Plant Gene Expression Genome Genes Expression

Differential gene expression Spatial: organs

Temporal : time

Same genome in somatic cells Different structures and functions

Differential Gene Expression

Same proteins for Common processes

Some abundant proteins in certain cell types

Some minor proteins in certain cells at certain times

- 1 0 ,0 0 0 2 0 ,0 0 0 proteins in cells- - 1520% as tissue specific proteins

Control of Gene Expression

Regulation at steps:

Transcription : which gene, when, howoften Post transcription : RNA processing and transport

Translation : which transcript and for howlong Post translation : activity and stability of proteins

Control of Gene Expression

******

*

Transcriptional Control

I Gene control regions & Regulatory protein components of genetic switch

Gene control regions 5 3’ Promoter and ’ Terminator 5’ Regulatory sequence

3(some inside or at ’)

Proximal promoter

5’ promoter or proximal promoter

Common sequence: TATA / CAAT box

Binding site for transcription machinery

RNA Pol and general transcription factors

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

Distal promoter

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

Hydrogen bond Ionic bond

Hydrophobic interaction

Strong and Specific binding Compatible structure of DNAand Protein

- 2030 contacts per match

Gene Activating Protein

DNA binding motif Helix Turn Helix

Zn fingerβ sheet

Leucine zipper Helix Loop Helix

Helix Turn Helix

Helix Turn Helix

trp repressor dimer

Helix Turn Helix

Zn Finger

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 structureHeterochromatin

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

Eukaryote: more often in CG dinucleotide Vertebrate: only in CG

DNA methylation

Cytosine Methylation

Methyl group on position #5 of Cytosine ring 5meC

Maintenance of methylation pattern

- Methylation induced Mutation

Deamination of Cytosine / 5meCytosine Cytosine deamination (U)

Repair 5me Cytosine deamination (T)

Mutation

- Methylation induced Mutation

Methylation

Plants: - 34~ %of genome = CGAnimals: ~0.5-1 % of genome = CG

- Methylation found in 70 80% of CG

Wheat Germ DNA

Highly methylated on CG or CNG 8 2 % of CG 19% of CA / CT

7 % of CC 80+ % of CAG / CTG

- 4 % of CAT

- Post transcriptional Control I Attenuati

on Bacterial regulation of polycistronic RNA Complete/Incomplete RNAproduction

mRNA inhibit RNA polymerase incomplete transcription

mRNA interact with Regulatory protein complete transcription

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

- Post transcriptional Control

Alternative Splicing

Exon skipping / Optional exon

Intron retention / Optional intron

Mutually exclusion exon

Alternative 5’ / 3’ splice site

Alternative selection of promoter / PolyAsite

Alternative Splicing

Alternative Splicing

Alternative Splicing

III Varied C terminus

- Post transcriptional Control

Polyadenylation at different sites Same proteins of Different lengths

w wwwww wwwwww wwwwwwwwwww w wwww wwww. /IV RNA transport

Half of primary transcripts: destroyed Parts of transcripts to be processed Most mature RNA: out to cytoplasm

V RNA editing (modification) Addition or Deletion of U

Deamination of C to U (plant mt)

- Post transcriptional Control

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

Petunia - Transformed with pigment producing gene

Expected deep purple color Appeared variegated to white

- Transgene induced gene silencingwwww ww wwwwwwwwwwwww

- gene specific methylation

- Transgene induced PTGS

- Viral induced PTGS

Introduction of certain viruses to host plants

dsRNA as trans- acting factor responsible for PTGS

w wwwwwwww w w w wwwwwwwwww for gene silencing

P T P T

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

application

RNAi

Much more efficient than using either strand as in cosuppression or antisense technology Good for Gene Knockout studies

wwwwww www wwwwwww wwwwww wwww-ww-wwwwwwww wwwwwwwwww Tool for Functional Genomics

wwwww ww wwwwwwwww w wwwwww ww w wwww w

I mRNA stability - Bacterial RNA: 3 minute half life

Eukaryote: more stable esp. housekeeping mRNA except for tx of regulatory proteins

AU stretch at3’ UTR activates RNAdegradation by removing Poly A tail

Translational Control

Translational Control

II Translational recoding / frameshifting mechanism found in virus

1 1mRNA for > proteinNNNNNNNNNNNNNNNNNN

- Post translational Control I Protein activity

Folding: helix/ sheet Assembly: Subunits

Reversible phosphorylation

II Protein stability Degradation of unassemble, misfolded, or

damage protein

Protein structure