosidase lacy = galactoside permease laca = galactoside transacetylase All 3 genes are transcribed together producing 1 mrna, a polycistronic message that starts from a single promoter Each cistron, or gene, has its own ribosome binding site Each cistron can be translated by separate ribosomes that bind independently of each other 7-2

3 Control of the lac Operon The lac operon is tightly controlled, using 2 types of control Negative control, like the brake of a car, must remove the repressor from the operator - the brake is a protein called the lac repressor Positive control, like the accelerator pedal of a car, an activator, additional positive factor responds to low glucose by stimulating transcription of the lac operon 7-3

4 Negative Control of the lac Operon Negative control indicates that the operon is turned on unless something intervenes and stops it The off-regulation is done by the lac repressor Product of the laci gene Tetramer of 4 identical polypeptides Binds the operator just right of promoter 7-4

5 Negative Control of the lac Operon 7-5

6 Inducer of the lac Operon The repressor is an allosteric protein Binding of one molecule to the protein changes shape of a remote site on that protein Altering its interaction with a second molecule The inducer binds the repressor Causing the repressor to change conformation that favors release from the operator The inducer is allolactose, an alternative form of lactose 7-6

7 Inducer of the lac Operon The inducer of the lac operon binds the repressor The inducer is allolactose, an alternative form of lactose 7-7

8 Discovery of the Operon During the 1940s and 1950s, Jacob and Monod studied the metabolism of lactose by E. coli Three enzyme activities / three genes were induced together by galactosides Constitutive mutants need no induction, genes are active all the time Created merodiploids or partial diploid bacteria carrying both wild-type (inducible) and constitutive alleles 7-8

9 Discovery of the Operon Using merodiploids or partial diploid bacteria carrying both wild-type and constitutive alleles distinctions could be made by determining whether the mutation was dominant or recessive Because the repressor gene produces a repressor protein that can diffuse throughout the nucleus, it can bind to both operators in a meriploid and is called a trans-acting gene because it can act on loci on both DNA molecules Because an operator controls only the operon on the same DNA molecule it is called a cis-acting gene 7-9

10 Effects of Regulatory Mutations: Wild-type and Mutated Operator with Defective Binding 7-10

11 Effects of Regulatory Mutations: Wild-type and Mutated Operon binding Irreversibly 7-11

12 Effects of Regulatory Mutations: Wild-type and Mutated Repressor 7-12

13 The Mechanism of Repression The repressor does not block access by RNA polymerase to the lac promoter Polymerase and repressor can bind together to the lac promoter Polymerase-promoter complex is in equilibrium with free polymerase and promoter 7-13

14 lac Repressor and Dissociation of RNA Polymerase from lac Promoter Without competitor, dissociated polymerase returns to promoter Heparin and repressor prevent reassociation of polymerase and promoter Repressor prevents reassociation by binding to the operator adjacent to the promoter This blocks access to the promoter by RNA polymerase 7-14

15 Mechanism Summary Two competing hypotheses of mechanism for repression of the lac operon RNA polymerase can bind to lac promoter in presence of repressor Repressor will inhibit transition from abortive transcription to processive transcription The repressor, by binding to the operator, blocks access by the polymerase to adjacent promoter 7-15

16 lac Operators There are three lac operators The major lac operator lies adjacent to promoter Two auxiliary lac operators - one upstream and the other downstream All three operators are required for optimum repression The major operator produces only a modest amount of repression 7-16

17 Catabolite Repression of the lac Operon When glucose is present, the lac operon is in a relatively inactive state Selection in favor of glucose attributed to role of a breakdown product, catabolite Process known as catabolite repression uses a breakdown product to repress the operon 7-17

18 Positive Control of lac Operon Positive control of the lac operon by a substance sensing lack of glucose that responds by activating the lac promoter The concentration of a nucleotide, cyclic-amp (camp), rises as the concentration of glucose drops 7-18

19 Catabolite Activator Protein camp added to E. coli can overcome catabolite repression of the lac operon The addition of camp leads to the activation of the lac gene even in the presence of glucose Positive controller of lac operon has 2 parts: camp A protein factor is known as: Catabolite activator protein or CAP Cyclic-AMP receptor protein or CRP Gene encoding this protein is crp 7-19

20 The Mechanism of CAP Action The CAP-cAMP complex binds to the lac promoter Mutants whose lac gene is not stimulated by complex had the mutation in the lac promoter Mapping the DNA has shown that the activatorbinding site lies just upstream of the promoter Binding of CAP and camp to the activator site helps RNA polymerase form an open promoter complex 7-20

21 Lac Operon: three operators one promoter and one CAP binding site

22 CAP Binding sites for CAP in lac, gal and ara operons all contain the sequence TGTGA Sequence conservation suggests an important role in CAP binding Binding of CAP-cAMP complex to DNA is tight CAP-cAMP activated operons have very weak promoters Their -35 boxes are quite unlike the consensus sequence If these promoters were strong they could be activated even when glucose is present 7-22

23 Proposed CAP-cAMP Activation of lac The CAP-cAMP dimer binds to its target site on the DNA The αctd (α-carboxy terminal domain) of polymerase interacts with a specific site on CAP Binding is strengthened between promoter and polymerase Transcription 7-23

24 Summary CAP-cAMP binding to the lac activator-binding site recruits RNA polymerase to the adjacent lac promoter to form a closed complex CAP-cAMP causes recruitment through protein-protein interaction with the αctd of RNA polymerase 7-24

25 7.3 The trp Operon The E. coli trp operon contains the genes for the enzymes the bacterium needs to make the amino acid tryptophan The trp operon codes for anabolic enzymes, those that build up a substance Anabolic enzymes are typically turned off by a high level of the substance produced This operon is subject to negative control by a repressor when tryptophan levels are elevated trp operon also exhibits attenuation 7-25

26 Negative Control of the trp Operon Without tryptophan no trp repressor exists, just the inactive protein, aporepressor If aporepressor binds tryptophan, changes conformation with high affinity for trp operator Combine aporepressor and tryptophan to form the trp repressor Tryptophan is a corepressor 7-26

27 Attenuation in the trp Operon 7-27

28 Mechanism of Attenuation Attenuation imposes an extra level of control on an operon Operates by causing premature termination of the operon s transcript when product is abundant In the presence of low tryptophan concentration, the RNA polymerase reads through the attenuator so the structural genes are transcribed In the presence of high tryptophan concentration, the attenuator causes premature termination of transcription 7-28

29 Defeating Attenuation Attenuation operates in the E. coli trp operon as long as tryptophan is plentiful If amino acid supply low, ribosomes stall at the tandem tryptophan codons in the trp leader trp leader being synthesized as stalling occurs, stalled ribosome will influence the way RNA folds Prevents formation of a hairpin This is part of the transcription termination signal which causes attenuation 7-29

30 Overriding Attenuation 7-30

31 Temporal control of Transcription in phage SPO-1 infected B. subtilis

32 T7 RNA Polymerase.

33 Ester Lederberg UV treated bacteria discovered λ virus (Phage)

34 Lambda Life Cycle.

35 Lambda

36 Lambda Genome

37 Plaques are turbid

38 Lytic Life Cycle.

39 N anti-terminates transcription.

40 Protein complexes involved in N-mediated anti-termination.

41 Lytic Growth Summary P L and P R are used by RNAP no ci present Cro and N are made N anti-terminates tx at t L and t R1 Polymerase transcribes ciii and recombination genes (L) Polymerase transcribes cii, DNA replication genes and Q (R) Cro binds to O R and O L blocking the P RM If cii levels are low then lytic growth continues because the P RE is not activated

42 Lambda

43 Cro vs ci Cro and ci are DNA binding proteins that bind to the operator sequences OR and OL There are three binding sites for each protein in the operator Cro and ci bind with opposite affinities to these three sites ci binds first to site 1, then 2 and at high concentrations site 3 Cro binds to site 3 first, then site 2 followed by site 1 ci binds to sites adjacent to its own promoter P RM activating its own transcription Cro binds to the P RM and prevents transcription from this promoter

44 The Battle between cro and ci.

45 Q-mediated anti-termination of the late genes. Recent data support the idea that the Q binding site is actually upstream of the -35 box

46 Q- anti-termination Q binds to the late promoter Pr (a unique promoter for Q) through the Q utilization site Holoenzyme binds to the Pr (PQ) promoter (Pr ) and sigma interacts tightly with the -10 region of this promoter Q associates with the sigma factor in the 4 domain (-35 region) releasing sigma from the polymerase Q continues to associate with the polymerase and late genes are transcribed by the Q-containing core enzyme

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50 Establishing Lysogeny If cii levels are high then lysogenic growth will proceed cii activates P RE (repressor establishment) cii activates P I (int) cii activates P antiq Resulting in the synthesis of ci and the turn off of P R eliminating cro synthesis ci binds to the O L and O R preventing further P L and P R transcription ci activates its own promoter P RM and lysogeny is established

51 Lambda

52 Establishing Lysogeny.

53 cii activates three promoters: P int P re P antiq

54 Maintaining lysogeny.

55 The Life-Cycle Decision The levels of cii and ciii are critical they sense the health of the cell Healthy rapidly growing cells have high levels of proteases which degrade ciii and cii ciii tries to block the proteases from cleaving cii Hfl (high frequency lysogen) is a bacterial gene that greatly influences this decision When Hfl is absent or mutated lysogeny is highly favored as this protease cleaves ciii When cells are starved and not dividing protease levels are low and cii and ciii levels are stabilized thus favoring lysogenic growth

56 Lambda Integration Once cii levels are established cii activates the P Int promoter allowing the synthesis of int P Int is located in the xis gene so only int is made and integration results

57 The importance of P int for integration and lysogeny

58 Lambda induction DNA damage results in the activation of the SOS system in bacteria and the synthesis of reca The reca protein causes ci to cleave and dissociate from DNA When O R and O L no longer have ci bound P R and P L become active and lytic gene expression proceeds

59 Inducing the Lambda prophage.

60 λ induction

61 Integration and excision of λ DNA

62 Excision of Lambda DNA Integration of lambda DNA into the genome results in the physical separation of the b- region (sib) from int and xis Under these circumstances the synthesis of both xis and int occur from PL When both int and xis are present excision will occur and the prophage is excised from the genome

63 Lytic replication Lytic replication of λ DNA occurs both when the initial decision was to grow lyticly and after excision from the bacterial chromosome P R transcription results in the synthesis of the O, P and Q genes For the first few replication cycles the λ genome is replicated circle to circle Circular DNA cannot be packaged into phage particles O protein binds to the ori site internal to the O gene and P binds to O as well as host DNA polymerase Rolling circle replication then proceeds resulting in a long concatemer of λ genomes The concatemer is cleaved and linear DNAis packaged

64 Why are plaques turbid?