Chapter 16Gene Regulation

in Prokaryotes

Outline Part 1 Principles of Transcriptional Regulation

Part 2 Regulation of Transcription Initiation

Part 3 Examples of Gene Regulation after Transcription Initiation

Part 1 Principles of Transcriptional Regulation

1-1 Gene Expression is Controlled by Regulatory Proteins

• Genes are very often controlled by extracellular singals.The singals are communicatedto genes by regulateory proteins :

• Postive regulators or activators • Increase the transcription• Negative regulators or repressors

• Decrease or eliminates the transcription

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

a. Absence of Regulatory Proteins(operator)

b. To Control Expression

c. To Activate Expression

Fig 16-1

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

Fig 16-2

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

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

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)

Cooperative binding: the activator interacts simultaneously with DNA and polymerase and so recruits the enzyme to the promoter

Part 2: Part 2: Regulation of Regulation of Transcription Transcription Initiation : Initiation : Examples Examples from Bacteriafrom Bacteria

2-1 Example from bacteria:Lac operon

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

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 PlacOlac

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

An Activator and a Repressor Together Control the lac Genes

The activator is called CAP( Catabolite Activator Protein ) .CAP can bind DNA and activate the lac genes only in the absence of glucose.

The lac repressor can bind DNA and repress transcrition only in the absence of lactose.

Both CAP and lac repressor are DNA-binding proteins and each binds to a specific site n DNA at or near the lac promoter.

Fig 16-6

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

1.1.Lac operatorLac operator ------the site bound by lac repressor

This 21 bp sequence is twofold summetric and is recognized by two subunits of lac repressor, one binding to each half-site.

Fig 16-7

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

Fig 16-8

Fig 16-9

CTD: C-terminal domain of the subunit of RNAP

2-3 CAP has separate activating and DNA-binding surfaces

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

Cap use the strucure called helix-turn-helix

The helix-turn-helix

lac repressor alse use the same mechanism

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

• Cap and Lac repressor are differences in detail

• Lac repressor binds as a tetramer not a dimer

• Lac repressor ,other regions of protein ,outside the helix-turn-helix domain interact with the DNA.

• In many cases ,binding of the protein does not alter the stricture of the DNA

The Difference

•Lac repressor binds as a tetramer, with each operator is contacted by a repressor dimer.

Fig 16-13

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

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

Response to glucose

• 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

EXAMPLE TWO---- ALTERNATIVE σσFACTORSFACTORS

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

Recall from Chapter 12 that it is the σsubunit of RNA polymerase that recognizes the promoter suquences.

Promoter recognition

•Different σ factors bind to the RNA recognize the promoter sequence ,for example σ70. σ32

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

2-8 NtrC and Mert: Transcriptional Activators that Work by Allostery Rather than by Recruitment

NtrC controls expression of genes involved in nitrogen metabolism, such as the glnA gene. At the glnA gene, Ntrc induces a conformational change in the RNA Polymerase, triggering tansition to the open complex.

MerR controls a gene called merT. Like NtrC, MerR induces a conformational change in the inactive RNA polymerase-promoter complex, and this change can trigger open complex formation

NtrC Has ATPase Activity and Works from DNA Sites Far from the Gene

NtrC has separate activating and DNA-binding domains, and binds DNA when the nitrogen levels are low. The phosphorlated by a kinase. NtrC change the structure and display the activator domain

Fig 16-15 activation by NtrC

The major process:

Low nitrogen levels

NtrB phosphorylates NtrC

NtrC’s DNA-binding domain revealed

NtrC binds four sites located some 150 base pairs upstream of the promoter

NtrC interacts with 54

ATP hydrolysis and conformation change in polymerase

Trigger polymerase to initiate transcription

2-9: MerR activates transcription by twisting promoter DNA

MerR bound to the single DNA-binding site, in the presence of mercury MerR activates the MerT gene. And the Mert twists the DNA.

Fig 16-15 Structure of a merT-like promoter

2-10 Some repressors hold RNA polymerase at the promoter rather than excluding it

Repressors work in different ways

• By binding to a site overlapping the promoter, it blocks RNA polymerase binding. (lac repressor)

• The protein holds the promoter in a conformation incompatible with tanscription initiation.(the MerR case)

• 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)

Fourth example: Fourth example: araaraBADBAD operon operon

Fourth example: Fourth example: araaraBADBAD operon operon

2-11 AraC and control of the araBAD operon by antiactivation

•The promoter of araBAD operon form E.coli is activated in the presence of arabinose and the absence of glucose and directs expression of gene encoding enzyme required for required for arabinose metabolism.

Figure 16-18 control of the araBAD operon

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

Part three: Examples of gene regulation at steps after transcription initiation

3-1 Amino acid biosynthetic operons are controlled by premature transcription termination

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.

Fig 16-19

The trp operon encodes five structural genes required for tryptophan synthesis.These genes are regulated to efficiently express only when tryptophan is limiting.There are two layers of regulation involved: (1) transcription repression by the Trp repressor (2) attenuation

The Trp repressor When tryptophan is present, it

binds the Trp repressor and induces a conformational change in that protein, enabling it to bind the trp operator and prevent transcription.When the tryptophan concentration is low, the Trp repressor is free of its corepressor and vacates its operator , allowing the synthesis of trp mRNA to commence from the adjacent promoter

Attenuation

a regulation at the transcription termination step & a second mechanism to confirm that little tryptophan is available

The using of the Repressor and Attenuation

•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 hairpin loop is followed by 8 uridine residues. At this so-called attenuator , transcription usually stops,yielding a leader RNA 139 nucleotides long

Figure 16-20 trp operator leader RNA

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.

Figure 16-21 transcription at the trp attenuator

3-2 Ribosomal Protein Are Translational Repressors of their Own Synthesis

Control of ribosome protein genes is simplified by their organization to several operons , each containing genes for up to 11 ribisomal proteins. 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 is at the level of translation, not transcription

Ribosomal protein operons

•Ribosomal protein are repressors of their own translation

•One ribosomal proteins binds the messenger near the translation initiation sequence of one the first genes in the operon ,preventing ribosomes from binding and initiating translation .repressing translation of the first genes also prevents expression of some or all of the rest.

How to overcome the challenges:

For each operon,one ribosomal protein binds the messenger near the translation initiation sequence of the first genes in the operon, preventing ribosomes 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.

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

Part four The case of phage λ: layers of regulation

lysogeny

• The alternative propagation pathway –involves integration of the phage DNA into the bacterial chromosome where it is passively replicated at each division –just as though it were a legitimate part of the bacterial genome

• When the cell is exposed to agents that damage DNA. This switch from Lysogenic to lytic growth is called lysogenic induction.

Lysogenic induction

Lytic cycleand Establishment of lysogeny

1.

Figure

16

-2

4

4-1 Alternative patterns of gene expression control lytic and lysogenic growth

1.

Figure

16

-2

5

Promoters in the right and left control regions of phage λ

Transcription in the λcontrol regions in lytic and lysogenic

Arrows indicate which promoters are active at the decisive period during lytic and lysogenic growth , respectively .the arrows also show the direction of transcription from each promoter

4-2 Regulatory Proteins and Their Binding Sites

λrepressor, a protein of two domains joined by a flexible linker egion. λrepressor can both activate and repress trandcription Cro only represses transcription

4-3 Repressor and Cro Bind in Different Patterns to Control Lytic and Lysogenic Growth

Repressor bound to OR1and OR2 turns off transcription from PR . And Repressor bound at OR2 contacts RNA polymerase at PRM, activating expression of the cI gene. OR3 lies with PRM; Cro bound there represses transcription of cI.

Relative positions of promoter and operator sites in OR

1.

Figure

16

-3

1

The action of λ repressor and Cro

Lysogenic induction requires proteolytic cleavage

• Postiive autoregulation: when the level is too low the repressor activates its own repression.

• Negative autoregulation: when the level is too high the repressor will bind to Or3 and repressing Rrm.

Negative autoregulation of repressor require long-distance interations and a large DNA loop

Another Activator, λcII, Controls the Decision between Lytic and Lysogenic Growth upon Infection of a new Host

cII is a transcriptional activator. It binds to a site upstream of a promoter called PRE and stimulates transcription of the cI gene from that promoter.

Interaction between the c-terminal domain of λ repressors

1.

Figure

16

-3

3

Growth conditions of E.coil control the stability of CII protein and thus/lysogenic choice

►Transcriptional Antitermination in λ DevelopmentThe transcripts controlled by λN and Q proteins are initiated perfectly well in the absence of those regulators. But the transcripts terminate a few hundred to a thousand nucleotides downstream of the promoter unless RNA polymerase has been modified by the regulator; λN and Q protein are therefore called antiterminators.

1.

Figure

16

-3

6

Retoregulation:an interplay of control on RNA synthesis and stability determines int gene expression

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