(第1章第2部分)Prok.regulation(trp operon).pdf

Amino acid synthesis operons
• When certain AA is depleted from the
medium, the bacteria can start a specific
operon and make what it needed.
• When that AA is then added to the medium,
it will cause the operon for its synthesis to
turn off.
• This type of operons are called the -----
repressible operons

Anabolic enzymes that synthesize tryptophan
are in the same operon

分支酸 → 邻氨基苯甲酸 → 磷酸核糖基 → CDRP → 吲哚甘油-磷酸 → 色氨酸
邻氨基苯甲酸
邻氨基苯甲酸合成酶吲哚甘油色氨酸合成酶
硼酸合成酶
β链 α链
60，000 60，000 4 5，000 50，000 29，000
P O l a trpE trpD P trpC trpB trpA t t’
1560 1620 1353 1191 804 36
P：起动子；O：操纵子； l：前导序列； a：衰减子； t,t’ ：终止子
图 16-15 E.coli trpO 的结构及其产物所催化的色氨酸合成反应
羧苯氨基-脱氧核糖-5-磷酸

Regulation of trp Operon
two mechanisms
• The repressor/operator system: aporepressor coded
by trpR gene. its expression is separately controlled
(reduce transcription 70 times)
• The attenuation system: determines whether an
initiated transcription will be aborted or allows to
finish (reduce transcription 8-10 times)
• Together, trp operon can be regulated 560-700 times

Regulation of trp Operon
two mechanisms

trp Operon
The repressor/operator system:
1. Without tryptophan,
only aporepressor exits.
2. Tryptophan helps the
trp repressor bind to its
operator. Tryptophan is a
corepressor.
70-fold repression

Control of the trp Operon by Attenuation
after transcription is initiated
10-fold repression
The combination of repressor and attenuation shows 700-fold repression.

trp expression in Trp starvation
• Expression level depends on the severity of Trp
starvation: maximum expression under severe
starvation.
• Accomplished by a mechanism (attenuation) that
controls the ratio of full length mRNA (6962 bp)
to the truncated leader mRNA (140 bp)
• The more Trp there is, the more truncated RNA

The molecular model of attenuation
• Crucial to the attenuation model is the fact
that transcription and translation are
tightly coupled in prokaryotes, made
possible by the absence of a nuclear
envelope and the lack of processing of
mRNA transcripts.

The molecular events involved
the tight coupling of T/C and T/L
• trp mRNA T/C is paused when region 1 and 2 are synthesized
(which form a hairpin structure upon synthesis)
• The subsequent ribosome binding and synthesis of leader
peptide allows RNA polymerase to continue transcription
• The position of ribosome on leader transcript regulates the T/C
termination of attenuator

the organization the leader region

the organization the leader region
The 5’end of
leader mRNA
codes for a short
leader peptide,
and has multiple
Trp codons
Four regions of the leader mRNA can form alternative
internal hairpin structure that have different effects on
trp structure gene transcription.

Two Structures
available to the
leader-attenuator
transcript.
trp
trp
trp trp
Stop
codon
Stop
codon
(a)The more stable structure
(b) The less stable structure
(b)
(a)

Models for attenuation in the trp operon of E.coli

Trp starvation
• When Trp is scarce,
Trp-tRNATrp is not
available, which results
ribosome to stall at the
multiple Trp codon in
region 1
• Region 2 & 3 of leader
RNA then form a hairpin
structure, and prevents
the 3:4 hairpin structure
formation
• T/C continues.

Trp abundance
• Trp-tRNATrp is
available, leader peptide
synthesis is allowed to
finish at the stop codon.
Physically prevents the
2:3 hairpin formation.
• 3:4 hairpin structure
forms when region 4 is
synthesized. This is a
T/C termination signal--
--the attenuator
• T/C stops (aborted)

The genetic evidence
• Base substitutions in the regions 3 and 4.
• Mutations that makes
the hairpin structure less
stable, results in less
efficient T/C termination
at the attenuator

The genetic evidence
• Site directed mutagenesis of the multiple
Trp codons in the lead sequence
• when Trp codon is replaced with another
AA codon, the trp attenuation do not
respond to Trp concentration change,
instead, it will respond to the other AA’s
concentration.

Attenuation regulation is common
in many amino acid
biosynthetic operons

Gene regulation in
bacteriophages

The discovery of bacteriophage

1969年诺贝尔生理医学奖——
病毒的遗传结构

Nature. 2016. 533(7603):346-52.
噬菌体复杂的尾部结构包含有可以围绕在类似管状结构周围的收缩性鞘
状结构，这些鞘状结构可以在纳米尺度拉伸形成“线圈”结构，当噬菌
体吸附到细胞表面时，鞘状结构就会收缩并且驱动管状结构穿过细胞膜，
而所有的过程都是由噬菌体尾部百万个原子基底结构所完成的。

Phage therapy
• a bacteria eating submicroscopic agent
that can cure bacterial disease
• feasibility: use phage to cure infections
of bacteria?
• Gene-therapy

Proc. Natl. Acad. Sci. U.S.A. 104, 11197 (2007)
细菌生物被膜（或称细菌生物膜 Bacterial biofilm，
BF），是指细菌粘附于接触表面，分泌多糖基质等，
将其自身包绕其中而形成的大量细菌聚集膜样物。

Lambda Can Have Two Different Life Cycles
• In lysogenic cell, the
lytic pathway gene
are turned off by a
repressor-operator-
promoter circuit
How the initial
choice is made?

Is a bacteriophage codes for all the genes
it will need for any life cycle?
ØSmall genome, limited nucleic acid content
ØMost essential reproduction components are
provided by the host bacteria
ØPhage gene products take over the control
of host gene expression for its own use
ØPhage also code for genes that are related to
its life cycle and progeny production

The organization of  genome

Timed Expression of  Genes
There are three groups of  genes :
1 Immediate early genes: N, Cro (regulators for
delayed early genes)
2 Delayed early genes: replication, recombination,
and regulation (cII, O, P, Q and cIII)
3 Late genes: head tail proteins, cell lysis related
proteins

proteins

The  switch: lytic pathway or lysogeny
The delicate balance between:
 repressor vs Cro protein
Who will occupy the OL ,OR
 Repressor lysogeny
Cro protein  lytic growth

1. Immediate early genes: N, Cro (regulators for delayed early genes)
Rightward (PR): the Cro protein (control of repressor and
other)setting the genetic switch to the lytic pathway
leftward (PL): the N protein (transcription antiterminator) allow
RNA polymerase to read through N and Cro genes (produce all the
early genes )cII, O. P.and Q (antiterminator of late gene).
N and Cro proteins are
not stable--(one way of
post tranlational
regulation)

Effects of N on leftward
transcription
a. Map of N region of 
genome
b. Transcription in the absence
of N, only N is produced
c. Transcription in the presence
of N, delayed early genes are
produced besides the N protein

2. Delayed early genes: replication, recombination,

Delayed early genes
cII turns on:
cI (encodes the λ repressor),
Int (encodes the integrase required for
integrating the lambda chromosome in the
host chromosome during lysogenic pathway),
cII protein performs this function only when
the phage follows the lysogenic pathway

Delayed early genes
Gene O and P encode two DNA replication proteins:
Gene Q encodes a protein needed to turn on late genes
for lysis and phage particle proteins.
The Q protein is another antiterminator, permitting
transcription to continue into the late genes involved
in the lytic pathway.
However, only when the switch is set to the lytic
pathway and transcription continues from PR for a
sufficient time does enough Q protein accumulate to
function effectively

There are three groups of  genes :
and regulation (cII and cIII)
proteins

The First Genes to Be Expressed
there is no repressor cI in the beginning
L: leftward T/C. early
R: rightward T/C. Early
cro: control of repressor
and others
c: clear plaque
§ PL and PR is on
different DNA strands

Choice of the lysogenic pathway
• Early expression  CII
and CIII  activates CI
transcription (from PRE)

• CII protein is unstable
(by host protease)
• CIII protein stabilizes
CII protein by forming
complex with it
• CIII protein
stabilization of CII is a
function of growth
condition

CII and CIII Are Both Required
for Transcription Initiation at PRE

Choice of the lysogenic pathway
• CI gene product represses the
expression of genes involves in DNA
replication, phage assembly, and cell
lysis. establish repression

Repression of
 lytic Genes
in a Lysogenic
Cell
OL /OR overlap with their
corresponding PL/PR
Repressor binding sites:
17bp, two fold symmetry

cI Protein: the  Repressor
• C-terminal
domain for
dimerization

The lysogenic pathway
cI gene product: the  repressor
§ Binds to OR’ and OL’ , blocks PR and PL promoters (N and
Cro proteins not made).
§ Lack of N protein leads to the termination of O, P, Q
transcription lytic pathway is blocked.
§ OR1 binding cI product stimulates PRM and produce
more cI product (repressor)  maintain repressor Conc.
stay lysogenic

Autogenous Regulation of
 Repressor Synthesis
Has highest affinity for OL1
and OR1
Binding Cooperativity
betweem OL1 /OR1 and OL2
/OR2
Repressors at OR1 & OR2
Stimulate its own production
This autogenous regulation
enables the  lysogenic
state to be very stable
cIs at OR and OL can form a
long regulatory loop to
further tightly control cI
production

Change Lysogenic Cell to lytic Cell
Inducer: UV light  DNA
damage  SOS response:
 RecA activated (stimulates
the proteolytic autocleavage
of certain proteins: cI , LexA)
  repressor degradation 
PR repression is relieved 
Cro synthesized
 lytic pathway

Choice of the lytic pathway
When Cro is in
dominance, it binds to
OR3 & OL3,
ØPrevents repressor
synthesis,
ØAllows for the
transcription from PR
& PL  N, Cro, Q
synthesized  lytic

Cro protein and the OR sites
• Cro protein also binds to the
three OR sites as of cI protein
( repressor) , but with
reversed affinity
• Cro only acts as a negative
regulator
• Cro at OR3 site blocks 
repressor synthesis (PRM)--
switching to lytic pathway
• At very high [Cro], it blocks
PR , PL : cII, N and Cro (the
right early genes) Cro self regulation

(第1章第2部分)Prok.regulation(trp operon).pdf

(第1章第2部分)Prok.regulation(trp operon).pdf

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