lecture 12

(1)

Genetics: Analysis and Principles

(2)

INTRODUCTION



The term

gene regulation

means that the level of

gene expression can vary under different conditions



Genes that are unregulated are termed

constitutive

 They have essentially constant levels of expression

 Frequently, constitutive genes encode proteins that are

necessary for the survival of the organism



The benefit of regulating genes is that encoded

proteins will be produced only when required

14-2

(3)

INTRODUCTION



Gene regulation is important for cellular processes

such as

 1. Metabolism

 2. Response to environmental stress

 3. Cell division



Regulation can occur at any of the points on the

pathway to gene expression

 Refer to Figure 14.1

14-3

(4)

14-4

(5)

 The most common way to regulate gene expression in

bacteria is at the transcriptional level

 The rate of RNA synthesis can be increased or decreased

 Transcriptional regulation involves the actions of two main

types of regulatory proteins

 Repressors  Bind to DNA and inhibit transcription

 Activators  Bind to DNA and increase transcription

 Negative control refers to transcriptional regulation by

repressor proteins

 Positive control to regulation by activator proteins

14.1 TRANSCRIPTIONAL

REGULATION

(6)

 Small effector molecules affect transcription regulation

 However, these bind to regulatory proteins and not to DNA directly

 In some cases, the presence of a small effector molecule

may increase transcription

 These molecules are termed inducers  They function in two ways

 Bind activators and cause them to bind to DNA

 Bind repressors and prevent them from binding to DNA

 Genes that are regulated in this manner are termed inducible

 In other cases, the presence of a small effector molecule

may inhibit transcription

 Corepressors bind to repressors and cause them to bind to DNA  Inhibitors bind to activators and prevent them from binding to DNA  Genes that are regulated in this manner are termed repressible

(7)

Figure 14.2

14-7

 Regulatory proteins have

two binding sites

 One for a small effector molecule

(8)

Figure 14.2

(9)



At the turn of the 20

th

century, scientists made the

following observation

 A particular enzyme appears in the cell only after the cell

has been exposed to the enzyme’s substrate

 This observation became known as enzyme adaptation



François Jacob and Jacques Monod at the Pasteur

Institute in Paris were interested in this phenomenon

 They focused their attention on lactose metabolism in

E. coli to investigate this problem

The Phenomenon of Enzyme

Adaptation

(10)



An operon is a regulatory unit consisting of a few

structural genes under the control of

one

promoter

 It encodes polycistronic mRNA that contains the coding

sequence for two or more structural genes

 This allows a bacterium to coordinately regulate a group

of genes that encode proteins with a common function



An operon contains several different regions

 Promoter; terminator; structural genes; operator

The

lac

Operon

(11)

14-11

 Figure 14.3a shows the organization and transcriptional

regulation of the lac operon genes

 There are two distinct transcriptional units

 1. The actual lac operon

 a. DNA elements

 Promoter  Binds RNA polymerase

 Operator  Binds the lac repressor protein

 CAP site  Binds the Catabolite Activator Protein (CAP)

 b. Structural genes

 lacZ  Encodes -galactosidase

 Enzymatically cleaves lactose and lactose analogues

 Also converts lactose into allolactose (an isomer)

 lacY  Encodes lactose permease

 Membrane protein required for transport of lactose and analogues

 lacA  Encodes transacetylase

 Covalently modifies lactose and analogues

 Its functional necessity remains unclear

(12)

14-12

 Figure 14.3a shows the organization and transcriptional

regulation of the lac operon genes

 There are two distinct transcriptional units

 2. The lacI gene

 Not considered part of the lac operon

 Has its own promoter, the i promoter

 Constitutively expressed at fairly low levels

 Encodes the lac repressor

 The lac repressor protein functions as a tetramer

 Only a small amount of protein is needed to repress the lac operon

(13)

14-13

(14)



The

lac

operon can be transcriptionally regulated

 1. By a repressor protein

 2. By an activator protein



The first method is an inducible, negative control

mechanism

 It involves the lac repressor protein

 The inducer is allolactose

 It binds to the lac repressor and inactivates it

The

lac

Operon Is Regulated By

a Repressor Protein

(15)

14-15

Figure 14.4

Therefore no allolactose

Constitutive expression

RNA pol cannot access

the promoter

The lac operon is now

(16)

14-16

The conformation of the repressor is now altered

Some gets converted to allolactose Repressor can no longer

bind to operator

Translation The lac operon is now

(17)

14-17

The cycle of lac operon induction and repression Figure 14.5

Repressor does not completely inhibit transcription

(18)



In the 1950s, Jacob and Monod, and their colleague

Arthur Pardee, had identified a few rare mutant

strains of bacteria with abnormal lactose adaptation



One type of mutant involved a defect in the

lacI

gene

 It was designated lacI–

 It resulted in the constitutive expression of the lac operon

even in the absence of lactose

 The lacI– mutations mapped very close to the lac operon

Experiment 14A: The

lacI

Gene

Encodes a Repressor Protein

(19)



Jacob, Monod and Pardee proposed two different

functions for the

lacI

gene

Figure 14.6



Jacob, Monod and Pardee applied a genetic

(20)



They used bacterial conjugation methods to

introduce different portions of the

lac

operon into

different strains



They identified F’ factors (plasmids) that carried

portions of the

lac

operon



For example: Consider an F’ factor that carries the

lacI

gene

 Bacteria that receive this will have two copies of the lacI

gene

 One on the chromosome and the other on the F’ factor

(21)



Merozygotes were instrumental in allowing Jacob

et al

to elucidate the function of the

lacI

gene



There are two key points

 1. The two lacI genes in a merozygote may be different

alleles

 lacI– on the chromosome  lacI+ on the F’ factor

 2. Genes on the F’ factor are not physically connected to

those on the bacterial chromosome

 If hypothesis 1 is correct

 The repressor protein produced from the F’ factor can diffuse and regulate the lac operon on the bacterial chromosome

 If hypothesis 2 is correct

(22)

The Hypothesis



The

lacI

gene either/or

 1. Encodes a regulatory protein (the lac repressor)

that can diffuse throughout the cell

 2. Acts as a binding site for a repressor protein

 Thus it can only act on a physically connected lac operon

Testing the Hypothesis



Refer to Figure 14.7

(23)

14-23

(24)

(25)

(26)

The Data

14-26

Strain Addition of lactose Amount of -galactosidase (percentage of parent strain)

Mutant No 100%

Mutant Yes 100%

Merozygote No <1%

(27)

Interpreting the Data

14-27

Strain Addition of lactose Amount of -galactosidase (percentage of parent strain)

Mutant No 100%

Mutant Yes 100%

Merozygote No <1%

Merozygote Yes 220%

Expected result because of constitutive expression in the

lacI–_strain

In the absence of lactose, both lac

operons are repressed

In the presence of lactose, both lac

operons are induced, yielding a higher level of enzyme activity

This result is consistent with hypothesis 1

(28)

Interpreting the Data

14-28



The interaction between regulatory proteins and DNA

sequences have led to two definitions

 1. Trans-effect

 Genetic regulation that can occur even though DNA segments are

not physically adjacent

 Mediated by genes that encode regulatory proteins

 Example: The action of the lac repressor on the lac operon

 2. Cis-effect or cis-acting element

 A DNA sequence that must be adjacent to the gene(s) it regulates  Mediated by sequences that bind regulatory proteins

(29)

Interpreting the Data

14-29



Table 14.1 summarizes the effects of

lacI

gene

mutations versus

lacO

(operator) in merozygotes



Overall

 A mutation in a trans-acting factor is complemented by

the introduction of a second gene with a normal function

(30)

14-30

(31)



The

lac

operon can be transcriptionally regulated in

a second way, known as

catabolite repression



When exposed to both lactose and glucose

 E. coli uses glucose first, and catabolite repression

prevents the use of lactose

 When glucose is depleted, catabolite repression is

alleviated, and the lac operon is expressed



The sequential use of two sugars by a bacterium is

termed

diauxic growth

The

lac

Operon Is Also Regulated

By an Activator Protein

(32)



The small effector molecule in catabolite repression

is not glucose



This form of genetic regulation involves a small

molecule,

cyclic AMP

(cAMP)

 It is produced from ATP via the enzyme adenylyl cyclase

 cAMP binds an activator protein known as the Catabolite

Activator Protein (CAP)

 Also termed the cyclic AMP receptor protein (CRP)

The

lac

Operon Is Also Regulated

By an Activator Protein

(33)



The cAMP-CAP complex is an example of genetic

regulation that is inducible and under positive control

 The cAMP-CAP complex binds to the CAP site near the

lac promoter and increases transcription



In the presence of glucose, the enzyme adenylyl

cyclase is inhibited

 This decreases the levels of cAMP in the cell

 Therefore, cAMP is no longer available to bind CAP

 Transcription rate decreases

The

lac

Operon Is Also Regulated

By an Activator Protein

(34)

Figure 14.8

(35)

(36)



Detailed genetic and crystallographic studies have

shown that the binding of the

lac

repressor is more

complex than originally thought



In all, three operator sites have been discovered

 O

1  Next to the promoter

 O

2  Downstream in the lacZ coding region

 O

3  Slightly upstream of the CAP site



Refer to Figure 14.9

The

lac

Operon Has Three

Operator Sites for the

lac

Repressor

(37)

14-37

The identification of three lac operator sites Figure 14.9

Repression is 1,300 fold

Therefore, transcription is 1/1,300 the level when lactose is present

(38)



The results of Figure 14.9 supported the hypothesis

that the

lac

repressor must bind to two of the three

operators to cause repression

 It can bind to O

1 and O2 , or to O1 and O3

 But not O

2 and O3

 If either O

2 or O3 is missing maximal repression is not

achieved



Binding of the

lac

repressor to two operator sites

requires that the DNA form a loop

 A loop in the DNA brings the operator sites closer together

 This facilitates the binding of the repressor protein



Refer to Figure 4.14

(39)

14-39

Figure 14.10

Each repressor dimer binds to one

operator site Each repressor

(40)



Another operon in

E. coli

that is involved in sugar

metabolism is the

ara

(arabinose) operon



It contains

 Three structural genes involved in arabinose metabolism

 These are designated araB, araA and araD

 A single promoter, P

BAD

 A CAP site, which binds the catabolite activator protein



Refer to Figure 14.11

The

ara

Operon

(41)

14-41

 The araC gene is adjacent to the ara operon

 It has its own promoter, P

C

 It encodes a regulatory protein, AraC

 AraC can bind to three different operator sites

 Designated araI, araO

1 and araO2

The AraC protein can act as either a negative or positive regulator of

transcription

(42)

 AraC protein binds to all three operators

 AraC dimer bound to araO

1inhibits transcription of the araC gene

 This keeps AraC protein levels fairly low

 AraC monomers bound to araO

2and araIrepress the ara operon

 They bind to each other (via looped DNA), and block RNA pol access to P

BAD

araO₁

Figure 14.12

(43)

 Arabinose binds to the AraC proteins

 The interaction betweem the AraC proteins at the araO

2 and araI is broken

 This breaks the DNA loop

 Another, AraC protein binds to araI

 This AraC dimer at araI activates transcription

14-43

CAP-cAMP activation occurs when glucose levels are low

(44)



The

trp

operon (pronounced “trip”) is involved in the

biosynthesis of the amino acid tryptophan

 The genes trpE, trpD, trpC, trpB and trpA encode

enzymes involved in tryptophan biosynthesis

 The genes trpR and trpL are involved in regulation

 trpR  Encodes the trp repressor protein

 Functions in repression

 trpL  Encodes a short peptide called the Leader peptide

 Functions in attenuation

The

trp

Operon

(45)

14-45

Organization of the trp operon and regulation via the trp

repressor protein Figure 14.13

Cannot bind to the operator site RNA pol can bind

(46)

14-46

(47)

14-47

repressor protein Figure 14.13

(48)

 Attenuation occurs in bacteria because of the coupling of

transcription and translation

 During attenuation, transcription actually begins but it is

terminated before the entire mRNA is made

 A segment of DNA, termed the attenuator, is important in facilitating

this termination

 In the case of the trp operon, transcription terminates shortly past

the trpL region (Figure 14.13c)

 Thus attenuation inhibits the further production of tryptophan

 The segment of trp operon immediately downstream from

the operator site plays a critical role in attenuation

 The first gene in the trp operon is trpL

 It encodes a short peptide termed the Leader peptide

(49)

14-49

Sequence of the trpL mRNA produced during attenuation Figure 14.14

These two codons provide a way to sense if there is sufficient

tryptophan for translation

The 3-4 stem loop is followed by a sequence

of Uracils

 Region 2 is complementary to regions 1 and 3  Region 3 is complementary to regions 2 and 4

 Therefore several stem-loops structures are possible

(50)



Therefore, the formation of the 3-4 stem-loop

causes RNA pol to terminate transcription at the

end of the

trpL

gene



Conditions that favor the formation of the 3-4

stem-loop rely on the translation of the

trpL

mRNA



There are three possible scenarios

 1. No translation

 2. Low levels of tryptophan

 3. High levels of tryptophan

(51)

14-51

Possible stem-loop structures formed from trpL mRNA under different conditions of translation

Figure 14.15

Therefore no coupling of transcription and translation Most stable form of

mRNA occurs

(52)

14-52

Figure 14.15

Insufficient amounts of tRNAtrp

Region 1 is blocked

3-4 stem-loop does not form

(53)

14-53

Figure 14.15

Sufficient amounts of tRNAtrp

Translation of the trpL mRNA progresses until stop codon

Region 2 cannot base pair with any other region

3-4 stem-loop forms

(54)



The study of many operons revealed a general trend

concerning inducible versus repressible regulation

 Operons involved in catabolism (ie. breakdown of a

substance) are typically inducible

 The substance to be broken down (or a related compound) acts

as the inducer

 Operons involved in anabolism (ie. biosynthesis of a

substance) are typically repressible

 The inhibitor or corepressor is the small molecule that is the

product of the operon

Inducible

vs

Repressible Regulation

(55)



Genetic regulation in bacteria is exercised

predominantly at the level of transcription

 However, there are many examples of regulation that

occur at a later stage in gene expression



For example, regulation of gene expression can be

 Translational

 Posttranslational

14.2 TRANSLATIONAL AND

POSTTRANSLATIONAL

REGULATION

(56)



For some bacterial genes, the translation of mRNA

is regulated by the binding of proteins



A

translational regulatory protein

recognizes

sequences within the mRNA



In most cases, these proteins act to inhibit

translation

 These are known as translational repressors

Translational Regulation

(57)



Translational repressors inhibit translation in one of

two ways

 1. Binding next to the Shine-Dalgarno sequence and/or

the start codon

 This will sterically hinder the ribosome from initiating translation

 2. Binding outside the Shine-Dalgarno/start codon region

 They stabilize an mRNA secondary structure that prevents initiation



Translational repression is also found in eukaryotes

Translational Regulation

(58)



A second way to regulate translation is via the

synthesis of

antisense RNA

 An RNA strand that is complementary to mRNA



Consider, for example, the trait of osmoregulation

 The ability to control the amount of water inside the cell

 The protein ompF in E. coli is important in osmoregulation

 This outer membrane protein is encoded by the ompF gene  OmpF protein is preferentially produced at low osmolarity

 At high osmolarity its synthesis is decreased

Translational Regulation

(59)

14-59

Figure 14.16

 The expression of another gene, termed micF, is responsible

for inhibiting the ompF gene at high osmolarity

 micF RNA does not code for a protein

 It is, however, complementary to ompF mRNA

 It is thus termed antisense RNA

(60)



A common mechanism to regulate the activity of

metabolic enzymes is

feedback inhibition



The final product in a pathway often can inhibit the

an enzyme that acts early in the pathway



Refer to Figure 14.17

Posttranslational Regulation

(61)

14-61

 Enzyme 1 is an allosteric enzyme,

with two different binding sites  Catalytic site  binds substrate

 Regulatory site  binds final

product of the pathway

 If the concentration of product 3

becomes high

 It will bind to enzyme 1

 Thereby inhibiting its ability to

(62)



A second strategy to control the function of proteins

is by the covalent modification of their structure



Some modifications are irreversible

 Proteolytic processing

 Attachment of prosthetic groups, sugars, or lipids



Other modifications are reversible and transiently

affect protein function

 Phosphorylation (–PO

4)

 Acetylation (–COCH

3)

 Methylation (–CH

3)

Posttranslational Regulation

(63)



Bacteriophages are viruses that infect bacteria

 Their study has greatly advanced our basic knowledge of

genetic regulation



The structural genes of bacteriophages are often in

an operon arrangement

 Like bacterial operons, phage operons can be controlled

by repressor proteins or activator proteins



To understand how this works, we will examine the

two life cycles of phage



(lambda)

14.3 GENE REGULATION IN THE

BACTERIOPHAGE LIFE CYCLE

(64)



Phage



can bind to the surface of a bacterium and

inject its genetic material into the bacterial cytoplasm



The phage will then proceed along only one of two

alternative life cycles

 Lytic cycle

 Lysogenic cycle



Let’s review Figure 6.9

Life Cycles of Phage



(65)

Figure 6.9

Virulent phages only undergo a lytic cycle

Temperate phages can follow both cycles

14-65

Prophage can exist in a dormant

state for a long time

It will undergo the lytic cycle

This process is termed

(66)

 Figure 14.18 shows the genome of phage 

 Inside the viral head, phage  DNA is linear

 After injection into the bacterium, the two ends attach covalently to

each other forming a circle

 The organization of the genes within this circular structure

reflects the two alternative life cycles of the virus

 The genes in the top center are transcribed very soon after infection,

at the beginning of either life cycle

 The pattern of their expression determines which of the two cycles prevails

 The genes on the left side of the viral genome encode proteins that are

responsible for the lysogenic infection

 The genes on the right side of the viral genome encode proteins that

are responsible for the lytic infection

(67)

14-67

(68)



So how is the decision made between the lytic and

lysogenic cycles?



The choice depends on the actions of several

genetic regulatory proteins



The process is quite detailed

 It involves a series of intricate steps in which these

proteins bind to several different sites in the  genome



Refer to Figure 14.19

Life Cycles of Phage



(69)

14-69

Encoding two proteins: N and cro The N protein is an antiterminator

It binds to RNA polymerase and prevents transcriptional termination

cII gene encodes an activator protein The O and P genes

encode enzymes needed to initiate DNA synthesis

The Q gene encodes another antiterminator needed for the lytic cycle

cIII gene encodes a protein that helps stabilize the

cII activator protein

(70)

14-70

cII/cIII activates transcription from P_I and P_RE

P_RE= Promoter for  Repressor during

Establishment of lysogenic cycle

int gene encodes the protein integrase, which integrates  DNA into the bacterial chromosome

The  repressor binds to operators that are adjacent to P_R and P_L

It thus inhibits the expression of genes required for the lytic cycle

The  repressor also activates P_RM

This is sufficient to make enough

Repressor to Maintain the lysogenic cycle

(71)

14-71

Figure 14.19

The cro protein binds to two operators O_R and O_L

Binding to O_L inhibits transcription from P_L

Binding to O_R has several effects

1. It inhibits transcription from P_RM in the

leftward direction

•This prevents the expression of the cI

gene which encodes the  repressor 2. It allows a low level of transcription

from P_R in the rightward direction

• This enables the transcription of the O,

P and Q genes

The Q protein is an antiterminator that permits transcription through another

promoter, P_R’

P_R’ controls a very large operon that encodes the proteins necessary for the assembly of the phage coat, packaging of the DNA and lysis of the bacterial cell The O and P proteins are necessary

for the replication of 

(72)



The activity of the cII protein plays a key role in

directing



to the lysogenic or lytic cycle



The cII protein is easily degraded by cellular

proteases produced by

E. coli



Whether or not these proteases are produced

depends on the environmental conditions

Cellular Proteases Influence the

Choice Between the Two Cycles

(73)



If the growth conditions are very favorable, the

intracellular levels of the proteases are high

 The cII protein tends to be degraded

 Therefore, P

RE cannot be activated and the  repressor is not made

 Instead, the cro protein slowly accumulates to high levels

 The binding of the cro protein to O

R prevents transcription of the 

repressor from P_RM

 At the same time, the cro protein allows the lytic cycle to proceed



Thus, environmental conditions that are favorable for

growth promote the lytic cycle

 This makes sense because a sufficient supply of nutrients

is necessary to synthesize new bacteriophages

(74)



If the nutrients are limiting (starvation conditions),

the cellular proteases are relatively inactive

 The cII protein builds up much more quickly than cro

 Therefore, the cII protein will turn on P

RE

 The  repressor is made



Thus, environmental conditions that are unfavorable

for growth promote the lysogenic cycle

 This makes sense because there may not be sufficient

nutrients for the production of new bacteriophages

(75)



After lysogeny is established, certain environmental

conditions can also favor induction to the lytic cycle



For example, exposure to UV light

 recA (a cellular protein normally involved in DNA

recombination) detects the DNA damage

 It is activated to become a protease

 It cleaves the  repressor and inactivates it

 This allows transcription from P

R

 Therefore, the cro protein will accumulate

 Favoring the lytic cycle

 This makes sense, because the exposure to UV light may

have already damaged the bacterium to the point where further bacterial growth and division are prevented

(76)



To understand how this switch works, we need to

take a closer look at the

O

_R

region

 The O

R region contains three operator sites, designated

O_R1, O_R2, and O_R3

 These operator sites control two promoters, P

R andPRM, which

transcribe in opposite directions

 The  repressor protein or the cro protein can bind to any

or all of the three operator sites

 This binding governs the switch between the lysogenic and the

lytic cycles

The

O

_R

Region Provides a Genetic

Switch Between the Two Cycles

(77)



Two critical issues influence this binding

 1. The relative affinities that the regulatory proteins have

for these operator sites

 2. The concentrations of these regulatory proteins in the

cell



Refer to Figure 14.20

The

O

_R

Region Provides a Genetic

Switch Between the Two Cycles

(78)

14-78

Figure 14.20

 repressor has the highest affinity to O_R1

then O_R2 then O_R3

 repressor is a dimer

cooperative interactionvia

This binding inhibits transcription from P_R

So the lytic cycle is switched off

 repressor falls off

O_R3 first

Cro protein has the highest affinity to O_R3

and simiar affinity to

O_R2 then O_R1

cro protein is a dimer

This binding blocks transcription from P_RM

So the lysogenic cycle is switched off

P_R is not needed in

the later stages of

(79)



Genetic switches, like the one just described in

phage



, are also important in the developmental

pathways of bacteria and eukaryotes



For example

 The choice between sporulation and vegetative growth in

bacteria

 Initiation of cell differentiation during development in

eukaryotes