RNA Transcription

• Pre-Initiation Complex (PIC) binds promoter

• PIC recruits RNA Polymerase II

• Pol2 transcription elongation complex (TEC) transcribes sequence

• 7-methyl guanosine cap

• Spliceosome ribozymes remove introns

• Polyadenylation factors recruit poly-A polymerase

Transcription regulation

• Initiation– Chromatin structure– Promoter elements– Enhancer elements

• Elongation

• Stability– miRNA

Conformational control of transcription

• Transcription initiation depends on– DNA accessibility– Affinity for Pol2

• DNA structure– Supercoiling– Kinks– Nucleosomes

• Matrix association

Intron Intron IntronExon ExonCoreEnhancer

Promoter

-4000 -500 -40-+50 10000+

MAR MAR

TranscriptionElongation Complex

NucleosomeCore Particle

TranscriptionBubble

Chromatin remodeling

• Nucleosome remodeling– SWI/SNF , ISWI, CHD– Displace nucleosome relative to DNA– Displace histone proteins

• Steric regulation of transcription factor binding

• Histone modifications– Methylation– Acetylation

Histone code

• Histone tails have many lysines– Terminal amine H-bonds with PO4 backbone– Subject to multiple methylation/acetylation– Interactions between nucleosomes– Interaction btw histone & DNA

• Histone acetyltransferase (HAT)– p300/CBP,P/CAF– Transcribed genes

• Histone deacetylase (HDAC)– Sin3, NuRD– Silenced genes

Histone structure

• Amino terminal of H3/H4 link adjacent nucleosomes

• Acetylation (-CO-CH3) blocks this association

• HATs activate genes

• HDACs repress

• Epigenetic inheritance

Gene specific transcription

• Promoter proximal region– Reporter construct– Canonical response elements

• Remote enhancer/insulator elements– Clusters of regulatory elements– Chromatin loops of 50+kb

Exon Exon ExonIntron IntronCoreEnhancer

Promoter

-4000 -500 -40-+50 10000+

MAR MAR

Gene specific transcription factors

• TFs may be as much as 6% of the genome• Specific regulation requires teamwork

– Combinatorial control– Synergistic, greater than additive interaction

• Common features– Basic, DNA binding domain– Phosphate binding– Dimer/oligomer-ization

• Options– Major groove– Over the top

Characteristics of Gene-specific TFs

Brivanlou & Darnell 2002

Transcriptional activators

ConstitutiveSp1

CCATNF1

Conditional

DevelopmentalGATAHNFPit1

MyoDBicoidHox

Signal Dependent

Steroid ReceptorGRERPRTR

Internal SignalsSREBP

p53ChoREB

NFkB

Surface Receptor

Nuclear ResidentETS

CREBSRF

FOS-JUN

Latent CytoplasmicSMADSTATNFAT

DNA binding

• Charged AAs– Positive (blue N) groups (lys, arg, his)– Negative (red O) groups (asp glu)– O+N (asn, gln)

• Protein -helix 3-4 NTPs c-jun

jun side-chains compliment nucleotide polarity to give sequence recognition

Jun bindingJun (leucine zipper): 1 mtakmettfy ddaLnasfLp sesgpygysn pkiLkqsmtL nLadpvgsLk phLraknsdL 61 LtspdvgLLk LaspeLerLi iqssnghitt tptptqflcp knvtdeqegf aegfvralae 121 lhsqntlpsv tsaaqpvnga gmvapavasv aggsgsggfs aslhseppvy anlsnfnpga 181 lssgggapsy gaaglafpaq pqqqqqpphh lpqqmpvqhp rlqalkeepq tvpempgetp 241 plspidmesq erikaerkrm rnriaaskcr krkleriarl eekvktlkaq nselastanm 301 lreqvaqlkq kvmnhvnsgc qlmltqqlqt f

Jun DNA binding: kaerkrm rnriaask

DNA Consensus: TCA

Dimer palindrome: TGA X tca

Other side chains interact with DNA PO4 backbone

TF Mechanisms

• PIC interaction

• Mediator interaction

• Target histone modifiers

GMSA shows increase in PIC assembly with CREB activation domain (CRG) but not CREB DNA binding domain alone (DBD)

Felinski et al., 2001

CREBActivation domain

DNA binding

MyoD

• “Master control switch” for myogenesis

• Associates with muscle specific promoters– Recruits P/CAF HAT– Trigger differentiation

• Recruits HDAC1– Represses myogenesis

Dual specificity chromatin-IP shows MyoD complexed with HDAC1 during growth and P/CAF during differentiation

Growth Differentiation

Cooperativity

• Most promoters contain dozens of TF domains

• Most TFs capable of binding dozens of cofactors

• Hydrophobic protein-protein interaction

• Combinatorial/network control of transcription

Protein-protein binding map for MyoD

TFIIOther TF

HATs

Other TF

Enhanceosome

• Combination of GTFs & GSEs

• Family of xscription factors produce specific molecular surface

• Highly nonlinear

• Interferon-beta– ATF+jun– IRF-1– p50+p65– HMG-1– Only effective as combined group

Kim & Maniatis (1997)

Mediator

• Helps recruit Pol2 to PIC

• GTF cofactor

• Integrates GTF & gene specific TFs

• CTD kinase

• Many subunits (30+)– Head– Middle– Tail

Chadick & Austurias, 2005

Mediator

• GTF interactions– TBP– TFIIH

• Cdk activity targets Pol2 CTD– CDK8/CDK11 + cyclin D– Gene specific activation/repression

• Gene specific TFs may act through mediator to recruit/activate pol2 and through HDAC/HATs to recruit GTFs

Regulation of elongation

• TEC velocity• Pause/arrest• Negative Elongation Factor (NELF)

– Binds DNA & blocks PolII progress– NELF-B = Cofactor of BRCA1 (COBRA1)

• Reversible Block– Binds PolII/DSIF complex– Released by P-TEFb phosphorylation of

PolII– NELF-P-TEFb competition

P-TEFb releases pause

• Cyclin dependent kinase– Cdk9– Cyclin T/K

• Phosphorylates DSIF

• Phosphorylates CTD– Stimulates elongation– Blocks NELF binding

Pol IIDSIF

NELF

P-TEFb

PO4

Transcription Blocked

Transcription Allowed

Yamaguchi et al., 1999 Cell 97:41

Chromatin remodeling

Set1/PAF methylate histone, loosen structure

Chd1 displaces H2A/B, allowing transcription

Competition displaces Set1, downstream histones less methylated

Turner 2003

miRNA

• Short, noncoding RNA

• Drosha forms hairpins

• Dicer form short dsRNA

• Steric translation block

• Slicer dsRNA degradation

Drosha

Dicer

Slicer

Mature slicer (Ago) with ssRNA

Regulatory Circuits

• Cascade

• Feedback/Regulatory Inhibition

• Combinatorial activation

One or more external signals allow activation

Product inhibits expression of promoter

External signalExternal signal

External signal

Modulation of TF activity

• Many TFs are constitutively expressed

• Secondary control mechanisms– Phosphorylation (phos-myoD binds HDAC)– Ligand/dimer binding (steroid)– Inhibitor dissociation (NF-kb)– Subcellular localization (MEF2 dephos)

• Some TF form cascades– “IEG”s– Delayed Early Genes– Cyclins/cdks

Example: c-Jun

• Activator Protein-1 component– Phosphorylation by JNK/ERK

• Ser 63, S73 (protein binding domain)• Required for activity

– Phosphorylation by GSK• Thr231, Thr239, Ser243, Ser249 (DNA binding domain)• Prevents DNA binding

– Association with JNK• Facilitates ubiquitination

• Binds TATA-binding protein-Associated Factor-1• Cell cycle regulation

– CyclinD/cdk4 transcription after mitogens– Repression by GSK during starvation