PLANT GENETICS and MOLECULAR BIOLOGY
Department of Cell Biology & Molecular Genetics
Inducible Systems- Gene regulation in response to internal or external cues
I. Levels of regulation
Transcription initiation or elongation
RNA processing, RNA export
mRNA stability
Translation initiation or elongation
Protein stability
Enzymatic activity
II. Identifying induced genes
A. Induced trait -> clone gene
B. Differential cloning (e.g. subtractive library)
C. cDNA array screening
III. Generalized Components- in transcriptional regulation
A. Receptor- protein or molecule which can perceive a chemical or physical
stimulus into a biochemical
-often will bind chemical signals
-in many systems, dimerization of two receptors when they bind the
compound starts the signal pathway
B. transcriptional activator- protein which can bind to the appropriate gene to
be turned on and influence the transcription of that gene
Important properties:
-Ability to specifically bind DNA- amino acid motifs which allow close amino acid-DNA
nucleotide contact for hydrogen bonding recognition of sequence
e.g. helix-turn-helix, Zn finger, Basic/ leucine zipper (dimerization)
-Ability to influence transcription initiation
-bound factors may recruit certain parts of RNA polymerase to that promoter
e.g. TATA binding protein (TFIID)
-may act through an intermediary protein
-bound factor may influence histone deacetylase in surrounding sequences
-acetylated histones are more loosened,
-deacetylated histones tighter, associated with repression of genes
Often act in synergy with other transcription facotrs on a promoter
C. Intermediary signaling- how signal gets from receptor to transcriptional activator
- influence by protein-protein interactions- ability of protein to recognize
specific other proteins and bind
-protein phosphorylation- modifies enzymatic activity of
phosphorylated/dephosphorylated protein (e.g. turn on DNA binding activity
of a transcriptional activator)
-kinase- adds phosphate phosphatase- removes phosphate
-secondary messengers- small molecules which can carry message
-e.g. cAMP, Ca++, Inositol 3-Phosphate
- receptor influences production of secondary messenger
-level of secondary messenger influence other component-e.g. a protein kinase
IV. Example- Ethylene Perception in plants
A. Plant responses influenced by ethylene
-fruit ripening, senescence, mechanical stress,
-cell elongation
-triple response
B. Genetic approach to identify components
Ethylene-insensitive mutants- ETR1, EIN2-7
Constitutive mutants: CTR1
Epistasis: ethylene -> ETR1 -| CTR1 -| EIN2 -> [EIN3, EIN5] -> response
Cloning- Map-based cloning
Sequence: ETR1- homology to histidine kinase receptors in bacteria
CTR1- sequence similarity to mitogen activated protein kinases
C. Biochemical test of model
1. ethylene binding- radioactive ethylene
2. kinase activity- ability to phosphorylate specific protein in the test tube
3. testing for protein-protein interactions
-Yeast Two-Hybrid System
-yeast GAL4- two parts:
i) DNA binding domain, ii) Transcriptional activator domain
-need to be physically linked to work
-clone fusion of each of your proteins to the two different GAL4 domains
-if proteins interact- then the GAL4 domains are joined and induces a reporter gene- yeast turns blue
D. Action at the gene level
1. cloning genes induced by ethylene treatment- subtractive DNA hybridization
2. Identifying the regulatory element (specific DNA sequence)
a. system developed for following promoter activation- GUS reporter gene
b. deletion studies- removal of the ethylene-dependent activation
c. addition studies- which sequences can confer ethylene induction to another
promoter
d. seeing protein-DNA sequences
-gel-shift assays
-DNAaseI protection
3. Results: two different elements in different ethylene-induced genes
GCC box ; binding protein is EREBP; found in pathogenesis-related gene
-DNA binding is influenced by ethylene
non-GCC box; CEBP-1 protein; found in carnation flower senescence and tomato
fruit ripening genes
-DNA binding is not influenced by ethylene
4. Cloning of transcription factors- cDNA expression library screened for ability
to bind the DNA fragment
Links:
Control of plant gene expression- PLSC731 NDSU
Reporter genes for analysis of gene expression-PLSC731 NDSU
PLACE database- cis-acting elements in vascular plants
Kinase activity
of ETR1- Proceedings of the National Academy of Sciences 1998. Vol. 95,
pp 7825-7829. Gamble, Coonfield & Schaller
Two-hybrid system
for showing interaction between CTR1 and ETR1- Proceedings of the National
Academy of Sciences,1998 Vol. 95, pp 5401-5406. Clark, Larsen, Wang and
Chang
DNA binding of
a foactor at an ethylene-responsive cis element- Proceedings of the National
Academy of Sciences 1997 vol. 94 pp 5961-5966. Büttner and Singh
Last revised: April 12, 1999- Straney