Download I + Z

Regulation of gene expression
11-30-2015
Liver Cells
Brain Cells
In differentiated cell, the non-expressed genetic information is
deleted out (totally lost) or switch off (temporally inactivate)?
完全分化的體細胞仍然擁有完整的遺傳資訊！
Gurdon, J.B. & Uehlinger, V. Nature 210, 1240–1241 (1966)
它是誰？
Regulation of gene expression
• Transcriptional level
– control of transcription initiation.
• Post transcriptional level
– Alterative splicing
– Stability of mRNA
• Translational level
– control of translation initiation
• Post translational level
– Protein stability
– Reversible chemical modification
– Irreversible modification
The first case of gene expression regulation
at transcriptional level in E. coli
How lac operon was discovered
"diauxic
growth"
J. Monod, Recherches sur la Croissance des
Cultures Bactériennes, Hermann, Paris, 1941.
Beta-galactosidase activity
Two question: 1, how lactose induce beta-galactosidase activity?
2, how glucose suppress lactose induce enzyme activity?
Take home question:
What message you learn from this experiment?
Regulatory mutations in the lacI gene
Genotype
(lac Z )
b-galactosidase
-IPTG +IPTG
(lac A )
transacetylase
-IPTG +IPTG
Conclusion
I+Z+A+
I+Z-A+
<0.1 100
<0.1 <0.1
<1
<1
100
100
Inducible
I Z +A +
I+Z -A+ /F' I -Z +A+
100 100
<0.1 100
100
<1
100
200
Constitutive
I+ >I - action in trans
•The lacI gene encodes a trans-acting factor (protein)
needed for repression (repressor).
•Most lacI - mutants are constitutive.
Regulatory mutations in the operator
Genotype
I +o + Z +
I +o C Z +
I +o C Z + / F' I +o + Z
I +o C Z - / F' I +o + Z +
b-galactosidase
-IPTG +IPTG
<0.1
100
100
100
Conclusion
Inducible
Constitutive
100
<0.1
Constitutive
Inducible
100
100
Loss-of-function alleles of the operator confer a constitutive
phenotype on the operon. They are called oC.
The operator acts in cis, i.e. it affects the allele to which it is linked.
The allele of the operator that is in cis to the active reporter
gene is the dominant allele. The operator shows cis -dominance.
Regulatory mutations in the lacI gene
(lac Z )
b-galactosidase
-IPTG +IPTG
Genotype
s
I Z +A+
s
I Z +A+ /F' I+Z +A+
<0.1 <1
<0.1 1
(lac A )
transacetylase
-IPTG +IPTG
<1
<1
<1
1
Conclusion
Noninducible
s
I >I+ in trans
The lacI S allele is noninducible.
What is the possible mechanism?
Lactose Operon
• Structural genes
– lac z, lac y, & lac a
– Promoter
– Polycistronic mRNA
• Regulatory gene
– Repressor
• Operator
• Operon
• Inducer - lactose
Regulatory
Gene
i
Operon
p
o
z
y
a
DNA
m-RNA
Protein
Transacetylase
-Galactosidase
Permease
Lactose Operon
• Inducer -- lactose
– Absence
Absence of lactose
i
p
y
a
No lac mRNA
– Presence
• Negative control
z
Active
• Active repressor
• No expression
• Inactivation of
repressor
• Expression
o
Presence of lactose
i
p
o
z
y
a
Inactive
-Galactosidase Permease Transacetylase
Catabolite Repression
(Glucose Effect)
• Definition:
Control of an
operon by
glucose
• Catabolic
operons
Units of -galactosidase
- glucose
Glucose
added
+ glucose
Time (hr)
+ lactose
Mechanism of Catabolite Repression
• c-AMP
• CAP (CRP)
protein
• CAP-cAMP
complex
– Promoter
activation
• Positive
control
Absence of glucose
Adenyl cyclase
c-AMP
CAP
i
p
ATP
z
o
y
a
Active
Inactive
-Galactosidase Permease Transacetylase
Maximum expression
Mechanism of Catabolite Repression
• Glucose:cAMP
• CAP (CRP) protein
• No CAP-cAMP
complex
– No Promoter
activation
Presence of glucose
Adenyl cyclase
i
p
X
cAMP
CAP
o
z
ATP
y
a
Inactive
-Galactosidase Permease Transacetylase
Low level expression
lac regulatory region
Activator binding site
-72
Promoter
-52
TTTACA
TATGTT
-35
-10
a
cAMP-CAP
Operator
s
b
RNA polymerase
+1
b'
+11
Repressor
There are 3 lac operators and all three operators are important!
Lac repressor binds as a tetramer, with each operator is
contacted by a repressor dimer.
Why tetrameric repressor is better than dimeric
repressor ?
How eucaryotic gene expression
is regulated?
Same principle but more complicated!
Promoters, enhancers, silencers etc.
RNA polymerase is responsible for transcription,
but transcription factors turn genes on and off.
Transcription factors are proteins that recognize
and bind to a specific base sequence in DNA.
…AGCCTACCAAAAAAGGTTCCACG…
…TCGGATGGTTTTTTCCAAGGTGC…
Regulatory RNAs: Riboswitch;
RNA interference; microRNA and lncRNA
How RNA interference was
discovered?
An unexpected observation from
control experiment!
How to isolate genes for RNA interference in C. elegant?
Normal worm die
after a few days
Isolation of mutants which is alive under the same situation and then
identify which gene is mutated that make RNA interference lost !
What is miRNA?
37
The first miRNA was discovered in C. elegans.
lin-14(lf) mutations caused precocious development.
lin-14(gf) and lin-4 mutations caused retarded development.
wild type: embryo
preccious: embryo
retarded: embryo
L1
L2
L1
L2
L3
L1
L3
L4
L2
L4 adult
adult
L3…..
lin-14(lf); lin-4(lf) mutants are precocious.
LIN-14 is a repressor of embryo development
LIN-4 is a negative regulator of LIN-14
lin-14 gene (simplified)
loss-of-function
gain-of function
mutations in coding sequences mutations in 3’UTR
lin-4 encodes no protein. Only an RNA.
LIN-14 RNA always present, but protein decreases through larval
development (lin-14 protein plays as negative regulator for larval
development).
lin-4 required for LIN-14 decrease.
lin-4 RNA increases though larval development.
lin-4 RNA potentially could base pair with lin-14 3’ UTR.
lin-4
lin-14
LIN-14 protein
LIN-14 protein in
lin-14(gf) or lin4(lf)
lin-4
RNA
LIN-14 protein in
wild type
Developmental time
miRNA complex
contains Risc proteins
Characteristics of miRNAs
•
•
•
•
•
•
Small non-coding double stranded RNAs
Approximately 19-22 nt long
Repressive activity of complementary mRNAs
Regulate 30% of mammalian gene products
1 miRNA = hundreds of mRNAs
To date, nearly 8600 miRNA genes have been identified
among 73 eukaryotic organisms (plants and animals) and 15
viruses
• There are, for example 132 C. elegans, 78 Drosophila, 377
mouse, and 474 human miRNA genes
• Many are conserved between vertebrates and invertebrates
Conservation of miRNA sequence and structure
• Certain miRNAs are
highly conserved and
thus evolutionarily
ancient (e.g. let-7)
• Sequence conservation
must fulfill the require to
form a dsRNA hairpin
from which the miRNA is
processed by Dicer
Genomic Organization
miRNA processing
Microprocessor
Complex
Differences in miRNA Mode of Action
CORRELATION OF MIR EXPRESSION WITH PROGRESSION AND
PROGNOSIS OF GASTRIC CANCER*
PATIENTS: 181 patients from 2 cohorts (Japan)
CLASSIFICATION: Stages I-IV
Diffuse vs. Intestinal type
ANALYSIS:
• Custom miR microarray chip (Ohio State Univ.)
• miR expression in 160 paired samples
(tumor vs. non-tumor)
• Correlations of miR expression vs. stage,
type and prognosis (survival)
* Lancet Oncol. 11,136, 2010
MiRs AS PROGNOSTIC FACTORS: GASTRIC
CANCER SURVIVAL*
Intestinal-Type Gastric Cancer
miR-495
HAZARD RATIO
(disease free survival)
5
4
3.2
3
2
1
0
Stages
I-II
Stages
III-IV
HAZARD RATIO
(disease free survival)
10
9
8
7
6
5
4
3
2
1
0
miR-199
Let-7g
high
low
high
low
low
high
I-II
III-IV
I-II
III-IV
I-II
III-IV
What is lincRNAs?
Large intergenic noncoding RNAs (lincRNAs)
are emerging as key regulators of diverse cellular
processes.
Determining the function of individual lincRNAs
remains a challenge.
Recent advances in RNA sequencing (RNA-seq)
and computational methods allow for an
unprecedented analysis of such transcripts.
Cell nucleus is a highly organized structure just like a Rome city!
Long Noncoding RNAs May Alter Chromosome’s 3D Structure
24 MAY 2013 VOL 340 SCIENCE page 910
RNA world
• Carry information (DNA)
• Catalyze chemical reaction (protein enzyme)
• Nutrient sensor to control gene expression
(protein receptor)
• Broadly control gene expression through
mRNA stability, translational efficiency etc.
(protein activator or repressor)
• Global control nuclear and chromosome
structure.(Histone code)
Non-Coding RNA: Formerly known as “JUNK”
A Key to Eukaryotic Complexity?
Cell July 3, 2013. Page 26
The evolution of RNAi as a defence against
viruses and transposable elements
Phil. Trans. R. Soc. B (2009) 364, 99–115
RNAi, Antiviral After All! Science 342: 207; 2013
www.sciencemag.org/cgi/content/full/322/590
0/442/DC1