welcome each of you to my molecular biology class

Welcome Each of You to My Molecular

Biology Class

Welcome Each of You to My Molecular

Biology Class

2

Molecular Biology of the Gene, 5/E --- Watson et al. (2004)

Part I: Chemistry and Genetics

Part II: Maintenance of the Genome

Part III: Expression of the Genome

Part IV: Regulation

Part V: Methods

2005-5-10

3

Part IV Regulation

Ch 16: Regulation in prokaryotes

Ch 17: Regulation in eukaryotes

Ch 18: Regulation during development

Ch 19: Comparative genomics and evolution of animal diversity

4

Housekeeping genes : expressed constitutively, essential for basic processes involving in cell replication and growth.

Inducible genes : expressed only when they are activated by inducers or cellular factors.

Expression of many genes in cells are regulated

5

Chapter 16 Regulation principles and How genes are regulated in bacteria

Chapter 17 Basic mechanism of gene expression in eukaryotes

Chapter 18 How genes are regulated to bestow cell type specificity in a group of genetically identical cells

Chapter 19 How different animals diverse in genomes, why human is so special?

6

Surfing the contents of Part IV

7

Some of the peoples who significantly contribute to

the knowledge of gene regulation

10

Chapter 16Gene Regulation

in Prokaryotes

Chapter 16Gene Regulation

in Prokaryotes

•Molecular Biology Course

11

TOPIC 1 Principles of Transcriptional Regulation [watch the animation]

TOPIC 2 Regulation of Transcription Initiation: Examples from Bacteria (Lac operon, alternative factors, NtrC,MerR, Gal rep, araBAD operon)

TOPIC 3 Examples of Gene Regulation after Transcription Initiation (Trp operon)

TOPIC 4 The Case of Phage λ: Layers of Regulation

12

Topic 1: Principles Topic 1: Principles of Transcription of Transcription

Regulation Regulation

Topic 1: Principles Topic 1: Principles of Transcription of Transcription

Regulation Regulation

CHAPTER 16 Gene Regulation in Prokaryotes

5/10/2005

13

1-1 Gene Expression is Controlled by Regulatory Proteins

Gene expression is very often controlled by Extracellular Signals, which are communicated to genes by regulatory proteins :

Positive regulators or activators INCREASE the transcription

Negative regulators or repressors

DECREASE or ELIMINATE the transcription

Prin

cip

les o

f P

rincip

les o

f Tra

nscrip

tion

Reg

ula

tion

Tra

nscrip

tion

Reg

ula

tion

14

What are the transcription steps

targeted by the regulators ?

15

Fig 12-3-initiation

Promoter Binding (closed complex)Promoter Binding (closed complex)

Promoter “melting” (open complex)

Initial transcription

16

Fig 12-3-Elongation and termination

Termination

Elongation

17

1-2 Targeting promoter binding: Many promoters are regulated by activators that help RNAP bind DNA and by repressors that block the binding

At many promoters, RNAP binds weakly Lac operon is a good example

Prin

cip

les o

f P

rincip

les o

f Tra

nscrip

tion

Reg

ula

tion

Tra

nscrip

tion

Reg

ula

tion

18

a. Absence of Regulatory

Proteins(operator)

b. To Control Expression

c. To Activate

Expression

Fig 16-1

19

1-3 Targeting transition to the open complex: Some Activators Work by Allostery and Regulate Steps after RNA Polymerase Binding

Fig 16-2

Examples: Activator promoter

NtrC glnAMerR merT

20

Some promoters are inefficient at more than one step and can be activated by more than one mechanism

Repressors can work in ways other than just blocking the promoter binding. For example, inhibition of the transition to the open complex.

21

1-6 Targeting termination and beyond: Antitermination and Beyond

The bulk of gene regulation takes place at the initiation of transcription.Some involve transcriptional elongation/termination, RNA processing, and translation of the mRNA into protein.

Prin

cip

les o

f P

rincip

les o

f Tra

nscrip

tion

Reg

ula

tion

Tra

nscrip

tion

Reg

ula

tion

22

1-4 Action at a Distance and DNA Looping. Some proteins interact with each other even when bound to sites well separated on the DNA

Fig 16-3

23

Fig 16-4 DNA-binding protein can facilitate interaction between DNA-binding proteins at a distance

Fig 16-4

24

1-5 Cooperative Binding and Allostery have Many Roles in Gene Regulation

Cooperative binding: the activator interacts simultaneously with DNA and polymerase and so recruits the enzyme to the promoter Group of regulators often bind DNA cooperatively: (1) produce sensitive switches to rapidly turn on a gene expression, (2) integrate signals (some genes are activated when multiple signals are present)

25

Allostery is not only a mechanism of gene activation , it is also often the way that regulators are controlled by their specific signals.

26

Topic 2: Regulation Topic 2: Regulation of Transcription of Transcription

Initiation : Initiation : Examples Examples

from Bacteriafrom Bacteria

Topic 2: Regulation Topic 2: Regulation of Transcription of Transcription

Initiation : Initiation : Examples Examples

from Bacteriafrom Bacteria


5/10/2005

27

OperonOperon:: a unit of prokarytoic gene expression and regulation which typically includes: 1. Structural genes for enzymes in a specific biosynthetic pathway whose expression is coordinately controlled. 2. Control elements, such as operator sequence. 3. Regulator gene(s) whose products recognize the control elements.Sometimes are encoded by the gene under the control of a different promoter

28

Control element

Structural genes

29

First example: Lac First example: Lac operonoperon

First example: Lac First example: Lac operonoperon

5/10/2005

Regulation of Transcription Regulation of Transcription Initiation in Bacteria Initiation in Bacteria

30

The lactose (The lactose (LacLac) Operon ) Operon (( 乳糖操纵子乳糖操纵子 ))

The enzymes required for the use of lactose as a carbon source are only synthesized when lactose is available as the sole carbon source.

Fig 16-5

The LAC operon

31

Lactose operon: a regulatory gene and 3 stuctural genes, and 2 control elements

lacI

Regulatory gene

lacZ lacY lacA DNA

m-RNA

β -GalactosidasePermease

Transacetylase

Protein

Structural GenesCis-acting elements

PlacI Plac Olac

The LAC operon

32

lacY encodes a cell membrane protein called lactose 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 )to get rid of the toxic thiogalacosides

The LAC operon

33

1. The lacZ, lacY, lacA genes are transcribed into a single lacZYA mRNA (called polycistronic message) under the control of a signal promoter Plac .

2. LacZYA transcription unit contains an operator site Olac

position between bases -5 and +21 at the 3’-end of Plac

Binds with the lac repressor

The LAC operon

34

2-1: An activator and a repressor together control the lac genes

The activator: CAP (Catabolite Activator Protein) or CRP (cAMP Receptor Protein); responses to the glucose level.The repressor: lac repressor that is encoded by LacI gene; responses to the lactose.

The LAC operon

35

Fig 16-6

The LAC operon

36

2-2: CAP and lac repressor have opposing effects on RNA polymerase binding to the lac promoter

The LAC operon

37

The site bound by lac repressor is called the lac operator.

The LAC operon

38

The lac operator overlaps promoter, and so repressor bound to the operator physically prevents RNA polymerase from binding to the promoter.

Fig 16-8

The LAC operon

39

CAP binds to a site with the similar structure as the operator, which is 60 bp upstream of the start site of transcription.

CAP also interacts with the enzyme and recruit it to the promoter.

Fig 16-9

CTD: C-terminal domain of the subunit of RNAP

40

2-3: CAP has separate activating and DNA- binding surface

CAP binds as a dimer

CTD

Fig 16-10

The LAC operon

41

2-4: CAP and lac repressor bind DNA using a common structural motif

The LAC operon

42

Both CAP and lac repressor bind DNA using a helix-turn-helix motif.

One is the recognition helix that can fits into the major groove of the DNA.

Fig 16-11

The LAC operon

43

DNA binding by a helix-turn-helix motif

Fig 16-12 Hydrogen Bonds between repressor and the major groove of the operator

The LAC operon

44

Lac repressor binds as a tetramer, with each operator is contacted by a repressor dimer. In addition to the primary operator, there are two other lac operators located 400 bp downstream and 90 bp upstream, respectively.

Not all the binding use a helix-turn-helix motif

Fig 16-13

45

2-5: The activity of Lac repressor and CAP are controlled allosterically by their signals

Binding of the corresponding signals alter the structure of these two regulatory proteins

The LAC operon

46

i p o z y a

Very low level of lac mRNA

Absence of lactose

Active

i p o z y a

-Galactosidase

PermeaseTransacetylase

Presence of lactose

Inactive

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.

Response to lactose

47

Response to glucose

The LAC operon

48

A regulator (CAP) works together with different repressor at different genes, this is an example of Combinatorial Control.

In fact, CAP acts at more than 100 genes in E.coli, working with an array of partners.

2-6: Combinatorial Control ( 组合调控 ): CAP controls other genes as well


49

Second example: Second example: Alternative Alternative factor factor

Second example: Second example: Alternative Alternative factor factor

5/10/2005


50

2-7: Alternative factor direct RNA polymerase to alternative site of promoters

Alternative Alternative factors factors

51

factor:

factor subunit bound to RNA polymerase for transcription

initiation

52

Promoter recognition

Different σfactors binding to the same RNA Pol

Confer each of them a new promoter specificity

70 factors is most common one in E. coli under the normal growth condition


53

Many bacteria produce alternative sets of σfactors to meet the regulation requirements of transcription under normal and extreme growth condition

E. coli : Heat shock 32

Sporulation in Bacillus subtilis

Bacteriophage σfactors


54

Heat shock 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.


55

Many bacteriophages synthesize

their own σfactors to endow the

host RNA polymerase with a

different promoter specificity and

hence to selectively express their

own phage genes .

Bacteriophages


56

B. subtilis SPO1 phage expresses a cascade of σfactors which allow a defined sequence of expression of different phage genes .

Fig 16-14Alternative Alternative factors factors

57

Normal bacterial holoenzyme

Express early genes

Encodeσfactor for transcription of late genes

Encode σ28

Express middle genes (gene 34 and 33 )


58

Third example: NtrC Third example: NtrC and MerR and and MerR and

allosteric activationallosteric activation

Third example: NtrC Third example: NtrC and MerR and and MerR and

allosteric activationallosteric activation

5/10/2005


59

2-7: NtrC and MerR:Transcriptional activators that work by allostery rather than by recruitment

NtrC and MerR and allosteric NtrC and MerR and allosteric activationactivation

60

The majority of activators work by recruitment, such as CAP. These activators simply bring an active form of RNA polymerase to the promoter

In this case of allosteric activation, RNAP initially binds the promoter in an inactive complex, and the activator triggers an allosteric change in that complex to activate transcription.

61

In the absence of these activators, the RNAP binds to the corresponding promoter to form a closed stable complex.

NtrC activator induces a conformational change in the enzyme, triggering transition to the open complex

MerR activator causes the allosteric effect on the DNA and triggers the transition to the open complex

62

2-8: NtrC has ATPase activity and works from DNA sites far from the gene


NtrC controls expression of genes involved in nitrogen metabolism, such as the glnA gene

NtrC has separate activating and DNA-binding domains, and binds DNA only when the nitrogen levels are low.

63

Low nitrogen levelsNtrC phosphorylation and conformational changeThe DNA binding domain binds DNA sites at ~ -150 positionNtrC interacts with 54

(glnA promoter recognition)ATP hydrolysis and conformation change in polymerase transcription STARTs

Fig 16-15 activation by NtrC

64

2-8: MerR activates transcription by twisting promoter DNA


MerR controls a gene called merT, which encodes an enzyme that makes cells resistant to the toxic effects of mercury ( 抗汞酶 )

In the presence of mercury, MerR binds to a sequence between –10 and –35 regions of the merT promoter and activates merT expression.

65

As a 70 promoter, merT contains 19 bp between –10 and –35 elements (the typical length is 15-17 bp), leaving these two elements neither optimally separated nor aligned.

66Fig 16-15 Structure of a merT-like promoter

67

Fig 16-15

When Hg2+ is absent, MerR binds to the promoter and locks it in the unfavorable conformationWhen Hg2+ is present, MerR binds Hg2+ and undergo conformational change, which twists the promoter to restore it to the structure close to a strong 70

promoter

68

2-9: Some repressors hold RNA polymerase at the promoter rather than excluding it


69

Repressors work in many ways :Blocking RNA polymerase binding through binding to a site overlapping the promoter. Lac repressorBlocking the transition from the closed to open complex. Repressors bind to sites beside a promoter, interact with polymerase bound at that promoter and inhibit initiation. E.coli Gal repressorLocking the promoter in a conformation incompatible with transcription initiation

70

Fourth example: Fourth example: araaraBADBAD operon operon

Fourth example: Fourth example: araaraBADBAD operon operon

5/10/2005


71

2-10: AraC and control of the araBAD operon by antiactivation

The araBAD The araBAD operonoperon

The promoter of the araBAD operon from E. coli is activated in the presence of arabinose ( 阿拉伯糖 ) and the absence of glucose and directs expression of genes encoding enzymes required for arabinose metabolism. This is very similar to the Lac operon.

72

Different from the Lac operon, two activators AraC and CAP work together to activate the araBAD operon expression

Fig 16-18

CAP site

194 bp

73

Because the magnitude of induction of the araBAD promoter by arabinose is very large, the promoter is often used in expression vector.

If fusing a gene to the araBAD promoter, the expression of the gene can be easily controlled by addition of arabinose （阿拉伯糖） .

What is an expression vector ?

74

Topic 3: Examples of Topic 3: Examples of Gene Regulation Gene Regulation

at Steps After at Steps After Transcription Transcription

InitiationInitiation

Topic 3: Examples of Topic 3: Examples of Gene Regulation Gene Regulation

at Steps After at Steps After Transcription Transcription

InitiationInitiation


5/10/2005

75

3-1: Amino acid biosynthetic operons are controlled by premature transcription termination: the tryptophan operon

Examples of Gene Regulation at Steps After Examples of Gene Regulation at Steps After Transcription InitiationTranscription Initiation

76

The trp operon encodes five structural genes required for tryptophan synthesis.

These genes are regulated to efficiently express only when tryptophan is limiting.

Two layers of regulation are involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuation

The TRP operon

77

The Trp repressorThe Trp repressor （色氨酸阻遏物）（色氨酸阻遏物）

The TRP operon

78

1. Trp repressor is encoded by a separate operon trpR, and specifically interacts with the operator that overlaps with the promoter sequence

2. The repressor can only bind to the operator 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).

The TRP operon

Remember the lac repressor acts as an inducer

79

The TRP operon3. 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 )

80

trpR operon

trp operon

The TRP operon

81

Attenuation ( 衰减子 ) : a regulation at the transcription termination step & a second mechanism to confirm that little tryptophan is available

Attenuation ( 衰减子 ) : a regulation at the transcription termination step & a second mechanism to confirm that little tryptophan is available Repressor serves as the primary

switch to regulate the expression of genes in the trp operon

Attenuation serves as the fine switch to determine if the genes need to be efficiently expressed

The TRP operon

82

Fig 16-19

Transcription of the trp operon is prematurally stopped if the tryptophan level is not low enough, which results in the production of a leader RNA of 161 nt. (WHY?)

83

1. Transcription and translation in bacteria are coupled. Therefore, synthesis of the leader peptide immediately follows the transcription of leader RNA.

2. The leader peptide contains two tryptophan codons. If the tryptophan level is very low, the ribosome will pause at these sites.

3. Ribosome pause at these sites alter the secondary structure of the leader RNA, which eliminates the intrinsic terminator structure and allow the successful transcription of the trp operon.

84Fig 16-20 The leader RNA and leader peptide of the trp operon

85

Complementary 3:4 termination of transcription

Complementary 2:3 Elongation of transcription

Four regions (1,2,3,4) of the leader sequence can base pair and form different structures depending on the ribosome action

free leader RNA

The TRP operon

86

Low Trp

High Trp

Fig 16-21

87

Importance of attenuation Importance of attenuation 1. A typical negative feed-back regulation

2. Use of both repression and attenuation allows a fine tuning of the level of the intracellular tryptophan.

3. Attenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuation for their regulation.

4. Provides an example of regulation without the use of a regulatory protein, but using RNA structure instead.

88

Box 4Riboswitches

Riboswitches are regulatory RNA elements that act as direct sensors of small molecule metabolites to control gene transcription or translation.

Some operate at the level of transcription termination

Others operate at the level of translation

Another kind responds to the uncharged tRNA rather than responding to a metabolite. Antitermination mechanism.

90

3-2: Ribosomal proteins are translational repressors of their own synthesis: a negative feedback

Examples of Gene Regulation at Steps After Examples of Gene Regulation at Steps After Transcription InitiationTranscription Initiation

Challenges the ribosome protein synthesis

1. Each ribosome contains some 50 distinct proteins that must be made at the same rate

2. The rate of the ribosome protein synthesis is tightly closed to the cell’s growth rate

91

Strategies to meet the challenges:1. Organization of the ribosomal

proteins to several operons, each containing up to 11 ribosomal protein genes

2. Some nonribosomal proteins whose synthesis is also linked to growth rate are contained in these operons, including those for RNAP subunits , and ’.

3. The primary control is at the level of translation, not transcription.

92

Ribosomal protein operons

The protein that acts as a translational

repressor of the other proteins is shaded

red.

Fig 16-22

93

Strategies to meet the challenges (cont):

4. For each operon, one (or two) ribosomal proteins binds the mRNA near the translation initiation sequence, preventing the ribosome from binding and initiating translation.

5. Repressing translation of the first gene also prevents expression of some or all of the rest. Are proteins processed after translation?

6. The strategy is very sensitive. A few unused molecule of protein L4, for example, will shut down synthesis of that protein and other proteins in this operon.

94

The mechanism of one ribosomal protein also functions as a regulator of its own translation: the protein binds to the similar sites on the ribosomal RNA and to the regulated mRNA

Fig 16-23

95

1.Principles of gene regulation. (1) The targeted gene expression events; (2) the mechanisms: by recruitment/exclusion or allostery

2.Regulation of transcription initiation in bacteria: the lac operon, alternative factors, NtrC, MerR, Gal rep, araBAD operon

3.Examples of gene regulation after transcription initiation: the trp operon, riboswitch, regulation of the synthesis of ribosomal proteins

Key points of the chapter

welcome each of you to my molecular biology class

Documents

layers of regulation

regulated slide

regulation principles

examples of gene regulation

molecular biology class

prokaryotes chapter

transcription steps

expression fig