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 10, ,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 *** *** *

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

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

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 DNA and Protein contacts per match

Gene Activating Protein DNA binding motif Helix Turn Helix Zn finger β sheet Leucine zipper Helix Loop 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

Heterochromatin Genetically inactive Inaccessible for transcription machinery Euchromatin Loose nucleosome / movable histones Available for protein binding Genetically active

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 5me C

Maintenance of methylation pattern

Methylation-induced Mutation Deamination of Cytosine / 5me Cytosine Cytosine deamination (U) Repair 5me Cytosine 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

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 / PolyA site

Alternative Splicing

III Varied C terminus Post-transcriptional Control 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

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 silencing also at transcription gene-specific methylation Transgene-induced PTGS

Viral-induced PTGS Introduction of certain viruses to host plants dsRNA as trans-acting factor responsible for PTGS Antisense RNA technology for gene silencing PT PT

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 easily and quickly create Loss-of-Function phenotypes Tool for Functional Genomics Study of Expressed Regions of Genome

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:  helix/  sheet Assembly: Subunits Reversible phosphorylation II Protein stability Degradation of unassemble, misfolded, or damage protein

Protein structure