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Plant Gene Expression
Genome
Genes
Expression
Same genome in somatic cells
Different structures and functions
Differential gene expression
Spatial: organs
Temporal: time
Differential Gene Expression
Same proteins for Common processes
Some abundant proteins in certain cell types
Some minor proteins in certain cells at certain times
10,000 - 20,000 proteins in cells
15-20% as tissue-specific proteins
Control of Gene Expression
Regulation at steps:
Transcription: which gene, when, how often
Post transcription: RNA processing and transport
Translation: which transcript and for how long
Post translation: activity and stability of proteins
Control of Gene Expression
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Transcriptional Control
I Gene control regions & Regulatory protein
components of genetic switch
Gene control regions
5’ Promoter and 3’ Terminator
5’ Regulatory sequence
(some inside or at 3’)
Proximal promoter
5’ promoter or proximal promoter
Common sequence: TATA / CAAT box
Binding site for transcription machinery
RNA Pol and general transcription factors
Distal promoter
Regulatory sequences or distal promoter
or cis element or responsive element
Specific and consensus for gene expression
Binding site for gene regulatory proteins
or trans acting element or trans element
or specific transcription factor
Transcriptional Control
Genetic switch: turning genes on / off
Transcription Initiation
Transcription Initiation
Gene activating protein
DNA binding domain
Gene activation domain
Specific binding at major groove of DNA
Control rate of transcription initiation
except for general transcription factor TFIID:
TATA binding protein that binds minor groove
Major-Minor groove
DNA-Protein interaction
Strong and Specific binding
Compatible structure of DNA and Protein
20-30 contacts per match
Hydrogen bond
Ionic bond
Hydrophobic interaction
Gene Activating Protein
DNA binding motif
Helix Turn Helix
Zn finger
β sheet
Leucine zipper
Helix Loop Helix
Helix Turn Helix
Helix Turn Helix
trp repressor dimer
Helix Turn Helix
Zn Finger
b sheet
Leucine Zipper
Leucine zipper
Helix Loop Helix
Transcriptional control
II Chromatin structure
DNA packaging
Heterochromatin: condensed
Euchromatin: relaxed
Chromatin structure
Most organisms have both types
Except for
yeast: euchromatin
some algae and maize B chromosome:
heterochromatin
Chromatin structure
Chromatin structure
Heterochromatin
Genetically inactive
Inaccessible for transcription machinery
Euchromatin
Loose nucleosome / movable histones
Available for protein binding
Genetically active
Chromatin structure
Chromatin structure
Transcriptional control
III DNA methylation
Hypermethylation: Gene inactivation
Methyl group on Base
Methylase
DNA methylation
Eukaryote: more often in CG dinucleotide
Vertebrate: only in CG
Cytosine Methylation
Methyl group on position #5 of
5meC
Cytosine ring
Maintenance of methylation pattern
Methylation-induced Mutation
Deamination of Cytosine / 5meCytosine
Cytosine deamination (U)
Repair
5meCytosine deamination (T)
Mutation
Methylation-induced Mutation
Methylation
Plants:
~3-4 % of genome = CG
Animals: ~0.5-1 % of genome = CG
Methylation found in 70-80% of CG
Wheat Germ DNA
Highly methylated on CG or CNG
82 % of CG
19 % of CA / CT
7 % of CC
80+ % of CAG / CTG
4- % of CAT
Post-transcriptional Control
I Attenuation
Bacterial regulation of polycistronic RNA
Complete/Incomplete RNA production
mRNA inhibit RNA polymerase
incomplete transcription
mRNA interact with Regulatory protein
complete transcription
Post-transcriptional Control
II Alternative splicing
1 primary transcript / many mature transcript
1 gene / diversed mRNA / many proteins
May function in different organs
May function in different developmental stages
May have opposite functions
Alternative Splicing
Exon skipping / Optional exon
Intron retention / Optional intron
Mutually exclusion exon
Alternative 5’ / 3’ splice site
Alternative selection of promoter / PolyA site
Alternative Splicing
Alternative Splicing
Alternative Splicing
Post-transcriptional Control
III Varied C terminus
Polyadenylation at different sites
Same proteins of Different lengths
eg. Protein with/without hydrophobic C terminus
IV RNA transport
Half of primary transcripts: destroyed
Parts of transcripts to be processed
Most mature RNA: out to cytoplasm
Post-transcriptional Control
V RNA editing (modification)
Addition or Deletion of U
Deamination of C to U (plant mt)
VI Trans-Splicing
Exons from 2 independent transcripts
Post-transcriptional Gene Silencing
PTGS: transcription without translation
Found in plants and animals
Also in protozoa, insects and nematodes
Caused by transgene, virus or homologous dsRNA
Transgene-induced PTGS
Cosuppression
Silencing of Endogenous gene
Triggered by Transgene
Silencing occurs at post-transcriptional level
Homologous transcripts made & exported
Rapid degradation in cytoplasm
Transgene-induced PTGS
Petunia
Transformed with pigment-producing gene
Expected deep purple color
Appeared variegated to white
Transgene-induced gene silencing
also at transcription
gene-specific methylation
Viral-induced PTGS
Introduction of certain viruses
to host plants
dsRNA as trans-acting factor
responsible for PTGS
P
T
Antisense RNA technology
for gene silencing
P
T
application
RNA interference
dsRNA to knock out gene expression
so called RNA interference or RNAi
by initiating Small Interfering RNA (siRNA)
to induce silencing of
endogenous transcript
RNAi
Much more efficient than using either strand
as in cosuppression or antisense technology
Good for Gene Knockout studies
easily and quickly create Loss-of-Function phenotypes
Tool for Functional Genomics
Study of Expressed Regions of Genome
Translational Control
I mRNA stability
Bacterial RNA: 3-minute half life
Eukaryote: more stable esp. housekeeping mRNA
except for tx of regulatory proteins
AU stretch at 3’ UTR activates RNA degradation
by removing Poly A tail
Translational Control
II Translational recoding / frameshifting
mechanism found in virus
1 mRNA for > 1 protein
NNNNNNNNNNNNNNNNNN
Post-translational Control
I Protein activity
Folding: a helix/ b sheet
Assembly: Subunits
Reversible phosphorylation
II Protein stability
Degradation of unassemble, misfolded, or
damage protein
Protein structure