Gene Regulation

1
Chapter 16

• Gene Regulation
• in Prokaryotes

2
Outline
Part 1 Principles of Transcriptional Regulation
Part 2 Regulation of Transcription Initiation
Part 3 Examples of Gene Regulation after
Transcription Initiation
3
Part 1 Principles of Transcriptional Regulation
4
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

5

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

6
Fig 16-1
a. Absence of Regulatory Proteins (operator)
b. To Control Expression
c. To Activate Expression
7
1-3 Targeting transition to the open complex
Some Activators Work by Allostery and Regulate
Steps after RNA Polymerase Binding
Fig 16-2
8
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
9
Fig 16-4 DNA-binding protein can facilitate
interaction between DNA-binding proteins at a
distance
10
1-5 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
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)
11
Part 2 Regulation of Transcription Initiation
Examples from Bacteria
12
2-1 Example from bacteriaLac operon
The lactose (Lac) Operon (?????)
13
Lactose operon a regulatory gene and 3
stuctural genes, and 2 control elements
Regulatory gene
Structural Genes
Cis-acting elements
DNA
lacI
lacZ
lacY
lacA
PlacI
Olac
Plac
m-RNA
Protein
ß -Galactosidase
Transacetylase
Permease
14
codes for ß-galactosidase (?????) for lactose
hydrolysis
lacZ
encodes a cell membrane protein called lactose
permease (???????) to transport Lactose across
the cell wall
lacY
encodes a thiogalactoside transacetylase
(??????????)to get rid of the toxic
thiogalacosides
lacA
15
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.
16
Fig 16-6
17

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

1.Lac 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
18
The lac operator overlaps promoter, and so
repressor bound to the operator physically
prevents RNA polymerase from binding to the
promoter.
Fig 16-8
19
2-3 CAP has separate activating and DNA-binding
surfaces
Fig 16-9
a CTD C-terminal domain of the a subunit of RNAP
20
2-4 CAP and lac repressor bind DNA using a
common structural motif
21
Cap use the strucure called helix-turn-helix
The helix-turn-helix
22
lac repressor alse use the same mechanism
Fig 16-12 Hydrogen Bonds between l repressor and
the major groove of the operator
23

• 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

24
The Difference

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

Fig 16-13
25

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

26
Response to lactose
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, andß-galactosidase
catalyzes the conversion of some lactose to
allolactose. Allolactose acts as an inducer,
binding to the lac repressor and inactivate
it.
Presence of lactose
i
p
o
z
y
a
Inactive
Permease
Transacetylase
b-Galactosidase
27
Response to glucose
28
2-6 Combinatorial Control (????) CAP controls
other genes as well

• 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.

29
EXAMPLE TWO---- ALTERNATIVE sFACTORS
2-7 Alternative s factor direct RNA polymerase to
alternative site of promoters
30
Recall from Chapter 12 that it is the ssubunit of
RNA polymerase that recognizes the promoter
suquences.
31
Promoter recognition

• Different s factors bind to the RNA recognize the
  promoter sequence ,for example s70. s32

32
Third example NtrC and MerR and allosteric
activation
5/10/2005
33
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
34
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
35
The major process
Low nitrogen levels
Trigger polymerase to initiate transcription
NtrB phosphorylates NtrC
ATP hydrolysis and conformation change in
polymerase
NtrCs DNA-binding domain revealed
NtrC binds four sites located some 150 base pairs
upstream of the promoter
NtrC interacts with ?54
36

• 2-9 MerR activates transcription by twisting
  promoter DNA

37
MerR bound to the single DNA-binding site, in the
presence of mercury MerR activates the MerT gene.
And the Mert twists the DNA.
38
Fig 16-15 Structure of a merT-like promoter
39
2-10 Some repressors hold RNA polymerase at the
promoter rather than excluding it
40
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)

41
Fourth example araBAD operon
42
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.

43
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
44

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

45
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
46

• 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

47
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

48
Attenuation

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

49
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

50
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
51
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.
52
Figure 16-21 transcription at the trp attenuator
53
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

54
Ribosomal protein operons
55

• 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.

56
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.

57
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
58

• Part four
• The case of phage ? layers of regulation

59
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

60
Lysogenic induction

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

61
Lytic cycleand Establishment of lysogeny

• Figure 16-24

62
4-1 Alternative patterns of gene expression
control lytic and lysogenic growth

• Figure 16-25

63
Promoters in the right and left control regions
of phage ?
64
Transcription in the ?control 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
65
4-2 Regulatory Proteins and Their Binding Sites

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

66
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.
67
Relative positions of promoter and operator sites
in OR
68
The action of ? repressor and Cro

• Figure 16-31

69
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.

70
Negative autoregulation of repressor require
long-distance interations and a large DNA loop
71
Another Activator, ?cII, 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.

72
Interaction between the c-terminal domain of ?
repressors

• Figure 16-33

73
Growth conditions of E.coil control the stability
of CII protein and thus/lysogenic choice
74

• Transcriptional Antitermination in ? Development

The transcripts controlled by ?N 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 ?N and Q protein are
therefore called antiterminators.
75

• Figure 16-36

76
Retoregulationan interplay of control on RNA
synthesis and stability determines int gene
expression
77
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