Biochemistry, 4th Edition P96 pps

The lac operon includes the regulatory gene lacI; its promoter p; and three structural genes, lacZ, lacY, and lacA, with their own promoter p lac and operator O Figure 29.11.. lac Repres

transcribed determines whether transcription takes place This sequence is termed the

operator(Figure 29.8) The operator is located next to a promoter Interaction of a

regulatory proteinwith the operator controls transcription of the gene cluster by

con-trolling access of RNA polymerase to the promoter.3Such co-expressed gene clusters,

together with the operator and promoter sequences that control their transcription,

are called operons.

Transcription of Operons Is Controlled by Induction and Repression

In prokaryotes, gene expression is often responsive to small molecules serving as

sig-nals of the nutritional or environmental conditions confronting the cell Increased

synthesis of enzymes in response to the presence of a particular substrate is termed

(a)

RNA polymerase

ter site mRNA

 factor

(b)

(c)

(d)

mRNA

FIGURE 29.7 The rho factor mechanism of

tran-scription termination Rho factor (a) attaches to

a recognition site on mRNA and (b) moves along it behind RNA polymerase (c) When RNA

polymerase pauses at the termination site, rho factor unwinds the DNA ⬊RNA hybrid in the

transcription bubble, (d) releasing the nascent

mRNA.

DNA

Transcriptional control region

Operator

Promotor

Structural genes 1, 2, 3

FIGURE 29.8 The general organization of operons Operons consist of transcriptional control regions and a set

of related structural genes, all organized in a contiguous linear array along the chromosome The

transcrip-tional control regions are the promoter and the operator, which lie next to, or overlap, each other, upstream

from the structural genes they control Operators may lie at various positions relative to the promoter, either

upstream or downstream Expression of the operon is determined by access of RNA polymerase to the

pro-moter, and occupancy of the operator by regulatory proteins influences this access Induction activates

tran-scription from the promoter; repression prevents it.

3 Although this is the paradigm for prokaryotic gene regulation, it must be emphasized that many

prokaryotic genes do not contain operators and are regulated in ways that do not involve protein

⬊op-erator interactions.

induction.For example, lactose (Figure 29.9) can serve as both carbon and energy

source for E coli Metabolism of lactose depends on hydrolysis into its component

sug-ars, glucose and galactose, by the enzyme ␤-galactosidase In the absence of lactose,

E coli cells contain very little -galactosidase (less than 5 molecules per cell) How-ever, lactose availability induces the synthesis of -galactosidase by activating

transcrip-tion of the lac operon One of the genes in the lac operon, lacZ, is the structural gene

for-galactosidase When its synthesis is fully induced, -galactosidase can amount to almost 10% of the total soluble protein in E coli When lactose is removed from the

culture, synthesis of -galactosidase halts.

The alternative to induction—namely decreased synthesis of enzymes in response to

a specific metabolite—is termed repression For example, the enzymes of tryptophan

biosynthesis in E coli are encoded in the trp operon If sufficient Trp is available to the

growing bacterial culture, the trp operon is not transcribed, so the Trp biosynthetic enzymes are not made; that is, their synthesis is repressed Repression of the trp operon

in the presence of Trp is an eminently logical control mechanism: If the end product

of the pathway is present, why waste cellular resources making unneeded enzymes? Induction and repression are two faces of the same phenomenon In induction,

a substrate activates enzyme synthesis Substrates capable of activating synthesis of

the enzymes that metabolize them are called co-inducers, or, often, simply inducers.

Some substrate analogs can induce enzyme synthesis even though the enzymes are

incapable of metabolizing them These analogs are called gratuitous inducers A number of thiogalactosides, such as IPTG (isopropyl -thiogalactoside, Figure

29.10), are excellent gratuitous inducers of -galactosidase activity in E coli In

re-pression, a metabolite, typically an end product, depresses synthesis of its own

biosynthetic enzymes Such metabolites are called co-repressors

The lac Operon Serves as a Paradigm of Operons

In 1961, François Jacob and Jacques Monod proposed the operon hypothesis to

ac-count for the coordinate regulation of related metabolic enzymes The operon was considered to be the unit of gene expression, consisting of two classes of genes: the

structural genes for the enzymes and regulatory genes that control expression of the

structural genes The two kinds of genes could be distinguished by mutation Muta-tions in a structural gene would abolish one particular enzymatic activity, but muta-tions in a regulatory gene would affect all of the different enzymes under its control

Mutations of both kinds were known in E coli for lactose metabolism Bacteria with mutations in either the lacZ gene or the lacY gene (Figure 29.11) could no longer metabolize lactose—the lacZ mutants (lacZstrains) because -galactosidase activity

CH2OH

O HO

OH

O

OH

CH2OH O OH OH OH

Lactose

(O--D -galactopyranosyl (1 4) -D -glucopyranose)

FIGURE 29.9 The structure of lactose, a -galactoside.

CH2OH

O HO

OH

S

OH

CH

CH3

CH3

Isopropyl -thiogalactoside (IPTG)

FIGURE 29.10 The structure of IPTG (isopropyl

-thiogalactoside).

DNA

Polypeptide

p

Amino acids kD

kD

Function

360 38.6 Tetramer 154.4 Repressor

1023 116.4 Tetramer 465

-Galactosidase

417 46.5

46.5 Permease

203 22.7 Dimer 45.4 Trans-acetylase

Membrane protein

mRNA

FIGURE 29.11 The lac operon.The operon consists of two transcription units In one unit, there are three structural genes, lacZ, lacY, and lacA, under control of the promoter, p lac , and the operator O In the other unit, there is a

regulator gene, lacI, with its own promoter, p lacI lacI encodes a 360-residue, 38.6-kD polypeptide that forms a

tetrameric lac repressor protein lacZ encodes -galactosidase, a tetrameric enzyme of 116-kD subunits.lacY is the

-galactoside permease structural gene, a 46.5-kD integral membrane protein active in -galactoside transport

into the cell.The remaining structural gene encodes a 22.7-kD polypeptide, but the metabolic role of this protein in vivo remains uncertain.

was absent, the lacY mutants because lactose was no longer transported into the cell.

Other mutations defined another gene, the lacI gene lacI mutants were different

be-cause they both expressed -galactosidase activity and immediately transported

lac-tose, without prior exposure to an inducer That is, a single mutation led to the

expres-sion of lactose metabolic functions independently of inducer Expresexpres-sion of genes

independently of regulation is termed constitutive expression Thus, lacI had the

properties of a regulatory gene The lac operon includes the regulatory gene lacI; its

promoter p; and three structural genes, lacZ, lacY, and lacA, with their own promoter

p lac and operator O (Figure 29.11).

lac Repressor Is a Negative Regulator of the lac Operon

The structural genes of the lac operon are controlled by negative regulation.

That is, they are transcribed to give an mRNA unless turned off by the lacI gene

product This gene product is the lac repressor, a tetrameric protein (Figure

29.12) (Note that the language can be misleading: Inducers and co-repressors are

p lac

p lac

mRNA

DNA

lacI

O

No transcription

Repressor

monomer

Repressor

tetramer

mRNA

DNA

Transcription

Repressor monomer

Repressor tetramer Inducer

mRNA

Translation

-Galactosidase Permease Transacetylase

Without inducer

With inducer

FIGURE 29.12 The mode of action of lac repressor The structure of the lac repressor tetramer with bound IPTG

(purple) is also shown (pdb id  1LBH).

small molecules/metabolites; repressors are proteins.) The lac repressor has an

N-terminal DNA-binding domain; the rest of the protein functions in inducer

binding and tetramer formation In the absence of an inducer, lac repressor blocks lac gene expression by binding to the operator DNA site upstream from the lac structural genes The lac operator is a palindromic DNA sequence (Figure

29.13) Palindromes, or “inverted repeats,” provide a twofold, or dyad, symmetry,

a structural feature common at sites in DNA where proteins specifically bind

Despite the presence of lac repressor, RNA polymerase can still initiate tran-scription at the promoter (p lac ), but lac repressor blocks elongation of transcrip-tion, so initiation is aborted In lacI mutants, the lac repressor is absent or defec-tive in binding to operator DNA, lac gene transcription is not blocked, and the lac operon is constitutively expressed in these mutants Note that lacI is normally expressed constitutively from its promoter, so lac repressor protein is always avail-able to fill its regulatory role About ten molecules of lac repressor are present in

an E coli cell.

Derepression of the lac operon occurs when appropriate -galactosides occupy the inducer site on lac repressor, causing a conformational change in the protein that lowers the repressor’s affinity for operator DNA As a tetramer, lac repressor has

four inducer binding sites, and its response to inducer shows cooperative allosteric effects Thus, as a consequence of the “inducer”-induced conformational change, the inducer⬊lac repressor complex dissociates from the DNA, and RNA polymerase

transcribes the structural genes (see Figure 29.12) Induction reverses rapidly: lac

mRNA has a half-life of only 3 minutes, and once the inducer is used up through

metabolism by the enzymes, free lac repressor reassociates with the operator DNA, transcription of the operon is halted, and any residual lac mRNA is degraded.

In the absence of inducer, lac repressor binds nonspecifically to duplex DNA with an association constant, KA, of 2 106 M1 (Table 29.1) and to the lac operator DNA sequence with much higher affinity, KA 2  1013M1 Thus, lac

repressor binds 107times better to lac operator DNA than to any random DNA se-quence IPTG binds to lac repressor with an association constant of about 106M1 The IPTG⬊lac repressor complex binds to operator DNA with an association

con-stant, KA 2  1010M1 Although this affinity is high, it is 3 orders of magnitude

less than the affinity of inducer-free repressor for lac operator There is no differ-ence in the affinity of free lac repressor and lac repressor with IPTG bound for non-operator DNA The lac repressor apparently acts by binding to DNA and sliding along it, testing sequences in a one-dimensional search until it finds the lac opera-tor The lac repressor then binds there with high affinity until inducer causes this

affinity to drop by 3 orders of magnitude (Table 29.1)

CAP Is a Positive Regulator of the lac Operon

Transcription by RNA polymerase from some promoters proceeds with low efficiency

unless assisted by an accessory protein that acts as a positive regulator One such protein

is CAP, or catabolite activator protein Its name derives from the phenomenon of

catabolite repression in E coli Catabolite repression is a global control that

coordi-A T

T A

G C

T A

G C

A T

T A

T A

C G

A T DNA TAGCTAGCGC TAGCTAGC GC GCATTAATATGCATATTATATACGATCGATGCAT

Axis of symmetry

Protected bases

mRNA transcription

G C

T T

T

A T A

T

G T

A T

A T

T T T G

FIGURE 29.13 The nucleotide sequence of the lac

oper-ator This sequence comprises 36 bp showing nearly

palindromic symmetry The inverted repeats that

consti-tute this approximate twofold symmetry are shaded in

rose The bases are numbered relative to the 1 start

site for transcription The G ⬊C base pair at position 11

represents the axis of symmetry In vitro studies show

that bound lac repressor protects a 26-bp region from

5 to 21 against nuclease digestion Bases that

inter-act with bound lac repressor are indicated below the

operator Note the symmetry of protection at 1

through 4 TTAA to 18 through 21 AATT.

Repressor 

lac operator 2 1013M1 2 1010M1

All other 2 106M1 2 106M1

DNA

Specificity† 107 104

*Values for repressor ⬊DNA binding are given as association

constants, KA, for the formation of DNA⬊repressor complex

from DNA and repressor.

†Specificity is defined as the ratio (KAfor repressor binding to

operator DNA)/(KAfor repressor binding to random DNA).

TABLE 29.1 The Affinity of lac Repressor

for DNA*

nates gene expression with the total physiological state of the cell: As long as glucose

is available, E coli catabolizes it in preference to any other energy source, such as

lac-tose or galaclac-tose Catabolite repression ensures that the operons necessary for

me-tabolism of these alternative energy sources, that is, the lac and gal operons, remain

repressed until the supply of glucose is exhausted Catabolite repression overrides the

influence of any inducers that might be present

Catabolite repression is maintained until the E coli cells become starved of

glu-cose Glucose starvation leads to activation of adenylyl cyclase, and the cells begin to

make cAMP (In contrast, glucose uptake is accompanied by deactivation of adenylyl

cyclase.) The action of CAP as a positive regulator is cAMP-dependent cAMP is a

small-molecule inducer for CAP, and cAMP binding enhances CAP’s affinity for

DNA CAP, also referred to as CRP (for cAMP receptor protein), is a dimer of

iden-tical 22.5-kD polypeptides The N-terminal domains bind cAMP; the C-terminal

do-mains constitute the DNA-binding site Two molecules of cAMP are bound per

dimer The CAP–(cAMP)2complex binds to specific target sites near the promoters

of operons (Figure 29.14) Binding of CAP–(cAMP)2 to DNA causes the DNA to

bend more than 80° (Figure 29.15) This CAP-induced DNA bending near the

pro-moter assists RNA polymerase holoenzyme binding and closed propro-moter complex

formation Contacts made between the CAP–(cAMP)2complex and the -subunit of

RNA polymerase holoenzyme activate transcription

Negative and Positive Control Systems Are Fundamentally Different

Negative and positive control systems operate in fundamentally different ways

(al-though in some instances both govern the expression of the same gene) Genes

un-der negative control are transcribed unless they are turned off by the presence of a

repressor protein Often, transcription activation is merely the release from

nega-tive control In contrast, genes under posinega-tive control are expressed only if an acnega-tive

regulator protein is present The lac operon illustrates these differences The action

of lac repressor is negative It binds to operator DNA and blocks transcription;

ex-pression of the operon occurs only when this negative control is lifted through the

release of the repressor In contrast, regulation of the lac operon by CAP is positive:

Transcription of the operon by RNA polymerase is stimulated by CAP’s action as a

positive regulator

A DEEPER LOOK

The affinity of lac repressor for random DNA ensures that essentially

all repressor is DNA bound Assume that E coli DNA has a single

spe-cific lac operator site for repressor binding and 4.64 106base

pairs and any nucleotide sequence even one base out of phase with

the operator constitutes a nonspecific binding site Thus, there are

4.64 106nonspecific sites for repressor binding

The binding of repressor to DNA is given by the association

constant, KA:

KA where [repressor⬊DNA] is the concentration of repressor⬊DNA

complex, [repressor] is the concentration of free repressor, and

[DNA] is the concentration of nonspecific binding sites

Rearrang-ing gives the followRearrang-ing:

KA[DNA]

[repressor]

[repressor⬊DNA]

[repressor⬊DNA]

[repressor][DNA]

If the number of nonspecific binding sites is 4.64 106, there are (4.64 106)/(6.022 1023) 0.77  1017moles of binding sites contained in the volume of a bacterial cell (roughly 1015 liters) Therefore, [DNA] (0.77  1017)/(1015) 0.77  102M Since

KA 2  106M1(Table 29.1),

So, the ratio of free repressor to DNA-bound repressor is 6.5

105 Less than 0.01% of repressor is not bound to DNA! The behavior

of lac repressor is characteristic of DNA-binding proteins These

proteins bind with low affinity to random DNA sequences, but with much higher affinity to their unique target sites (Table 29.1)

1 (1.54 104)

1 (2 106) (0.77 102)

[repressor]

[repressor:DNA]

DNA binding and transcriptional activation

72 89 74 89 61 67 72 61 50

Binding region Upstream of RNA polymerase binding site at –41 or –61 or –71 bp

Inactive CAP

Active CAP

cAMP cAMP

A T T A A T N N G C T A G C A T N N T A N N N N N N N N T A C G A T N N A T T A T

A Consensus

% Occurrence

FIGURE 29.14 The mechanism of catabolite repression and CAP action Glucose instigates catabolite repression

by lowering cAMP levels cAMP is necessary for CAP binding near promoters of operons whose gene prod-ucts are involved in the metabolism of alternative en-ergy sources such as lactose, galactose, and arabinose The binding sites for the CAP–(cAMP) 2 complex are consensus DNA sequences containing the conserved pentamer TGTGA and a less well conserved inverted re-peat, TCANA (where N is any nucleotide).

Operons can also be classified as inducible, repressible, or both, depending on how

they respond to the small molecules that mediate their expression Repressible oper-ons are expressed only in the absence of their co-repressors Inducible operoper-ons are transcribed only in the presence of small-molecule co-inducers (Figure 29.16)

The araBAD Operon Is Both Positively and Negatively Controlled by AraC

E coli can use the plant pentose L-arabinose as sole source of carbon and energy Ara-binose is metabolized via conversion to D-xylulose-5-P (a pentose phosphate pathway intermediate and transketolase substrate [see Chapter 22]) by three enzymes

en-coded in the araBAD operon Transcription of this operon is regulated by both

catabolite repression and arabinose-mediated induction CAP functions in catabolite

repression; arabinose induction is achieved via the product of the araC gene, which

FIGURE 29.15 Binding of CAP–(cAMP) 2 induces a severe

bend in DNA about the center of dyad symmetry at the

CAP-binding site The CAP dimer with two molecules of

cAMP bound interacts with 27 to 30 base pairs of

du-plex DNA Two -helices of the CAP dimer insert into

the major groove of the DNA at the dyad-symmetric

CAP-binding site The two cAMP molecules bound by

the CAP dimer are indicated in yellow Binding of

CAP–(cAMP) 2 to its specific DNA site involves H bonding

and ionic interactions between protein functional

groups and DNA phosphates, as well as H-bonding

in-teractions in the DNA major groove between amino

acid side chains of CAP and DNA base pairs (pdb id 

1CGP).

Repressor

Co-inducer

Inactive repressor

DNA

mRNA Lactose operon

Repressor deletions are constitutive

Inactive inducer

Co-inducer

Active inducer

DNA

mRNA Catabolite repression

Inducer deletions are uninducible

DNA

mRNA Tryptophan operon

Repressor deletions are constitutive (de-repressed)

Active inducer

Corepressor

Inactive inducer

DNA

mRNA

Inactive repressor

Corepressor

Active repressor

Inducer deletions are uninducible

FIGURE 29.16 Control circuits

gov-erning the expression of genes.

These circuits can be either negative

or positive, inducible or repressible.

lies next to the araBAD operon on the E coli chromosome The araC gene product,

the protein AraC,4 is a 292-residue protein consisting of an N-terminal domain

(residues 1 to 170) that binds arabinose and acts as a dimerization motif and a

C-terminal (residues 178 to 292) DNA-binding domain Regulation of araBAD by

AraC is novel in that it acts both negatively and positively The ara operon has three

binding sites for AraC: araO1, located at nucleotides 106 to 144 relative to the

araBAD transcription start site; araO2(spanning positions 265 to 294); and araI,

the araBAD promoter The araI site consists of two “half-sites”; araI1 (nucleotides

56 to 78) and araI2(35 to 51) (The araO1site contributes minimally to ara

operon regulation.)

The details of araBAD regulation are as follows: When AraC protein levels are low,

the araC gene is transcribed from its promoter p c (adjacent to araO1) by RNA

poly-merase (Figure 29.17) araC is transcribed in the direction away from araBAD When

cAMP levels are low and arabinose is absent, an AraC protein dimer binds to two sites,

araO2and the araI1half-site, forming a DNA loop between them and restricting

tran-scription of araBAD (Figure 29.17) In the presence of L-arabinose, the monomer of

AraC bound to the araO2site is released from that site; it then associates with the

un-occupied araI half-site, araI2.L-Arabinose thus behaves as an allosteric effector that

al-ters the conformation of AraC In the arabinose-liganded conformation, the AraC

dimer interacts with CAP–(cAMP)2to activate transcription by RNA polymerase Thus,

AraC protein is both a repressor and an activator

4 Proteins are often named for the genes encoding them By convention, the name of the protein is

capitalized but not italicized.

(a) The araBAD operon

(b) Low [cAMP], no L -arabinose

(c) High [cAMP], L -arabinose present

DNA

RNA pol site

araC

araB araI 1

araC araO 2

CAP site

araO 1

pC

Regulatory gene

D -Xylulose-5-P

L -Ribulose-5-P

L -Ribulose

L -Arabinose

L -Arabinose

Ribulose-5-P epimerase

araI 2 araI 1

CAP

site

RNA polymerase

araB

araO 2 araO 1

araBAD mRNA araC araC

Structural genes

L -Arabinose

araC araC

RNA pol site araB araI 1

araC

araO 2

CAP site

araO 1

araC

ADP

ATP

FIGURE 29.17 Regulation of the araBAD operon by the

combined action of CAP and AraC protein.

Positive control of the araBAD operon occurs in the presence of L-arabinose and cAMP Arabinose binding by AraC protein causes the release of araO2, opening of

the DNA loop, and association of AraC with araI2 CAP–(cAMP)2binds at a site

be-tween araO1and araI, and together the AraC–(arabinose)2and CAP–(cAMP)2 com-plexes influence RNA polymerase through protein⬊protein interactions to create

an active transcription initiation complex Supercoiling-induced DNA looping may promote protein⬊protein interactions between DNA-binding proteins by bringing them into juxtaposition

The trp Operon Is Regulated Through a Co-Repressor–Mediated

Negative Control Circuit

The trp operon of E coli (and S typhimurium) encodes the five polypeptides, trpE through trpA (Figure 29.18), that assemble into the three enzymes catalyzing tryp-tophan synthesis from chorismate (see Chapter 25) Expression of the trp operon is

under the control of Trp repressor, a dimer of 108-residue polypeptide chains.

When tryptophan is plentiful, Trp repressor binds two molecules of tryptophan and

associates with the trp operator that is located within the trp promoter Trp

repres-sor binding excludes RNA polymerase from the promoter, preventing transcription

of the trp operon When Trp becomes limiting, repression is lifted because Trp re-pressor lacking bound Trp (Trp apo-rere-pressor) has a lowered affinity for the trp

pro-moter Thus, the behavior of Trp repressor corresponds to a co-repressor–mediated, negative control circuit (see Figure 29.16) Trp repressor not only is encoded by the

trpR operon but also regulates expression of the trpR operon This is an example of

autogenous regulation (autoregulation):regulation of gene expression by the prod-uct of the gene

Attenuation Is a Prokaryotic Mechanism for Post-Transcriptional Regulation of Gene Expression

In addition to repression, expression of the trp operon is controlled by transcription

attenuation. Unlike the mechanisms discussed thus far, attenuation regulates tran-scription after it has begun Charles Yanofsky, the discoverer of this phenomenon, has

DNA

L -Tryptophan Indole-3-glycerol-P

Enol-1-o-carboxy- phenylamino-1-deoxyribulose phosphate

N-(5 -Phospho- ribosyl)-anthranilate

Anthranilate synthase component I

N-(5 -Phosphoribosyl)-anthranilate isomerase Indole-3-glycerol phosphate synthase

trpC trpD

trpE trpL

Control sites

trp p,O

mRNA

Anthranilate synthase component II

Tryptophan synthase

-subunit

Tryptophan synthase

-subunit

Tryptophan synthase

(22 )

Anthranilate synthase (CoI2CoII2)

L -Serine

Glyceraldehyde-3-P

Anthranilate

P P

PRPP

Chorismate

Glutamine Glutamate

+ pyruvate

CO2 Attenuator

FIGURE 29.18 The trp operon of E coli.

defined attenuation as any regulatory mechanism that manipulates transcription termination

or transcription pausing to regulate gene transcription downstream In prokaryotes,

tran-scription and translation (see Chapters 10 and 30) are coupled, and the translating

ribosome is affected by the formation and persistence of secondary structures in the

mRNA In many operons encoding enzymes of amino acid biosynthesis, a transcribed

150- to 300-bp leader region is positioned between the promoter and the first major

structural gene These regions encode a short leader peptide containing multiple

codons for the pertinent amino acid For example, the leader peptide of the leu

operon has four leucine codons, the trp operon has two tandem tryptophan codons,

and so forth (Figure 29.19) Translation of these codons depends on an adequate

sup-ply of the relevant aminoacyl-tRNA, which in turn rests on the availability of the

amino acid When tryptophan is scarce, the entire trp operon from trpL to trpA is

tran-scribed to give a polycistronic mRNA But, as [Trp] increases, more and more of the

trp transcripts consist of only a 140-nucleotide fragment corresponding to the 5-end

of trpL Tryptophan availability is causing premature termination of trp transcription,

that is, transcription attenuation Although attenuation occurs when tryptophan is

abundant, attenuation is blocked when levels of tryptophan are low and little

trypto-phanyl-tRNA is available The secondary structure of the 160-bp leader region

tran-script is the principal control element in trantran-scription attenuation (Figure 29.20)

This RNA segment includes the coding region for the 14-residue leader peptide

Three critical base-paired hairpins can form in this RNA: the 1 ⬊2 pause structure, the

Met

his Thr Arg Val Gln Phe Lys His His His His His His His Pro Asp

Met

ilv Thr Ala Leu Leu Arg Val Ile Ser Leu Val Val Ile Ser Val Val Val Ile Ile Ile Pro Pro Cys Gly Ala Ala Leu Gly Arg Gly Lys Ala

Met

leu Ser His Ile Val Arg Phe Thr Gly Leu Leu Leu Leu Asn Ala Phe Ile Val Arg Gly Arg Pro Val Gly Gly Ile Gln His

Met

pheA Lys His Ile Pro Phe Phe Phe Ala Phe Phe Phe Thr Phe Pro

Met

thr Lys Arg Ile Ser Thr Thr Ile Thr Thr Thr Ile Thr Ile Thr Thr Gln Asn Gly Ala Gly

Met

trp Lys Ala Ile Phe Val Leu Lys Gly Trp Trp Arg Thr Ser

FIGURE 29.19 Amino acid sequences of leader peptides in various amino acid biosynthetic operons regulated

by attenuation Color indicates amino acids synthesized in the pathway catalyzed by the operon’s gene

prod-ucts (The ilv operon encodes enzymes of isoleucine, leucine, and valine biosynthesis.)

A

50

G

G

U

G

C

G

U

C G C

U

A U U

G

A

A U C A G A U A C C C

UUUUUU

C U

G A

Trp

Trp

70

110

130

A G G U U G G U G G C G C A C U U C C

AA

A G U A C C

U

A C C

G

A

A C U A

C C U A

G A G

U C G G G C

UUUU

130 UU

50

110

70

Trp

codons

Stop codon

for leader

peptide

1 • 2

3 • 4

“Antiterminator”

Pause

Structure

U

G

A

FIGURE 29.20 Alternative secondary struc-tures for the leader region (trpL mRNA) of

thetrp operon transcript.

3 ⬊4 terminator, and the 2⬊3 antiterminator Obviously, the 1⬊2 pause, 3⬊4 terminator,

and the 2⬊3 antiterminator represent mutually exclusive alternatives A significant fea-ture of this coding region is the tandem UGG tryptophan codons

Transcription of the trp operon by RNA polymerase begins and progresses

un-til position 92 is reached, whereupon the 1⬊2 hairpin is formed, causing RNA polymerase to pause in its elongation cycle While RNA polymerase is paused, a ribosome begins to translate the leader region of the transcript Translation by the ribosome releases the paused RNA polymerase and transcription continues, with RNA polymerase and the ribosome moving in unison As long as tryptophan

is plentiful enough that tryptophanyl-tRNATrpis not limiting, the ribosome is not delayed at the two tryptophan codons and follows closely behind RNA poly-merase, translating the message soon after it is transcribed The presence of the ribosome atop segment 2 blocks formation of the 2⬊3 antiterminator hairpin, al-lowing the alternative 3⬊4 terminator hairpin to form (Figure 29.21) Stable

hair-pin structures followed by a run of Us are features typical of rho -independent

transcription termination signals, so the RNA polymerase perceives this hairpin

as a transcription stop signal and transcription is terminated at this point On the other hand, a paucity of tryptophan and hence low availability of tryptophanyl-tRNATrpcauses the ribosome to stall on segment 1 This leaves segment 2 free to pair with segment 3 and to form the 2⬊3 antiterminator hairpin in the transcript Because this hairpin precludes formation of the 3⬊4 terminator, termination is prevented and the entire operon is transcribed Thus, transcription attenuation

is determined by the availability of tyrptophanyl-tRNATrpand its transitory influ-ence over the formation of alternative secondary structures in the mRNA

DNA ⬊Protein Interactions and Protein⬊Protein Interactions Are Essential to Transcription Regulation

Quite a variety of control mechanisms regulate transcription in prokaryotes Several

organizing principles materialize First, DNA ⬊protein interactions are a central

fea-ture in transcriptional control, and the DNA sites where regulatory proteins bind commonly display at least partial dyad symmetry or inverted repeats Furthermore, DNA-binding proteins themselves are generally even-numbered oligomers (for ex-ample, dimers, tetramers) that have an innate twofold rotational symmetry Second,

Antiterminator

trp operon mRNA

Transcribing RNA polymerase

“Terminated”

RNA polymerase

Ribosome stalled at

tandem trp

codons

(b) Low tryptophan

High tryptophan

(a)

trpL mRNA

DNA encoding trp operon

Transcription terminator

Ribosome transcribing the leader peptide RNA

+

FIGURE 29.21 The mechanism of attenuation in the trp

operon.