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Transcription often is controlled at the stage of initiation. Transcription is not usually controlled at elongation, but may be controlled at termination.

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Presentation on theme: "Transcription often is controlled at the stage of initiation. Transcription is not usually controlled at elongation, but may be controlled at termination."— Presentation transcript:

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2 Transcription often is controlled at the stage of initiation. Transcription is not usually controlled at elongation, but may be controlled at termination to prevent transcription from proceeding past a terminator to the gene(s) beyond. This is the primary control strategy for bacterial gene expression. Molecular Biology Course Gene expression controls

3 In eukaryotic cells, processing of the RNA product can also be regulated at the stages of modification, splicing, transport, or stability. In bacteria, an mRNA is in principle available for translation as soon as it is synthesized, and these stages of control are not available. Molecular Biology Course

4 Translation may be regulated, usually at the stages of initiation and termination (like transcription). Regulation of initiation is formally analogous to the regulation of transcription: the circuitry can be drawn in similar terms for regulating initiation of transcription on DNA or initiation of translation on RNA. This regulation will not be detailed in this course. Molecular Biology Course

5 Regulation of Transcription in Prokaryotes Molecular Biology Course

6 Operon directed regulation LAC operon TRP operon  Factor directed regulation Regulation of Transcription in Prokaryotes

7 Operon Regulation of Transcription in Prokaryotes

8 In 1961, Jacob and Monod distinguished between two types of sequences in DNA: sequences that code for trans-acting products; and cis-acting sequences that function exclusively within the DNA. Gene activity is regulated by the specific interactions of the trans-acting products (usually proteins) with the cis-acting sequences (usually sites in DNA). Regulation of Transcription in Prokaryotes

9 A gene is a sequence of DNA that codes for a diffusible product. This product may be protein (as in the case of the majority of genes) or may be RNA (as in the case of bgenes that code for tRNA and rRNA etc.). The crucial feature is that the product diffuses away from its site of synthesis to act elsewhere. Any gene product that is free to diffuse to find its target is described as trans-acting.

10 The cis-acting sequence applies to any sequence of DNA that is not converted into any other form, but that functions exclusively as a DNA sequence in situ, affecting only the DNA to which it is physically linked.

11 Operon: Operon: a unit of prokarytoic gene expression which typically includes: 1. Structural genes for enzymes in a specific biosynthetic pathway whose expression is co-ordinately controlled 2. Control elements, such as operator sequence 3. Regulator gene(s) whose products recognize the control elements. Can be encoded by a gene in another operon Regulation of Transcription in Prokaryotes

12 Control element Structural genes

13 L1L1 The Lac Operon L1L1 The Lac Operon L2L2 The Trp Operon L2L2 The Trp Operon L3L3 Transcriptional regulation by alternative σ Factors L3L3 Transcriptional regulation by alternative σ Factors The operon, the lactose operon, the lac repressor, induction, cAMP receptor protein The trp operon, the trp repressor, the attenuator, leader RNA structure, the leader peptide, attenuation & its importance Sigma factor, promoter recognition, heat shock, sporulation in B. subtilis, bacteriophage factors

14 L1 The Lac Operon L1 The Lac Operon 1. The operon (done) 2. The lactose operon (乳糖操纵子) 3. The lac repressor (乳糖抑制蛋白) 4. Induction (诱导) 5. cAMP receptor protein ( CRP ) Regulation of Transcription in Prokaryotes

15 The lac operon Lac repressor Transcription blocked Inducer Activate the P lac transcription CRPCRP + cAMP (glucose repressed) High level of transcription (Lactose) Overview

16 L1-2 The Lactose Operon L1-2 The Lactose Operon E. coli can use lactose as a source of carbon. However, the enzymes required for the use of lactose as a carbon source are only synthesized when lactose is available as the sole carbon source. L1: The LAC operon

17 Lactose operon: a regulatory gene and 3 stuctural genes, and 2 control elements lacI Regulatory gene lacZ lacY lacA DNA m-RNA β -Galactosidase Permease Transacetylase Protein Structural Genes Cis-acting elements P lacI P lac O lac

18 lacY encodes a galactoside permease ( 半乳糖苷渗透酶 )to transport Lactose across the cell wall lacZ codes for β -galactosidase ( 半乳 糖苷酶 ) for lactose hydrolysis lacA encodes a thiogalactoside transacetylase ( 硫代半乳糖苷转 乙酰酶 ) for lactose metabolism L1: The LAC operon

19 The lacZ, lacY, lacA genes are transcribed from a single (lacZYA) transcription unit under the control of a signal promoter P lac. LacZYA transcription unit contains an operator site O lac position between bases -5 and +21 at the 3’-end of P lac Binds with the lac repressor L1: The LAC operon

20 L1-3 The Lac repressor L1-3 The Lac repressor Regulation of Transcription in Prokaryotes The repressor is encoded by LacI and active as a tetramer consisting of 4 identical subunits (has a symmetrical structure). It binds to occupies the operator-binding site O lac (28bp, palindromic) and blacks almost all transcription of lacZYA when lack of inducer (such as lactose).

21 P lac

22 The repressor and RNA polymerase can bind simultaneously to the lac promoter and operator sites. The lac repressor actually increases the binding of the polymerase to the lac promoter by two orders of magnitude. Thus, RNA polymerase binds very tightly to P lac but no transcription occur because of the bound repressor L1: The LAC operon

23 L1-4 Induction L1-4 Induction When lac repressor binds to the inducer (whose presence is dependent on lactose), it changes conformation and cannot bind to O lac site any more. This allows rapid induction of lacZYA transcription. L1: The LAC operon

24 i p o z y a Very low level of lac mRNA Absence of lactose Active i p o z y a  -Galactosidase Permease Transacetylase Presence of lactose Inactiv e Lack of inducer: the lac repressor block all but a very low level of trans- cription of lacZYA. Lactose is present, the low basal level of permease allows its uptake, andβ- galactosidase catalyzes the conversion of some lactose to allolactose. Allolactose acts as an inducer, binding to the lac repressor and inactivate it.

25 Allolactose causes a change in the conformation of the repressor tetramer, reducing its affinity for the lac operator. The lac operator is removed from the Olac and allows the polymerase to rapidly begin transcription of the lacZYA. L1: The LAC operon

26 Lactose (allolactose) is a native inducer to release RNA transcription elongation from P lac. IPTG, a synthetic inducer, can rapidly simulate transcription of the lac operon structural genes. IPTG is used to induce the expression of the cloned gene from LacZ promoter in many vectors, such as pUC19.

27 Amp r ori pUC18 (3 kb) MCS (Multiple cloning sites, 多科隆位点) Lac promoter lacZ’ Gene X No IPTG, little expression of X gene With IPTG, efficient expression of X gene. L1: The LAC operon

28 L1-5 cAMP receptor protein (CRP) L1-5 cAMP receptor protein (CRP) L1: The LAC operon CRP is a transcriptional activator which is activated by binding to cAMP. However, it is only active when cAMP bound, and cAMP is controlled by glucose. CRP activator mediates the global regulation of gene expression from catabolic operons in response to glucose levels.

29 L1: The LAC operon The P lac is a weak promoter, lacking a strong – 35 and –10 consensus sequences. High level expression from this promoter requires the activity of the specific activator, CRP.

30  When glucose is present The level of cAMP is low in cell, and CRP exists as a dimer which can’t bind to DNA to regulate transcription.  When glucose is absent The level of cAMP increase and CRP bind to cAMP. The CRP-cAMP complex binds to P lac just upstream from the site for RNA polymerase. Induces a 90°bend in DNA which enhances RNA polymerase binding to the promoter and thus the transcription by 50-fold.

31 CRP-binding site is an inverted repeat.

32 C A B Summary A: RNA polymerase B: lac repressor C: CRP-cAMP

33 The CRP (also called CAP) protein can bind at different sites relative to RNA polymerase. Supp.

34 L2L2 The Trp Operon L2L2 The Trp Operon 1.The trp operon 2.the trp repressor 3.the attenuator 4.Leader RNA structure 5.The leader peptide 6.Attenuation 7.Importance of attenuation Regulation of Transcription in Prokaryotes

35 L2-1 The Trp Operon Regulation of Transcription in Prokaryotes (色氨酸操纵子 ) Bacillus subtilis uses a different regulation mechanism from what is described here (see the reference of this class).

36 1. The trp operon encodes five structural genes required for tryptophan synthesis. 2. It encodes a signal transcription ( 7kb, polycistron ) downstream of O trp. 3. These genes are co-ordinately expressed when tryptophan is in short supply in the cell. A B C L2: The trp operon

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38 L2-2 The Trp repressor Regulation of Transcription in Prokaryotes (色氨酸阻遏物 )

39 1. Trp repressor is encoded by a separate operon trpR, and specifically interacts with O trp, a palindrome of 18 bp, and overlaps with the P trp sequence between base –21 and +3) L2: The trp operon 2. The repressor can only bind to the operator O trp when it is complexed with tryptophan. Therefore, try is a co-repressor and inhibits its own synthesis through end-product inhibition (negative feed-back regulation).

40 L2: The trp operon 3. The repressor reduces transcription initiation by around 70-fold, which is much smaller than the binding of lac repressor. 4. The repressor is a dimer of two subunits which has a structure with a central core and two flexible DNA- reading heads (carboxyl-terminal of each subunit )

41 L2: The trp operon trpR operon trp operon

42 L2-3 The attenuator Regulation of Transcription in Prokaryotes (衰减子 ) Repressor does not account for all the regulation: Deletion of a sequence between the operator and trpE gene coding region (attenuator) increase both the basal and the activated (derepressed) levels of transcription.

43 1. Lies at the end of the transcribed leader sequence that precedes the trpE initiator codon. 2. Is a ρ-independent terminator site (GC-rich palindrome) f0llowed by eight successive U residues. L2: The trp operon 3. Acts as a highly efficient transcription terminator if the hairpin structure is formed, and only a very short transcipt is synthesized.

44 L2-4 Leader RNA structure Regulation of Transcription in Prokaryotes (先导 RNA 的结构 )

45 Complementary 3:4 termination of transcription Complementary 2:3 Elongation of transcription The leader sequence contains four regions (sequence 1,2,3,4) of complementary sequence that can form different structures free leader RNA L2: The trp operon

46 L2-5 The leader peptide Regulation of Transcription in Prokaryotes (先导肽 ) The leader RNA contains an efficient ribosome binding site (RBS) and encodes a 14-amino-acid leader peptide (bases 27-68), Codons 10 and 11 of this peptide encode trp. Thus the availability of trp will affect the translation/ ribosome position, which in turn to regulate transcription termination.

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48 L2-6 Attenuation Regulation of Transcription in Prokaryotes (衰减作用 ) Transcription and translation in bacteria are coupled. Therefore, synthesis of the leader peptide immediately follows the transcription of leader RNA, and the attenuation is possible

49 High trp High trp (attunation) Lack of trp Lack of trp (proceeding through the whole operon ) Transcription of the trp operon During transcription of the RNA from trp operon, the RNA Polymerease pauses at the end of sequence 2 (sequences 1 and 2 form a hairpin) until a ribosome began to translate the leader peptide. L2: The trp operon

50 High trp Trp is inserted at the trp codons Translate to the end of leader message Ribosome occlude sequence 2 Terminate transcription because 3:4 hairpin formed L2: The trp operon

51 Lack of trp Lack of aminoacyl tRNA phe Ribosome pause at trp codons, occluding sequence 1 2:3 hairpin (anti-terminator ) forms Transcription into trpE and beyond L2: The trp operon

52 Low Trp High Trp

53 L2-7 Importance of attenuation Regulation of Transcription in Prokaryotes (衰减作用的重要性 )

54 A typical negative feed-back regulation Give rise to a 10-fold repression of the trp operon transcription ( 细 调), increasing the regulatory effect up to 700-fold combining the 70-fold repressor effect ( 粗调). Faster and more subtle regulation of trp metabolism in bacteria.

55 Additional: Distinguishing positive and negative control Regulation of Transcription in Prokaryotes

56 1.Positive and negative control systems are defined by the response of the operon when no regulator protein is present. 2.The characteristics of the two types of control system are mirror images: Genes under negative control are expressed unless they are switched off by a repressor protein Genes under positive control, expression is possible only when an active regulator protein is present.

57 repressor To exert a negative control, a trans- acting repressor either binds to DNA to to prevent RNA polymerase from initiating transcription (inhibits transcription), or binds to mRNA to prevent a ribosome from initiating translation. In prokaryotes, multiple genes can be controlled coordinately on the transcription level through interaction of repressor with the operator sites. (Lac and trp repressors)

58 The cis-acting operator/promoter sites are adjacent to the structural genes Genes are on because RNA polymerase initiates transcription at promoter Genes are turned off when repressor binds to operator

59 activators In positive control, trans-acting activators must bind to cis-acting sites in order for RNA polymerase to initiate transcription at the promoter (help transcription), which is opposite to negative control. (CRP activator) In prokaryotes, multiple genes can be controlled coordinately on the transcription level through interaction of activator with the DNA sites near promoter. (CRP activator)

60 Gene off by default Gene turned on by activator

61 Either positive or negative control could be used to achieve either induction (诱导) or repression (阻 遏) by utilizing appropriate interactions between the regulator protein and the small-molecule inducer or corepressor.

62 Induction Inducer Positive control Negative control 负控诱导系统 正控诱导系统

63 Negative control Positive control Repression Corepressor 负控阻遏系统正控阻遏系统

64 Regulation of Transcription in Prokaryotes L3L3 Transcriptional regulation by alternative σ Factors L3L3 Transcriptional regulation by alternative σ Factors 1.Sigma factor 2.Promoter recognition 3.Heat shock 4.Sporulation in B. subtilis 5.Bacteriophage factors

65 L3-1&2: Sigma factor and promoter recognition Transcriptional regulation by alternative σ Factors

66 σ factor subunit bound to RNA pol for transcription initiation Released core enzyme αββ’ω RNA elongation σ factors is bifunctional protein Bind to core RNA Pol Recognize specific promoter sequence (-35 and –10) in DNA Transcriptional regulation by alternative σ Factors

67   factor: Transcriptional regulation by alternative σ Factors

68 Many bacteria produce alternative sets of σfactors to meet the regulation requirements of transcription under normal and extreme growth condition E. coli: Heat shock Sporulation in bacillus subtilis bacteriophage σfactors Transcriptional regulation by alternative σ Factors

69 Different σfactors binding to the same RNA Pol Confer each of them a new promoter specificity, and allows the diversion of the cell’s basic transcription machinery to the specific transcription of different classes of genes σ 70 factors is the most common σfactor in E. coli under the normal growth condition Transcriptional regulation by alternative σ Factors

70 L3-3: Heat shock Transcriptional regulation by alternative σ Factors The response to heat shock is one example in E. coli where gene expression is altered significantly by the use of different  factors.

71 Around 17 proteins are specifically expressed in E.coli when the temperature is increased above 37ºC. These proteins are expressed through transcription by RNA polymerase using an alternative  factor  32 coded by rhoH gene.  32 has its own specific promoter consensus sequences. Transcriptional regulation by alternative σ Factors

72 Comparison of the heat-shock  32 and general  70 responsive promoter Consensus promoter –35 sequence –10 sequence Standard  70 Heat shock  TTGACA----16~18bp---TATAAT T-C-C--- CTTGAA--13~15bp--CCCCAT--T Transcriptional regulation by alternative σ Factors

73 Heat shock Transiently expression of the 17 heat shock proteins Increase in temperature is more extremely (50ºC) Heat shock proteins are the only proteins made in E. coli to maintain its viability From 37ºC to 42ºC

74 L3-4: Sporulation in B. subtilis Transcriptional regulation by alternative σ Factors Under non-optimal environmental conditions Bacillus subtilis cells from spores through a basic cell differentiation process involving cell partitioning into mother cell and forespore.

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76 The process of spore formation involves the asymmetrical division of the bacterial cell into two compartments, the forespore, which forms the spore, and the mother cell, which is eventually discarded.

77 1.Vegetative B. subtilis cell contains a diverse set of  factors 2. Sporulation is regulated by a further set of  factors 3. Different  factors are specifically active before cell partition occurs in the forespore and in the mother cell to cross regulate the transcription. 4. Cross-regulation of this compartmentalization permits the forespore and mother cell to tightly co-ordinate the differentiation process.

78 L3-5: Bacteriophage  factors Transcriptional regulation by alternative σ Factors Many bacteriophages synthesize their own  factors in order to ‘take over’ the host cell’s transcription machinery by substituting the normal cellular  factor and altering the promoter specificity of the RNA polymerase.

79 1. Many bacteriophages synthesize their own σfactors to endow the host RNA Pol with a different promoter specificity and hence to selectively express their own phage genes. 2. B. subtilis SPO1 phage expresses a cascade of σfactors which allow a defined sequence of expression of different phage genes. different phage genes

80 Normal bacterial holonzyme Express early genes Encode σfactor for transcription of late genes Encode σ 28 Express middle genes (gene 34 and 33)

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