1

Chapter 16

Gene Regulation

in Prokaryotes

Chapter 16

Gene Regulation

in Prokaryotes

•Molecular Biology Course

There are four major parts in this chapter:

* Principles of Transcriptional Regulation

* Regulation of Transcription Initiation

* Examples of Gene Regulation at Steps after Transcription Initiation

* The Case of Phageλ:Layers of Regulation

outline

Part One

Principles of Transcriptional

Regulation

CHAPTER 16 Gene Regulation in Prokaryotes

►Gene Expression is Controlled by Regulation Proteins: Activators and Repressors

1.Activators, or Positive regulators, increase transcription of the regulated gene;

Repressors, or negative regulators, decrease or eliminate that transcription.

2. Many Promoters Are Regulated by Activation that Help RNA Polymerase Bind DNA and by Repressors that Block that Binding.

a. Absence of Regulatory

Proteins(operator)

b. To Repress

Expression c. To

Activate Expression

Fig 16-1

3.Some Activators Work by Allostery and Regulate Steps after RNA Polymerase Binding:

In some cases, RNA Polymerase binds efficiently unaided and forms a stable closed complex, which does not spontaneously undergo transition to the open complex.

Activator that stimulate this kind of promoter wrk by triggering a conformational change in either RNA Polymerase or DNA.

That is, they interact with the stable closed complex and induce a conformational change that causes transition to the open complex.

This mechanism is an example of allostery.

Fig 16-2

►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

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

Fig 16-4

In this example, we also call the DNA-binding protein “architectural” proteins.

►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.

Two roles: IN RESPONSE TO SMALL CHANGES SENSITIVELY and SERVE TO INTEGRATE SIGNALS

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

►Antitermination and Beyond: Not All of Gene Regulation Targets Transcription Initiation

The bulk of gene regulation takes place at the initiation of transcription in both eukaryotes and bacteria.

But regulation is certainly not restricted to that step in either class of organism. In this chapter we will see examples, in bacteria, of gene regulation that involve transcriptional elongation, RNA processing, and translation of the mRNA into protein.

Part Two

CHAPTER 16 Gene Regulation in Prokaryotes

Regulation of Transcription Initiation: Examples From Bacteria

EXAMPLE ONE------LAC OPERON

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

Fig 16-5

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

The LAC operon

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

►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

The LAC operon

►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

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

Fig 16-8

2. CAP2. CAP

CAP binds as a dimer to a site similar in length to the lac operator, but different in sequence and location.

CAP has separate activating and DNA-binding surfaces.

Fig 16-9

At the promoter,where there is no UP-element, a CTD binds to CAP and adjacent DNA instead.

►CAP and lac repressor bind DNA using a common structural motif

1.The Same

A. The protein binds as a homodimer to a site that is an inverted repeat or near repeat.

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

One of the two аhelices in helix-turn-helix domain is the recognition helix that can fits into the major groove of the DNA.

Fig 16-11

The second helix of the helix-turn-helix domain sits across the major groove an makes contact with the DNA backbone , ensuring proper presentation of the recognition helix, and at the same time adding binding energy to the overall protein-DNA interaction.

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

2. The Difference

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

Fig 16-13

•In some cases, other regions of the protein, outside the helix-turn-helix domain, also interact with the DNA.

•In many cases, binding of the protein does not alter the structure of the DNA .In some cases, however, various distortions are seen in the protein-DNA complex.

►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

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

The LAC operon

►Combinatorial Control: CAP controls other genes as well

•The lac genes provide an example of signal integration: their expression is controlled by two signals, each of which is communicated to the genes via a single regulator—the lac repressor and CAP, respectively.

•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.

Combinatorial control is a characteristic feature of gene regulation. More complex organisms—higher eukaryotes in particular---tend to have more signal integration.

EXAMPLE TWO---- ALTERNATIVE σσFACTORSFACTORS

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 σfactors binding to the same RNA Pol

Confer each of them a new promoter specificity

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

Heat shock--- 32 When E.coliis subject to heat shock, the amount o

f this new σfactor increases in the cell, it displaces σ70 from a proportion of RNA polymerases ,and directs those enzymes to transcribe genes whose products protect the cell from the effects of heat shock. The level of 32 is increased by two mechanisms: first, its translation is stimulated---that is,its mRNA is translated with greater efficiency after heat shock than it was before; and second, the protein is transiently stabilized.

Many bacteriophages synthesizetheir own σfactors to endow thehost RNA polymerase with a different promoter specificity and hence to selectively express their own phage genes .

Bacteriophages

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

Fig 16-14

Normal bacterial holoenzyme

Express early genes

Encodeσfactor for transcription of late genes

Encode σ28

Express middle genes (gene 34 and 33 )

EXAMPLE THREE---NtrC and MerR

►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 only when the nitrogen levels are low.

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

►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

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

The merT promoter is unusual. The distance between the -10 and -35 elements is 19bp instead of the 15 to 17 bp typically found in an eddicient 70 promoter. So, these two elements recognized by are neither optimally seperated nor aligned.

Fig 16-15 a

The binding of MerR locks the promoter in the unpropitious conformation in the absence of Hg2+ .

Fig 16-15 b

When 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

Just like this :

Fig 16-15 c

In this new configuration , RNA polymerase can efficiently initiate transcription.

Fig 16-15

►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)

EXAMPLE FOUR----araBAD OPERON

►AraC and control of the araBAD operon by antiactivation

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.

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

Fig 16-18

The magnitude of induction of the araBAD promoter by arabinose is very large , and for this reason the promoter is often used in expression vectors.

Expression vectors are DNA constructs in which efficient synthesis of any protein can be ensured by fusing a gene to a strong promoter .

Part Three

Examples of Examples of Gene Regulation Gene Regulation at Steps After at Steps After Transcription Transcription InitiationInitiation

CHAPTER 16 Gene Regulation in Prokaryotes

►Amino acid biosynthetic operons are controlled by premature transcription termination

the tryptophan operon:

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 (initiation); (2) attenuation

The Trp repressor

---the first layer of regulation

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.

The ligand that controls the activity of the trp repressor acts not as an inducer but as a corepressor.

Attenuation

---the second layer of regulation

The key to understanding attenuation came from examining the suquence of the 5’ end of trp operon mRNA.

161 nucleotides of RNA are made from tryptophan promoter before RNA polymerase encounters the first codon of trpE.

Near the end of this leader sequence ,and before trpE , is a transcription terminator, composed of a characteristic hairpin loop in the RNA.

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

Fig 16-20

Thre features of the leader sequence:• There is a second hairpin (besides the terminator hairpin) that can form between regions 1 and 2 of the leader sequence.

• region 2 also is complementary to region 3; thus , yet another hairpin consisting of regions 2 and 3 can form and when it does prevent the terminator hairpin (3,4) from forming.

•The leader RNA contains an open-reading frame encoding a short leader peptide of 14 amino acids, and this open-reading frame is preceded by a strong ribosome binding site.

The sequence encoding the leader peptide has a striking feature : two tyrptophan codons in a row.

The fuction of these codons is to stop a ribosome attempting to translate the leader peotide.

Above all , how transcription termination at the trp operon attenuator is controlled by the availability of tryptophan

?

Fig 16-21

The Importance of Attenuation The Importance of Attenuation

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

2. 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.

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

4. A typical negative feed-back regulation.

►Ribosomal Protein Are Translational Repressors of their Own Synthesis

The ribosome protein synthesis has challenges:

• 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

How to overcome the challenges:

• 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.

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.

Ribosomal protein operons

The protein that acts as a translational

repressor of the other proteins is shaded

red.

Fig 16-22

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

CHAPTER 16 Gene Regulation in Prokaryotes

The Case of Phage λ: Layers of Regulation

Bacteriophage λis a virus that infects E.coli. Upon infection, the phage can propagate in either of two ways: lytically or lysogenically.

A lysogen is extremely stable under normal circumstances ,but the phage dormant within it---the prophage---can efficiently switch to lytic growth if the cell is exposed to agents that damaged DNA . This switch from lysogenic to lytic growth is called lysogenic induction.

Lytic cycle

and

Establishment of lysogeny

►Alternative Patterns of Genes Expression Control Lytic and Lysogenic Growth

►Regulatory Proteins and Their Binding Sites

The cI gene encodes λrepressor, a protein of two domains joined by a flexible linker egion. λrepressor can both activate and repress trandcription.

Cro only represses transcription.

►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.

►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.

►Transcriptional Antitermination in λ Development

The 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.

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