Generalities for Transcriptional Generalities for Transcriptional RegulationRegulation

• Activators and Repressors Act in ConcertActivators and Repressors Act in Concert

• Effector Binding Changes Protein ShapeEffector Binding Changes Protein Shape

• DNA Binding Occurs at Specific SequencesDNA Binding Occurs at Specific Sequences

• DNA Looping Common FeatureDNA Looping Common Feature

Eukaryotes vs. ProkaryotesEukaryotes vs. Prokaryotes

• Complexity of GenomeComplexity of Genome

• Multiple, Compartmentalized Multiple, Compartmentalized ChromosomesChromosomes

• Transcription Separated from Transcription Separated from TranslationTranslation

• Transcript ProcessingTranscript Processing

• Multi-CellularMulti-Cellular

WholeanimalRBCLiverSkinWholeanimalRBCLiverSkinWholeanimalRBCLiverSkinWholeanimalRBCLiverSkinNorthern Blot

(RNA)Northern Blot

(RNA)Western Blot

(Protein)Western Blot

(Protein)

TranscriptionalTranscriptionalRegulationRegulation

TranslationalTranslationalRegulationRegulation

Eukaryotic RNA PolymerasesEukaryotic RNA Polymerases

• RNA Polymerase IRNA Polymerase I– Ribosomal RNA GenesRibosomal RNA Genes

• RNA Polymerase IIRNA Polymerase II– Protein Coding GenesProtein Coding Genes

• RNA Polymerase IIIRNA Polymerase III– Transfer RNA GenesTransfer RNA Genes

Hsp 83 RNA Accumulation During Hsp 83 RNA Accumulation During DrosophilaDrosophila Embryogenesis Embryogenesis

EnhancersEnhancerscis-acting elementscis-acting elements

• Act at a distanceAct at a distance

• May be upstream or downstreamMay be upstream or downstream

• Bind Activators and/or RepressorsBind Activators and/or Repressors

• Can act on additional genesCan act on additional genes

Fig. 16.5

Hsp83-lacZ Reporter ConstructHsp83-lacZ Reporter Construct

lacZ genelacZ gene

-873.+1621 -873.+1009

EnhancerEnhancer

lacZ expression PatternlacZ expression Pattern

Activating Transcription FactorsActivating Transcription Factors

• Great DiversityGreat Diversity

• Protein DomainsProtein Domains– DNA Binding DomainDNA Binding Domain– Activator DomainActivator Domain

• DNA Binding Domain MotifsDNA Binding Domain Motifs– Helix-Turn-Helix (aka. Homeodomain)Helix-Turn-Helix (aka. Homeodomain)– Zinc FingerZinc Finger– Basic Leucine Zipper Basic Leucine Zipper – Helix-Loop-HelixHelix-Loop-Helix

HomeodomainHomeodomain(Helix-Turn-Helix)(Helix-Turn-Helix)

Zinc FingersZinc Fingers

Steroid--Steroid Receptor ComplexSteroid--Steroid Receptor Complex

HormoneHormone

ReceptorReceptor

Basic Leucine Zipper Basic Leucine Zipper (bZIP)(bZIP)

Fig. 16.7

Helix-Loop-HelixHelix-Loop-Helix

Myc/MaxMyc/MaxHelix Loop HelixHelix Loop Helix

ActivatorActivatorRepressorRepressor

Fig. 16.10

Fig. 16.8

Fig. 16.11

Chromatin Structure and Gene ExpressionChromatin Structure and Gene Expression

• Dampens Transcription to Low LevelsDampens Transcription to Low Levels• Major Means of Keeping Expression Turned Major Means of Keeping Expression Turned

OffOff– In contrast, prokaryotes utilize repressorsIn contrast, prokaryotes utilize repressors– Chromatin remodeling precedes activation Chromatin remodeling precedes activation

• Reflected by DNAase I hypersensitivityReflected by DNAase I hypersensitivity

• Heterochromatin Silences All TranscriptionHeterochromatin Silences All Transcription– Example: X-inactivation and formation of Barr Example: X-inactivation and formation of Barr

BodiesBodies

Sex Determination in Sex Determination in DrosophilaDrosophila

• XX Females, XY MalesXX Females, XY Males– Early Embryo “Counts” the X ChromosomesEarly Embryo “Counts” the X Chromosomes

• Sex Lethal (Sxl) Gene Critical RegulatorSex Lethal (Sxl) Gene Critical Regulator– Mutations in Sxl Result in NO Females!Mutations in Sxl Result in NO Females!

• Sex Lethal Acts at the Level of SplicingSex Lethal Acts at the Level of Splicing– RNA-Binding Splice Factor RNA-Binding Splice Factor

Early EmbryoEarly Embryo

Helix-Loop-Helix Helix-Loop-Helix HomodimersHomodimers

Males (XY)Males (XY) Females (XX)Females (XX)

NO Helix-Loop-Helix NO Helix-Loop-Helix HomodimersHomodimers

Fig. 16.16

Sex lethal regulation of Sex lethal regulation of transformertransformer RNA splicing RNA splicing

Sex LethalProtein

No Sex LethalProtein

Splicing OccursSplicing Occursat start of Exon 2at start of Exon 2

Stop codon

mRNA include a premature stop codon;mRNA include a premature stop codon;transformer protein nonfunctionaltransformer protein nonfunctional

Splicing OccursSplicing Occurswithin Exon 2within Exon 2

mRNA lacks premature stop codon;mRNA lacks premature stop codon;transformer protein functionaltransformer protein functional

Males (XY)Males (XY) Females (XX)Females (XX)

after Fig. 16.16

Regulation of Gene Expression via RNA StabilityRegulation of Gene Expression via RNA Stability

RNA RNA in situin situ

Northern BlotNorthern Blot

Mechanisms to Degrade mRNAMechanisms to Degrade mRNA

• Poly-A Tail LengthPoly-A Tail Length– Long tails confer stabilityLong tails confer stability• Tail shortening leads to mRNA turnoverTail shortening leads to mRNA turnover

• Degradation SequencesDegradation Sequences– Map to 3’ (or 5’) Untranslated RegionsMap to 3’ (or 5’) Untranslated Regions• Interact with protein factors that degrade Interact with protein factors that degrade

the “targeted” mRNAthe “targeted” mRNA

Reporter Constructs Are Used Reporter Constructs Are Used to Identify Degradation Sequencesto Identify Degradation Sequences