BacterialRegulation (2)

7/29/2019 BacterialRegulation (2)

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Regulation of Gene Expression

Bacteria regulate gene expression levels in response toenvironmental stimuli and in concert with cell cycling.

Genes that are expressed at relatively constant levels aresaid to be constitutive, and the concentrations of thesegene products can range between a few copies per cellto over 100,000 copies per cell.

Regulated genes can be influenced by a number ofdifferent control systems. One mechanism for gene regulation that we have already seen

is the selective expression ofalternate factors, which are

responsible for initiating transcription of different subsets of abacteriums genes. This does, of course, raise the question whatis regulating the expression of the genes coding for the different factors. The answer is a signal transduction network onlypartly understood and beyond the scope of this course.


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Positive and Negative

Control Systems Any regulatory system can be either positive or

negative. Apositive control system is mediated by a

protein that directly increases transcriptionalfrequency.

Anegative control system is mediated by aprotein that directly inhibits transcriptional

frequency. In bacteria, regulated genes are usually affected

by at least one negative control system.


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Induction and Repression

A regulated gene can be either inducible or repressible.

An inducible gene is upregulated in the presence ofsome small effector molecule, called the inducer. For example, the gene coding for an enzyme might be induced

by that enzymes substrate.

Arepressible gene is downregulated in the presence ofsome small effector molecule. For example, the gene coding for an enzyme might be repressed

by that enzymes product.

Inducible and repressible regulation can both be undereither positive or negative control.


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General Terminology Regulatory transcription factors bind specific DNA sequences

sometimes called response elements. Transcription factors that increase the likelihood of transcription are

called activators, and their response elements are called enhancers. Transcription factors that decrease the likelihood of transcription are

called repressors, and their response elements are called silencers.

This terminology is not universal. For example, many silencers inbacteria are termed operators, and the genes they regulate arecollected together into an operon.

control mode transcription factor response element effector molecule

inducible systems

positive activator enhancer inducer

negative repressor silencer inducer

repressible systems

positive activator enhancer inhibitor

negative aporepressor silencer corepressor


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Operons

An operon is a group of genes regulated and expressed as a unit. Coordinate regulation of genes whose products act together is a regular

theme in all cellular life.

It includes two or more protein-coding or structural genes that allshare a single promoter and a single terminator.

An operon also includes at least one operator, a silencer elementthat serves as a binding site for a specific repressor protein.

The transcript is said to be polycistronic because it includes multipleprotein-coding sequences, each with its own start and stop codons.

The genes of a polycistron are translated sequentially. Note that the definition of the word gene is context-dependent

and differs from one person to the next. Since an operon is a singletranscriptional unit, some would refer to it as a gene. Others referto each coding segment within the operon as a gene, as I am doinghere.


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A Generic Operon


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The lacOperon

The lacoperon was the first genetic regulatory system to be welldescribed.

It is a negative inducible system.

The expression oflacis maximal in the presence of lactose and inthe absence of glucose.

The operon encodes three proteins involved in lactose metabolism.

Not shown in the above diagram are three distinct operators withvarying binding efficiencies for the repressor. All three are requiredfor maximal repression of the operon.


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lacGenes The first gene, lacZ, encodes the enzyme

galactosidase, which hydrolyzes the disaccharide lactoseinto its two constituent monosaccharides, galactose andglucose. A steady-state side product, allolactose, is alsoproduced.

The second gene, lacY, encodes the transport moleculelactose permease, which allows uptake of lactose fromthe environment across the cell membrane.

The final gene, lacA, encodes the enzyme galactosidetransacetylase. This enzyme acetylates substrates that

contain a galactose moiety. This may detoxifysubstances brought in by the permease, but really noone knows how it benefits the bacterium evidence thatevolution works but does not have to make sense.


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-Galactosidase Activity

The enzyme galactosidase hydrolyzesthe disaccharide lactoseinto its two constituent

monosaccharides,galactose and glucose.

A steady-state sideproduct, allolactose, isalso produced.

Galactose is isomerized byanother enzyme intoglucose, which is the cellspreferred carbon energysource.


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lacRepressor Protein The lacrepressor protein is

encoded by the constitutive genelacI, which happens to beimmediately upstream of the lacoperon.

This protein acts as ahomotetramer. Two copies of thepolypeptide form a DNA-bindingdomain. The mature protein istherefore able to bind two of thelacoperons three operators

simultaneously, inhibiting initiation. Each polypeptide has a second

binding site for the disaccharideallolactose. Binding of allolactoseallosterically inhibits the repressorsability to bind DNA.


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Catabolite ActivatorProtein

There is an additional level of control, aninducible positive system shared by manyoperons involved with alternate food sources forthe cell.

Operons like lachave CAP sites in theirpromoters. The CAP element is the binding sitefor the catabolite activator protein (CAP), acAMP-dependent activator.

Upon glucose starvation, the signal transductionenzyme adenylyl cyclase increases the cellularconcentration of cAMP. Via CAP, this signal

increases the likelihood of expression of enzymesable to utilize other carbon sources.


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Positive Regulation oflacOperon

In the absence of 3,5-cyclic AMP (cAMP), thecatabolite activatorprotein (CAP) is inactive.

When bound to cAMP,CAP becomes a positiveregulatory transcriptionfactor and increases thelikelihood of transcription

initiation at the lacoperon and dozens ofother operon promoters.


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The lacPromoter

This sequence depicts the lacpromoter and surroundingregion for E. coli.

The -35 and -10 sequences are the key elements in abacterial promoter for transcription initiation.

Two of the operons three operators are shown. One operator, o3, overlaps with the end of the lacIgene, which

otherwise is not considered to be a part of the operon. The operator not shown, o2, is near the end of the lacZgene.

The CAP binding site is also depicted.


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Behavior of the lacOperon You have seen that the lacoperon is not actually induced by lactose,

but rather, allolactose, and this induction is negatively mediated bythe repressor.

You might ask how allolactose is available to bind the repressor whenthe operon encoding permease, which brings lactose into the cell, and-galactosidase, which isomerizes lactose into allolactose, has not

been induced yet. Unlike eukaryotic genes, which can be silenced completely, even the

most repressed bacterial operon is expressed at some low basal level.So the gene products of the lacoperon, including permease and -galactosidase, are present in a few copies in every cell.

In a wild-type E. colistrain, the lacoperon is repressed in the absence

of lactose. Catabolite repression also prevents the active expression of the lac

operon in the presence of glucose. In the presence of lactose and the absence of glucose, the operon is

maximally expressed. Derepression by allolactose frees the promoter,and cAMP-bound CAP stimulates transcription.


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Mutational Analysis of

the lacOperon Jacob and Monod first described this regulatory

system by collecting E. colimutants withaberrant lacregulation and studying their

behavior. These experiments are best performed by using

an artificial inducer of the operon. IPTG(isopropyl-thio--galactoside) is able to bind the

repressor protein and induce the operon. It isnot a substrate for galactosidase. It is alsomembrane-permeable, so it is not dependentupon the presence of active permease.


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Problem Solving with Mutants In the genotypes below, I= lacI(the repressor gene), P= P

lac(the

operon promoter), O= Olac(the operators), Z= lacZ(the -galactosidase gene), and Y= lacY(the permease gene). As iscommon, a + superscript represents the wild-type allele, and asuperscript represents a nonfunctional allele. Oc is a nonfunctional setof operators not recognized by repressor protein and is called a

constitutive mutation. Is

is a super-repressor allele coding for arepressor protein that binds operator but can not bind inducer

IPTG is provided as an inducer in glucose-deficient medium.

genotype

inducer absent inducer present

-gal permease -gal permease

I+P+O+Z+Y+ low low high high

IP+O+Z+Y+ high high high high

ISP+O+Z+Y+ low low low low

I+

P+

Oc

Z+

Y+

high high high high


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Mutational Analysis of a

Merozygote When a plasmid contains chromosomal

genes, the host cell is called a merozygote

or partial diploid because it contains twocopies of those genes on separate DNA.

A cell, for example, that is diploid for lacIand the lacoperon can be designatedeither by I+P+O+Z+Y+/I+P+O+Z+Y+orI+P+O+Z+Y+

I+P+O+Z+Y+.


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Problem Solving with Merozygotes When evaluating these types of problems, first look at the lacIgenes, regardless

which molecule they are on. (This is a trans action.) Determine whether thecell has functional repressor or not and what form of the repressor is present inthe cell. Then consider both operons separately, considering the impact of theinducer on each. (The elements of the operon, like the promoter and operators,have cis effects.)

Here we are introducing a new mutant, lacI-d, the negative dominant. Thepolypeptide encoded by this allele is not only unable to bind operator, in formingtetramers with wild-type polypeptides it prevents them from functioning as well.

genotype

inducer absent inducer present

-gal permease -gal permease

I+PO+Z+Y+

IP+O+Z+Y+

low low high high

ISP+O+Z+Y

IP+OcZY+low high low high

I+P+O+Z+Y+

Id

P+

O+

Z+

Y+

high high high high


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Nonsense Mutations

Because of the sequential nature of translation of polycistronicmRNA, expression levels exhibit a polarity or positional effect to

premature STOP codons. Only the first gene is preceded by a high-efficiency Shine-Dalgarno

sequence. Subsequent genes rely on the proximity of their STARTcodons to the preceding STOP codon to attract a ribosome forinitiation of translation.

A premature STOP codon, or nonsense mutation, can drastically

reduce or prevent the expression of all following gene products.


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The araOperon The araoperon includes three genes, araB, araA, andaraD, which encode the enzymes that transform the sugar

arabinose into D-xylulose-5-phosphate, which can bemetabolized downstream.

This operon contains a CAP site and three operators,

which can be bound by the AraC protein. The araCgene is nearby and shares one of the operons

operators, so the AraC protein represses its own gene. The araoperon is inducible.

In the absence of the sugar arabinose, AraC represses the operon.

In the presence of arabinose, however, AraC acts as an activatorfor the araoperon, in concert with CAP.

This situation, where a single protein can act both asrepressor or activator in different circumstances, is rare inbacteria but is rather common in eukaryotic generegulation.


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The trpOperon


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trpRegulation The trpoperon encodes a small oligopeptide and five

enzymes responsible for tryptophan synthesis. The primary regulation of the trpoperon is negative and

repressible. The apo-repressor protein is encoded by the trpRgene, which is

nowhere near the trpoperon. When bound to its corepressor, tryptophan, the repressor binds

the trpoperator and downregulates the transcription of theoperon.

Therefore, when tryptophan is available from the environment,the synthetic enzymes are not expressed.

The small oligopeptide is encoded by the first operongene, trpL, or leader sequence. This peptide is notinvolved in tryptophan synthesis, but the coupling oftranscription and translation that occurs in bacteria allowsfor an additional level of regulation termed attenuation.


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The Leader Sequence and Attenuator The leader sequence, trpL, encodes an oligopeptide that is rich in

tryptophan. This sequence also include four self-complementary regions

followed by a string of AT pairs, collectively called the attenuator. For any bacterial mRNA, when the Shine-Dalgarno sequence at the

5 end is clear of the RNA polymerase, translation begins. When tryptophan, and therefore charged tryptophanyl-tRNAs, is

plentiful, the presence of the ribosome translating trpLinto theoligopeptide prevents the first two self-complementary regions fromhydrogen bonding. In this case the last two self-complementaryregions of the nascent mRNA and the following string of Unucleotides act as an intrinsic terminator, stopping transcription ofthe remainder of the operon.

When tryptophan is scarce enough to delay arrival of chargedtryptophanyl tRNAs, the ribosome is slowed at the first self-complementary region, allowing the second and third region tohydrogen bond. Their distance from the string of U nucleotidesdoes not allow this secondary structure to act as a terminator, andthe RNA polymerase continues transcribing the operon, leaving theattenuator region behind.


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Transcription without Attenuation


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Attenuation of Transcription


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Other Leader Peptides

Shown above are the leader peptide sequences for five operonsregulated in part by attenuation, taken from either E. colior

Salmonella typhimurium. phenylalanine histidine leucine threonine isoleucine and valine


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Other Attenuation Mechanisms

Not all early termination of transcription ismediated by the availability of charged tRNA forsynthesis of a leader peptide.

Some catabolic operons are regulated by anRNA-binding protein acting as an anti-terminatornear the 5 end of the nascent transcript.

Many genes encoding aminoacyl-tRNAsynthetases are regulated by attenuation.Uncharged tRNA stabilizes an anti-terminatorhairpin stem-loop in the mRNA.


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Other Gene Regulatory Systems

Regulation of viral gene expression is oftencomplex and exquisite, but it is also beyond thescope of this course.

Many Archaean genes are organized intooperons. Archaean DNA, however, is complexedwith histones, and the mechanisms of generegulation are more similar to those found ineukaryotes than those of bacteria.

Regulation of mitochondrial and chloroplasticgenes is minimal. Most are constitutivelyexpressed.

BacterialRegulation (2)

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