Chapter 10 The Operon Tsung-Luo Jinn. Gene expression controlled at:...

Chapter 10

The Operon

Tsung-Luo Jinn

Gene expression controlled at:

Transcription—initiation,termination, not elongation

Processing—in EuK RNA splicing, modification, transporting translation -- in ProK coupled transcription and translation Translation--like transcription

Genes products of:

Structural genes: functions as: structure proteins, enzymes

regulatory proteins

Regulatory genes: functions: a specific protein in regulation

of gene expression, by binding to particular sites on DNA

*cistrons

*constitutive expression!!

Gene regulatory mechanism:

Negative control: Repressor binds to operator, gene turn off

Positive control: Activator binds to operator, gene turn on

*inducible repressible

Negative control—Bacteria Repressor,Operator

**polycistronic mRNA

Positive control—ProK, EuKCis-acting sites, trans-acting factors

**monocistronic mRNA

Regulator bind to DNA ? Gene Expression

Positive regulation Negative regulation

YES

No ON

OFFON

OFF

Operon model Jacob and Monod, 1961The control of gene expression by induction and repression

How to control gene expression in Bacteria:

Two types of DNA sequences: Coding for protein—trans-acting proteins Non-coding—cis-acting sequences

** gene expression is controlled by: Interaction of the trans-factor with cis-element

Topics:

Regulation of Gene Expression

Metabolic regulation—Lac operone

Catabolite repression

Stringent response

Attenuation—Trp operon

Gene Regulation

Negative controlPositive control

Metabolic regulation

lec operonThe

The lac operon controls utilization of Lactose

Coding region

Control region

Regulatory gene

operon

4er dimer

How lac I gene control lac ZYA gene transcription ?

Negative regulation--

lac genes are transcribed unless it was turned off by a repressorprotein

Repressor–-lac I product

Inducer—lactose (allolactose), IPTG

*isopropyl thioglactoside

Induction of gene expression in lac operon

Inducers

Induction of lac genes expression --- by inducer

Induceable enzyme—From ~ 5 to 5,000 molecular

Shot life of mRNA—Half-life ~3 min

Stable of enzyme

* Is de novo synthesis !!!

Gratuitous inducer—IPTG

Mutations: O-type and I-type

Repressor:I---loss of function mutant --Constitutive expression --trnas-dominant

Opertor: Oc

--Constitutive expression-- Cis-dominant

LacIIS--abolish the ability of bind to inducer --Uninducible

I-d--loss of function to bind the operator --Constitutive expression

I- Z+/ I+Z+

O+ Z+/ OCZ+

OC Z+/ I-Z+

I+ Z+/ I-Z+

OC Z-/ O+Z+

OC Z+/ O+Z+

OC Z+/ O+Z-

Gene types Constitutive Inducible

-

+

++

+

-

-

-

-

-

-

++

+

*Interallelic complementation

Conclusions

Map of mutation sites

The operator is palindromic—with inverted repeats

TGTGTG------------------------------------------TGTGTG

Two models for repressor action

The crystal structure of a monomer of Lac repressor

HTHheadpiece

core

C-terminal Oligomeriation

Inducer biding

Operator biding

i

p

z

O3O2

CAP

O1

Three operator sequences

O2

O1

O3

: the original operator

: 410 bp downstream in lacZ

: 83 bp upstream in lacI

dimer

* This structure enhanced RNA polrmerase binding (100x)/store at promoter

Repressor tetramer binds to

The trp repressor controls three unlinked sets of genes

* Autogenous control

Operators lie on different positions relative to promoter

Catabolite repression

Medium with Glucose and Lactose—

It metabolizes glucose and represses the use of lactose—Catabolite repression * How??

ATP cAMPAdenyl cyclase

Glucose×

CAP

cAMP-CAP complex

Gene turn on

Positive control of gene expression

*mutations: I-,Oc

CAP: a product of Cap gene

No lac mRNA

cyclase: a product of Cya gene

promoter

Cya-, Cap-

The CAP dimer binding site with a conserved TGTAG pentamer

CAP biding to different region relative to promoter

CAP cause DNA to bend

The Stringent Response

Adverse growth condition induce

The Stringent Response

Massive reduction in the synthesis ofrRNA and tRNA (10-20X) Reduced mRNA synthesis (~3X)

Increase protein degradsation

Reduce synthesis of nucleotides, carbonhydrates and lipids, ect

Amino acid starvation

Accumulation o ppGpp and pppGpp nucleotides

The synthesis of ppGpp under Amino acid starvation

Function: is to bind to target proteins to alter their activity

Trigger:uncharged tRNA in the A site of ribosome

P AP A

What does ppGpp do?

Initiation is specifically inhibited at the promoters of operons coding for rRNA

The elongation of transcription of many or MOST template is reduced

*ppGpp is a broad spectrum inhibitors

Autogenous control at translation level

Autogenous control at translation level

Attenuation of gene expression

The Trp operon

Attenuation: a mechenism that controls the ability of RNA polymerase to read through an attenuator, which is an intrinsic terminator located at the beginning of a transcription unit.

The changes in secondary structure that control attenuation are determined by the position of the ribosome on mRNA.

The external circumstances is influenced ribosome movement in the leading sequence of mRNA.

The trp operon

Repressor: trpR gene coded in different locus（ inactive）

Corepressor: trytophane

With two different separate mechanism

Attenuation control

The trp operon—leader sequence

Chorismic acid to tryptophane

11 12

The structure on the leader region

W/ trpW/O trp

12 3 4

1 2

3 4

1 2 3

4

21

3 4

tRNAtrp

【 Trp 】

（ ）

Antisense RNA regulates transcription/translation

Prevent initiation of protein synthesis

Destabilization of dRNA by endonuclease

Premature termination of transcription

RNAi

PNAS (2,000) 97:4885-4990