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Molecular Biology. Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods. Part IV Regulation. Ch 16: Regulation in prokaryotes Ch 17: Regulation in eukaryotes

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Molecular biology

Molecular Biology

Part I: Chemistry and Genetics

Part II: Maintenance of the Genome

Part III: Expression of the Genome

Part IV: Regulation

Part V: Methods


Part iv regulation

Part IV Regulation

Ch 16: Regulation in prokaryotes

Ch 17: Regulation in eukaryotes

Ch 18: Regulation during development and in diseases (brief introduction)

Ch 19: Comparative genomics and evolution of animal diversity(Not covered in the lecture)


Molecular biology

Expression of many genes in cells are regulated

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.


Molecular biology

Chapter 16 Regulation principles and How genes are regulated in bacteria

Chapter 17 Basic mechanism of gene expression in eukaryotes

Chapter 18 The mechanism of RNAi and the role of miRNA in development and cancergenesis


Molecular biology

Surfing the contents of Part IV

--The heart of the frontier biological disciplines


Molecular biology

Some of the peoples who significantly contribute to the knowledge of gene regulation


Molecular biology

Chapter 16

Gene Regulation

in Prokaryotes

  • Molecular Biology Course


Molecular biology

TOPIC 1Principles of Transcriptional Regulation [watch the animation]

TOPIC 2 Regulation of Transcription Initiation: Examples from Bacteria (Lac operon, alternative s 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


Molecular biology

CHAPTER 16 Gene Regulation in Prokaryotes

Topic 1: Principles of Transcription Regulation


1 gene expression is controlled by regulatory proteins

Principles of Transcription Regulation

1. Gene Expression is Controlled by Regulatory Proteins (调控蛋白)

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


2 gene expression is controlled at different stages

Principles of Transcription Regulation

2. Gene expression is controlled at different stages(基因表达可以发生在不同时期)

  • 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.


Fig 12 3 initiation

Fig 12-3-initiation

Promoter Binding (closed complex)

Promoter “melting” (open complex)

Promoter escape/Initial transcription


Fig 12 3 elongation and termination

Fig 12-3-Elongation and termination

Elongation

Termination


Molecular biology

Principles of Transcription Regulation

3. Targeting promoter binding: many promoters are regulated by activators(激活蛋白)that help RNAP bind DNA (recruitment) and by repressors(阻遏蛋白)that block the binding.

RNAP binds many promoters weakly (?), activators that contain two binding sites to bind a DNA sequence and RNAP simultaneously can enhance the RNAP affinity with the promoters, and thus increases gene transcription.This is called recruitment regulation (招募调控).

On the contrary, Repressors can bind to the operator inside of the promoter region, which prevents RNAP binding and the transcription of the target gene.


Molecular biology

Fig 16-1

a. Absence of

Regulatory Proteins: basal level expression

b. Repressor binding to the operator represses

expression

c. Activator binding activates

expression


Molecular biology

Principles of Transcription Regulation

4 Targeting transition to the open complex: Allostery regulation (异构调控)after the RNA Polymerase Binding

In some cases, RNAP binds the promoters efficiently, but no spontaneous isomerization occurs to lead to the open complex, resulting in no or low transcription.

Some activators can bind to the closed complex, inducing conformational change in either RNAP or DNA promoter, which converts the closed complex to open complex and thus promotes the transcription.


Molecular biology

Allostery regulation

Fig 16-2

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


Molecular biology

Principles of Transcription Regulation

5 Targeting promoter escape by some repressors

Repressors can work in ways:

blocking the promoter binding.

blocking the transition to the open complex.

blocking promoter escape


Molecular biology

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

Activation mechanisms include recruitment(招募)and allostery (异构).


Molecular biology

6. Cooperative binding (recruitment) and allostery have many roles in gene regulation

Principles of Transcription Regulation

For example: group of regulators often bind DNA cooperatively (activators and/or repressors interact with each other and with the DNA, helping each other to bind near a gene they regulated) :

produce sensitive switches to rapidly turn on a gene expression,

integrate signals (some genes are activated when multiple signals are present).


Molecular biology

Principles of Transcription Regulation

7.Action at a Distance and DNA Looping. The regulator proteins can function even binding at a DNA site far away from the promoter region, through protein-protein interaction and DNA looping.

Fig 16-3


Molecular biology

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

Fig 16-4


Molecular biology

CHAPTER 16 Gene Regulation in Prokaryotes

Topic 2: Regulation of Transcription Initiation :

Examples from Bacteria


Molecular biology

Operon:a unit of prokarytoic gene expression and regulation which typically includes:

1.Structural genesfor 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


Molecular biology

Control element

Structural genes


Molecular biology

Regulation of Transcription Initiation in Bacteria

First example: Lac operon

The lactose Operon (乳糖操纵子)


Molecular biology

1. Lactose operon containsa regulatory gene and 3 structural genes, and 2 control elements.

Fig 16-5

The enzymes encoded by lacZ, lacY, lacA are required for the use of lactose as a carbon source. These genes are only transcribed at a high level when lactose is available as the sole carbon source.

The LAC operon


Molecular biology

codes for β-galactosidase (半乳糖苷酶) for lactose hydrolysis

lacZ

encodes a cell membrane protein called lactose permease (半乳糖苷渗透酶) to transport Lactose across the cell wall

lacY

encodes a thiogalactoside transacetylase (硫代半乳糖苷转乙酰酶)to get rid of the toxic thiogalacosides

lacA

The LAC operon


Molecular biology

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

LacZYA transcription unit contains anoperator site Olac

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

Binds with the lac repressor

The LAC operon


Molecular biology

2. An activator and a repressor together control the Lac operon expression

The activator:CAP (Catabolite Activator Protein,代谢产物激活蛋白) or CRP (cAMP Receptor Protein,cAMP受体蛋白); responses to the glucose level.

The repressor:lac repressor that is encoded by LacIgene; responses to the lactose.

Sugar switch-off mechanism

The LAC operon


Molecular biology

The LAC operon

Fig 16-6


Molecular biology

The LAC operon

3. Lac repressor bound to the operator prevents RNAP from binding to the promoter

The site bound by lac repressor is called the lac operator (Olac), and the Olacoverlaps promoter (Plac). Therefore repressor bound to the operator physically prevents RNA polymerase from binding to the promoter.

The LAC operon


Molecular biology

The LAC operon

Fig 16-8


Molecular biology

The LAC operon

4. CAP activates the Lac transcription through recruitment of RNAP to the weak Plac

CAP has two binding sites, one interacts with the CAP site on the DNA near promoter, and one interacts with RNAP. This cooperative binding ensures that RNAP effectively binds to Plac and initiates transcription of LacZYA.

The LAC operon


Molecular biology

  • CAPsite has the similar structure as the operator, which is 60 bp upstream of the start site of transcription.

  • CAP also interacts with the RNAP and recruit it to the promoter.

Fig 16-9

a CTD: C-terminal domain of the a subunit of RNAP


Molecular biology

The LAC operon

CAP binds as a dimer

a CTD

Fig 16-10. CAP has separate activating and DNA-binding surface


Molecular biology

5. CAP and Lac repressor bind DNA using a common structural motif: helix-turn-helix motif

Fig 16-11

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

The LAC operon


Molecular biology

DNA binding by a helix-turn-helix motif

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


Molecular biology

Lac operon contains three operators: the primary operator and two other operators located 400 bp downstream and 90 bp upstream.

Lacrepressor binds as a tetramer (四聚体), with each operator is contacted by a repressor dimer (二聚体). respectively.

Fig 16-13


Molecular biology

6 The activity of Lac repressor and CAP are controlled allosterically by their signals.

Allolactose: turn of Lac repressor

cAMP: turn on CAP

Lactose is converted to allolactose by b-galactosidase, therefore lactose can indirectly turn off the repressor.

Glucose lowers the cellular cAMP level, therefore, glucose indirectly turn off CAP.

The LAC operon


Molecular biology

Absence of lactose

z

y

a

i

p

o

Active

Very low level of lac mRNA

Response to lactose

Lack of inducer: the lac repressor block all but a very low level of trans-cription of lacZYA .

When Lactose is present, the low basal level of permease allows its uptake, and b-galactosidase catalyzes the conversion of some lactose to allolactose.

Allolactoseacts as an inducer, binding to the lac repressor and inactivate it.

Presence of lactose

z

y

a

i

p

o

Inactive

Permease

Transacetylase

b-Galactosidase


Molecular biology

Response to glucose


Molecular biology

7: Combinatorial Control (组合调控): CAP controls other genes as well

  • 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.


Molecular biology

Regulation of Transcription Initiation in Bacteria

Second example: Alternative s factor

Alternative s factor (可变s因子)direct RNA polymerase to alternative site of promoters


Molecular biology

factor subunit bound to RNA polymerase for transcription initiation (Ch 12)


Molecular biology

  • Different  factors binding to the same RNAP, conferring each of them a new promoter specificity.

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


Molecular biology

Many bacteria produce alternative sets of σfactors to meet the regulation requirements of transcription under normal and extreme growth condition. Bacteriophage has its own σfactors

E. coli : Heat shock 32

Bacteriophage σfactors

Sporulation in Bacillus subtilis


Heat shock

Heat shock (热休克)

  • 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. 32has its own specific promoter consensus sequences.

Alternative s factors


Molecular biology

Bacteriophages

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 .

Alternative s factors


Molecular biology

Alternative s factors

Fig 16-14

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


Molecular biology

Regulation of Transcription Initiation in Bacteria

Third example: NtrC and MerR and

allosteric activation

Transcriptional activators NtrC and MerR

work by allostery rather than by recruitment.


Molecular biology

Review

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

  • In the 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.


Molecular biology

  • In the absence of NtrC and MerR, 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


Molecular biology

NtrC and MerR and allosteric activation

  • NtrC controls expression of genes involved in nitrogen metabolism (氮代谢), such as the glnA gene

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

1. NtrC has ATPase activity and works from DNA sites far from the gene


Molecular biology

Low nitrogen levels (低水平氮)NtrC phosphorylation and conformational change NtrC (?) binds DNA sites at ~-150 positio as a dimer NtrC (?) interacts with 54 (glnA promoter recognition)  NtrCATPase activity provides energy needed to induce a conformation change in polymerase transcription STARTs

Fig 16-15 activation by NtrC


Molecular biology

NtrC and MerR and allosteric activation

  • 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 (汞), MerR binds to a sequence between –10 and –35 regions of the merT promoter and activates merT expression.

2. MerR activates transcription by twisting promoter DNA


Molecular biology

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


Molecular biology

Fig 16-15 Structure of a merT-like promoter


Molecular biology

When Hg2+ is absent, MerR binds to the promoter and locks it in the unfavorable conformation

When Hg2+ is present, MerR binds Hg2+ and undergoes conformational change, which twists the promoter to restore it to the structure close to a strong 70 promoter

Fig 16-15


Molecular biology

Repressors work in many ways-review

  • Blocking RNA polymerase binding through binding to a site overlapping the promoter. Lac repressor

  • Blocking 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 repressor

  • Blocking the promoterescape. P4 protein interaction with PA2c (bacteriophage f29 )


Molecular biology

Regulation of Transcription Initiation in Bacteria

Fourth example: araBAD operon


Molecular biology

The araBAD operon

  • 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.

1. AraC and control of the araBAD operon by anti-activation


Molecular biology

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

194 bp

CAP site

Fig 16-18


Molecular biology

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 thearaBAD promoter, the expression of the gene can be easily controlled by addition of arabinose(阿拉伯糖).

What is an expression vector ? [The answer is in the Methods part.]


Molecular biology

CHAPTER 16 Gene Regulation in Prokaryotes

Topic 3: Examples of

Gene Regulation

at Steps After

Transcription Initiation


Molecular biology

Examples of Gene Regulation at Steps After Transcription Initiation

First example: the tryptophan operon (色胺酸操纵子)


Molecular biology

1. Amino acid biosynthetic operons are controlled by premature transcription termination: the trp operon


Molecular biology

The TRP operon

  • The trp operon encodes five structural genes required for tryptophan (色胺酸)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


Molecular biology

The TRP operon

The Trp repressor

(色氨酸阻遏物 )


Molecular biology

The TRP operon

Trp repressor is encoded by a separate operon trpR, and specifically interacts with the operatorthat overlaps with the promoter sequence

The repressor can only bind to the operator when it is complexed with tryptophan. Therefore, Try is a co-repressorand inhibits its own synthesis through end-product inhibition (negative feed-back regulation).

Remember the lac repressor acts as an inducer


Molecular biology

The TRP operon

The repressor reduces transcription initiation by around 70-fold, which is much smaller than the binding of lac repressor.

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 )


Molecular biology

The TRP operon

trpR operon

trp operon


Molecular biology

The TRP operon

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


Molecular biology

  • 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


Molecular biology

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?)


Molecular biology

Transcription and translation in bacteria are coupled (细菌体内的转录和翻译是偶联的).Therefore, synthesis of the leader peptide immediately follows the transcription of leader RNA.

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

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.


Molecular biology

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


Molecular biology

High Trp

Complementary 3:4 termination of

transcription

Low Trp

Complementary 2:3 Elongation of transcription

Fig 16-21


Molecular biology

Importance ofattenuation

A typical negative feed-back regulation

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

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.

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


Molecular biology

Examples of Gene Regulation at Steps After Transcription Initiation

Second example: Riboswitches-a RNA structure control mechanism

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


Molecular biology

Box 4

  • Riboswitches operating at the level of transcription termination using an Antitermination mechanism.

  • Riboswitches operating at the level of translation, controlling the formation of an RNA structure that masks the ribosome binding site on mRNA.


Molecular biology

代谢物

代谢物

Tucker1 and Breaker, Current Opinion in Structural Biology 2005, 15:342–348


Molecular biology

The 2nd structures of 7 riboswitches and metabolites that they sense


Molecular biology

Examples of Gene Regulation at Steps After Transcription Initiation

Third example: Ribosomal proteins are translational repressors of their own synthesis: a negative feedback


Molecular biology

Challenges the ribosome protein synthesis

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

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


Molecular biology

Strategies to meet the challenges-Operon

Organization of the ribosomal proteins to several operons (操纵子), each containing up to 11 ribosomal protein genes

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

The primary control (主要调控)is at the level of translation, not transcription.


Molecular biology

Ribosomal protein operons

The protein that acts as a translational repressor of the other proteins is shaded red.

Fig 16-22


Molecular biology

Strategies to meet the challenges (cont):

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

Repressing translation of the first gene also prevents expression of some or all of the rest.

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.


Molecular biology

7. 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 regulatory RNA in its ownmRNA.

Fig 16-23


Molecular biology

Key points of the chapter

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

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

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


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