Chapter 15 Gene Regulation in Prokaryotes

Transcription

1 Chapter 15 Gene Regulation in Prokaryotes 17-1

2 Sections to study 15.1 The elements of prokaryotic gene expression 15.2 Regulation of transcription initiation via DNA-binding proteins 15.3 RNA-mediated mechanisms of gene regulation 15.4 Discovering and manipulating bacterial gene regulatory mechanisms 17-2

3 Bacteria respond to environmental changes by changing gene expression Vibrio cholerae causes cholera 17-3

4 15.1 The elements of prokaryotic gene expression RNA polymerase and the three phases of transcription. Initiation sigma subunit + core enzyme Binds to promoter, unwinds DNA, begins polymerization of bases Elongation core enzyme moves away from promoter, sigma subunit released, polymerization of ribonucleotides. 17-4

5 Termination Rho-dependent termination Rho factor recognizes sequence in mrna, binds to it, and pulls it away from RNA polymerase. Rho-independent termination stem-loop structure formed by sequence of 20 bases with a run of 6 or more U s U s signals release of RNA polymerase. 17-5

6 Polycistronic mrna: : One mrna contains the information of several genes. Translation in prokaryotes begins before transcription ends! Initiation sites for translation signal ribosomes to bind near 5 5 end of mrna while downstream transcription is still occurring. Ribosomes can initiate translation at several positions along a single mrna. 17-6

7 The regulation of gene expression can occur at many steps Transcription initiation Shift from initiation to elongation Release of mrna at termination Stability of mrna Efficiency of ribosomes to recognize translation initiation sites Stability of polypeptide product 17-7

8 15.2 Regulation of transcription initiation via DNA-binding proteins A model system for studying gene regulation: The utilization of lactose by E. coli Jacques Monod's bi-phasic growth curve 17-8

9 Bacteria grown with two different sugars often displayed two phases of growth. Phase I Use glucose Phase II Use lactose Glucose Lactose 17-9

10 The presence of lactose induces expression of the genes required for lactose utilization Induction: : A process by which a signal induces expression of a gene or set of genes. Inducer: : The molecule responsible for the induction of gene expression. Fig

11 Advantages of E. coli and lactose utilization system Culture large numbers of bacteria allow isolation of rare mutants. Lactose genes are not essential for survival. Induction increases protein level 1000-fold making mutant identification easy. Color changes using -galactosidase substrates (e.g., ONPG, X-X Gal) make measurement of expression levels efficient. Liquid -galactosidase assay using ONPG substrate Plate -galactosidase assay using X-Gal substrate 17-11

12 The 1965 Nobel Prize for Physiology or Medicine François Jacob André Lwoff Jacques Monod Working in the Pasteur Institute in Paris Revealed coordinate repression and induction of three genes by studying lactose-utilization mutants. Proposed the Operon Theory of gene regulation

13 Identification of lactose-utilization genes Jacques Monod and his collaborators isolated many Lac mutants unable to grow on lactose. Three genes, lacz, lacy,, and laca, were identified in a tightly linked cluster. Fig

14 Experimental evidence for a repressor protein Mutants of laci gene: Synthesize -galactosidase and lac permease all the time, even in the absence of inducer. Constitutive mutants: : Mutants M that synthesize certain enzyme all the time, irrespective of environmental conditions. Suggest that laci encodes a repressor cells would need laci protein product to prevent expression of lacz and lacy in the absence of inducer

15 Start with a laci lacz mutant. Transfer laci + and lacz + genes into the cells on a piece of DNA. -galactosidase was detected immediately after DNA transfer, and the synthesis stopped in an hour. If add lactose, - galactosidase keeps accumulating. PaJaMo experiment Fig

16 Explanation of experimental results: Initial lack of repressor allowed synthesis of LacZ enzyme. As LacI is produced, LacZ synthesis was shut off, unless an inducer is present to prevent repressor from repressing. Conclusion: laci encodes the negative regulator of the lac genes

17 Mutational and structural analysis of the repressor Fig. 15.7,

18 Changes in the operator DNA to which the repressor binds can also affect repressor activity Fig

19 The operon hypothesis: A repressor stops transcription by binding to an hypothetical operator site near promoter of lactose-utilization genes. An inducer can bind to the repressor and prevents it from binding to the operator. The repressor (allosteric( protein) ) can change shape when exposed to inducer.. Repressor bound to inducer can t t bind to DNA

20 The operon theory of gene regulation The operon theory: : A single signal can simultaneously regulate the expression of several genes that are clustered together on a chromosome and are involved in the same biological process. Operon ( 操纵子 ): A unit of DNA composed of several clustered genes, plus a promoter and/or operator. These genes are regulated in the same way in response to environmental changes and are transcribed into one single mrna. The genes clustered together on a chromosome are transcribed together as single mrna (polycistronic( polycistronic)

21 The lac operon The players lacz, lacy, laca genes that split lactose into glucose and galactose Promoter ( 启动子 ) to which RNA polymerase binds cis-acting operator site trans-acting acting repressor that can bind to operator (encoded by laci gene) Inducer that prevents repressor from binding to operator Fig

22 Repression In absence of lactose, repressor binds to operator which prevents transcription. Negative regulatory element Fig

23 Induction Inducer binds to repressor. Repressor changes shape and can not bind to operator. RNA polymerase binds to promoter and initiates transcription of polycistronic mrna. Fig

24 Allolactose,, but not lactose, is the true inducer of the lac operon. lacz mutants express lacy in the presence of IPTG, but not in the presence of lactose. Fig

25 Proteins act in trans; ; but DNA sites act only in cis Cis-acting elements: : Short DNA sequences that constitute the control elements adjacent to genes. They can only influence the expression of adjacent genes on the same DNA molecule. Trans-acting acting elements: : Genes that code for DNA-binding proteins. Their protein products can diffuse through cytoplasm and act at target DNA sites on any DNA molecule in the cell

26 Three experiments elucidate cis- and trans-acting acting elements using F F plasmid laci + gene encodes a diffusible element that binds to any operator it encounters regardless of chromosomal location. LacI + protein acts in trans. Fig

27 LacI s superrepressor can not bind inducer. All operator sites (O + ) are eventually occupied by superrepressor laci s mutant encodes a diffusible element that binds to operator regardless of chromosomal location (trans-acting acting element). LacI s protein acts in trans Fig

28 Presence of laco + plasmid does not compensate for laco c mutation on bacterial chromosome. Operator is cis-acting element laco c acts in cis Fig

29 17-29

30 Biochemical experiments support the operon hypothesis The lac repressor binds to operator DNA. Radioactive tag attached to lac repressor. Repressor from laci + cells purified and mixed with operator DNA; cosediment. Repressor from laci + mixed with mutant operator DNA; no cosediment. Fig

31 DNase I footprinting identifies DNA sequence to which a protein binds Mix radioactivelabeled target DNA with or without repressor. Partial digestion with DNase I. DNase I can t digest DNA regions covered with proteins. Separation of DNA fragments and autoradiography. Fig

32 Pink: lac repressor tetramer Fig

33 Summary of the repressor Repressor not bound to inducer can bind to the operator. Repressor bound to inducer can t bind to DNA. The binding of repressor to operator keeps RNA polymerase from recognizing the promoter. The repressor has separate domains corresponding to two functions. Defects in either domain as well as the presence or absence of the inducer affect the function of the repressor

34 Bacteria grown with two different sugars often displayed two phases of growth. Monod's "bi-phasic growth curve 17-34

35 The lac operon is also regulated by positive control Positive regulation of the lac operon by CRP-cAMP and catabolite repression by glucose. camp binds to CRP (camp receptor protein) when glucose is low. CRP-cAMP binds to regulatory region. Enhances activity of RNA polymerase at lac promoter. Fig

36 Maximum induction of lac operon occurs in media containing lactose but lacking glucose Regulation of the lac operon depends on at least two proteins: the repressor (a negative regulator) and CRP-cAMP (a positive regulator). Under these conditions, the repressor binds inducer and become unable to bind to the operator, while CRP-cAMP binds to a site near the promoter to assist RNA polymerase in the initiation of transcription. When glucose is present in the medium, there is little camp available to bind to CRP and therefore little induction of the lac operon,, even if lactose is present

37 Most regulatory proteins in transcription are oligomeric Contain more than one domain. Bind to each other to form oligomers. CRP-cAMP complex: dimer Fig

38 lac repressor: tetramer Blue: CRP-cAMP Pink: lac repressor tetramer Fig ,

39 How regulatory proteins interact with RNA polymerase? Repressor-binding site and RNA polymerase-binding site overlaps. Negative regulators, e.g. lac repressor, physically block RNA polymerase-binding site. Fig

40 Positive regulators establish physical contact with RNA polymerase enhancing enzyme s s ability to initiate transcription. Fig

41 Lactase persistence and milk drinking in humans 65% of the human population today has lactose intolerance: drinking milk makes them ill, with symptoms including cramps ( 腹痛 ) and bloating ( 腹胀 ). Lactase ( 乳糖酶 ) is required for breaking down lactose in milk

42 Humans express lactase only in infants but not in adults. Lactase persistence allows adults to drink milk. 35% of the humans carry a mutation that caused constitutive expression of lactase (lactase( persistence). Lactase Lactase Lactase Lactase persistence 17-42

43 Lactase persistence allows adults to drink milk. The mutation occurred between 7,000-9,000 years ago among several dairying communities in places like northern Europe and eastern Africa. Fig

44 15.3 RNA-mediated mechanisms of gene regulation 1. Diverse RNA leader devices act in cis to regulate gene expression. Attenuation Riboswitches 2. Regulatory small RNAs act in trans to regulate the translation of mrnas. 3. Genes can also be regulated by antisense RNAs

45 1. Diverse RNA leader devices act in cis to regulate gene expression (1) Attenuation in the trp operon of E. coli: trpr gene encodes repressor. Tryptophan functions as a corepressor tryptophan binds to trp repressor allowing it to bind to operator DNA and inhibit transcription. Fig

46 Fig

47 Repressor-independent regulation of the trp operon When tryptophan is present, trpr mutants are not completely de-repressed in the expression of trp genes. The presence of tryptophan can still inhibit the expression of trp genes in trpr mutants

48 Two alternative transcripts lead to different transcriptional outcomes. RNA leader sequence (~ 140 bases) can fold in two different stable conformations

49 Fig

50 Attenuation ( 弱化作用 ): Control of gene expression by premature termination of transcription

51 (2) Riboswitches: Allosteric RNA leaders that bind small molecule effectors to control gene expression. 17 different riboswitch aptamers have been identified in the E. coli genome. Aptamer: The region of a riboswitch that can bind an effector and thereby alter its stem-loop structures as well as those of the expression platform connected to it. Expression platform: The region of a riboswitch that controls gene expression by altering its stem-loop structures in response to the aptamer configuration

52 Fig

53 Temperature-sensing mrnas CspA: E. coli cold shock protein, essential for growth in cold. Only at low temperatures, cspa mrna is stable and is preferentially translated. 5 UTR: Forms stem-loop at high temperature. mrna half-life: 37 :20 s 10 :30 min

54 RNA thermometers: RNA leaders in some bacterial mrnas that regulate translation in response to temperature through stemloop structure whose stability is temperature-dependent. At low temperatures, stem-loop structures that occlude the ribosome binding site form. At high temperatures, the stem-loop unzips. The most well-studied RNA thermometer is found in the rpoh gene in E. coli. This thermosensor upregulates heat shock proteins under high temperatures through 32, a specialized heat-shock sigma factor (Storz G. Genes Dev. 1999, 13: ) Fig

55 2. Regulatory small RNAs act in trans to regulate the translation of mrnas Bacterial genomes encode many small RNA molecules (srnas, nt long) that regulate translation in trans by base pairing with mrnas. Most srnas inhibit translation of target mrnas by base pairing with the ribosome binding site (RBS). Some srnas activate translation of target mrnas by disrupting the formation of a stem-loop structure in the leader mrna that would otherwise block the ribosome binding site. Some srnas influence the expression of target mrnas by base pairing with mrna and promoting the degradation of the mrna by ribonucleases

56 Fig

57 3. Genes can also be regulated by antisense RNAs Antisense RNAs: Regulatory RNAs that are complementary in sequence to the mrnas they regulate because they are transcribed using the opposite strand of DNA as a template. They range in size from nt. Some antisense RNAs can block transcription or translation of their target mrnas. Some could inhibit translation of target mrnas by base pairing with the sense mrna and blocking the ribosome binding site. The double-stranded RNA formed by base pairing between antisense RNA and the target mrna can be degraded by ribonucleases. The transcription of antisense RNA can interfere with initiation of transcription of the sense gene in a yet unknown mechanism. Fig

58 15.4 Discovering and manipulating bacterial gene regulatory mechanisms Use of lacz gene as reporter of gene expression. Reporter gene: : A protein-coding gene whose expression in the cell is quantifiable by techniques of protein detection. Fig

59 Fusion of reporter gene to cis-acting regulatory regions allows assessment of gene activity by monitoring amount of reporter gene product. X-Gal staining of 13.5 days mouse embryo carrying Cecr2-lacZ fusion

60 Creating a collection of lacz insertions in the chromosome Fig

61 lac operon regulatory sequences help produce protein drugs in bacteria Fig

62 RNA-Seq is a general tool for characterizing transcriptomes and their regulation Transcriptome: : All the mrnas expressed in a single cell or organism. RNA-Seq: Method for analysis of the transcriptome of an organism in which millions of cdnas are sequenced. Each read is about 150 nt long