Lecture 6 - Eukaryotic Transcription

Mapping Transcriptional Start Sites

• DNA promoter elements that control transcription are often located near the start site of transcription of the gene and there are important regulatory elements in the 5’ UTR of mRNA

• to define the promoter for a gene you need to know the start of transcription

Similarities in Transcription and DNA Synthesis

• there is a multi-subunit complex for the enzyme which makes the nucleotide strand in 5’-3’ direction

• Mg2+ is a co-factor and is necessary to add to buffers for in vitro transcription

• both DNA strands can be templates for RNA synthesis

  • antisense strand = template 

Differences in Transcription and DNA Synthesis

• enzyme is RNAP which doesn’t need a primer

• promoter sequences where RNAP binds are asymmetrical - RNAP positioned so it can only transcribe one strand from one promoter

• DNA is unwound locally only vs energetically favourable state that doesn’t need ATP

• product is ssRNA that is released immediately so helix can re-form

• precursors use rNTPs

• less efficient in proofreading but less important than DNA mistakes since those stay over generations

Gene Expression

process in which information carried by a gene is converted into observable product

Transcription Definition

first step in gene expression where one strand of a DNA molecule is used as a template for synthesis of a complementary RNA (mRNA) which carries info for a specific protein

One-cell System of Gene Expression

in bacteria

cell has to survive and reproduce; gene expression is regulated to adjust to changes in nutritional environment to enable cell growth and division

Multicellular Organism Gene Expression

has to survive and reproduce but also grow and develop; different parts of the body have different functions

gene expression is regulated during:

• development (time)

• tissue differentiation (space and space/time combo where needs to be on sometimes but not always)

• stress (as a response to environmental stress - induction)

Eukaryotic vs. Prokaryotic Gene Expression

prok:

• transcription and translation are coupled in the same compartment

• mRNA is polycistronic without introns, and has a short half-life

euk:

• eukaryotic cell is compartmentalized and there is regulation in each compartment

• gene expression can be regulated at various levels

Eukaryotic Transcription

in a different compartment than translation

• pre-mRNAs are subject to extensive post-transcriptional mods → processing

• chromatic structure limits accessibility (only 0.01% of genes in a typical cell are undergoing transcription at any given moment)

• euk RNAP does not recognize binding site by itself → needs general transcription factors (GTF) to help

• mostly multicellular organisms with different cells/tissues

• 3 major RNAPs with different roles

Bacterial RNAP

called RNAP

transcribes all bacterial genes

Eukaryotic RNAPs

RNAP I - transcribes rRNA genes

RNAP II - transcribes mRNA, snoRNAs, some snRNAs, miRNAs

RNAP III - transcribes tRNA, 5S rRNA, some snRNAs

RNAP IV and V (plants only) - transcribes siRNA-directed DNA methylation and gene silencing

mitochondrial RNAP and chloroplast RNAPs also exist

RNAP I Intro 

located in nucleolus, where ribosomal genes are concentrated

responsible for transcribing rRNA genes

14 subunits

RNAP II Intro

located in the nucleus

responsible for transcribing the majority of genes: mRNA, snoRNAs, some snRNAs, miRNAs

12 subunits

RNAP III Intro

located in nucleus

responsible for transcribing tRNA, 5S rRNA, some snRNAs

17 subunits

Common Subunits in the RNAPs

rpb 5, 6, 8, 10, and 12

Separation and Purification of RNAPs

(1) rRNA genes:

• have high GC content (60%)

• are repetitive (up to 20 000 copies of the gene/cell)

• found in nucleolus

(2) RNA synthesis:

• high ionic concentration - RNA with low GC content

• low ionic concentration - RNA with high GC content (similar to rRNA)

• Mg2+ low ionic strength - most transcription in nucleolus

• Mn2+ high ionic strength - transcription throughout the nucleus

this shows us there is more than one RNAP, one works in the nucleolus, stimulated by low salt and Mg2+, and the other in nucleoplasm stimulated by high salt and Mn2+

Salt concentration in elution buffer will be ____ in each subsequent elution.

higher;

higher salt concentration displaces proteins with positive charge

different fractions will have proteins based on charge differences → use proteins from each fraction for in vitro transcription

this shows clear third RNAP 

RNAP I, II, and III and Ionic Strength

• RNAP I – active at low ionic strength, works with both Mg2+ and Mn2+

• RNAP II – more active at high ionic strength, works better with Mn2+

• RNAP III – active over a broad range of ionic strengths, works better with Mn2+

Biochemical Experiments

showed RNAP II is most sensitive to a-amanitin toxin from mushroom since it binds to rpb1 and prevents RNAP II translocation

RNAP I is most sensitive to actinomycin D antibiotic made by Streptomyces since it intercalates into GC rich regions and inhibits transcription

RNAP I

makes rRNA 45S precursor (13 000 nucleotide polymer) → 18S, 5.8S, 28S rRNAs

uses only 1 type of promoter: core promoter + upstream promoter element (UPE) where the efficiency of the core promoter is increased by UPE

• upstream binding factor (UBF) and selectivity factor 1 are ancillary factors needed for high-frequency initiation bind to UPE (increases promoter affinity and strength)

• RNAP I holoenzyme binds to UBF-SL1 complex at core promoter

• 2 UBFs bind to minor groove of G:C rich element in UPE and DNA turns to bring UPE and core promoter closer together

  • lets UBF stimulate binding of SL1 part of a complex → TBP + 3 RNAP I specific TBP-associated factors

  • TBP is a component of positioning factors needed for initiation by RNAP II and III

Core Promoter

shortest sequence at which RNAP can initiate transcription typically at low efficiency, interaction with more elements such as UPE increases it

The Nucleolus and RNAP I Transcription

human: clusters of ~40 copies of rRNA genes on 5 chromosome pairs = 400 gene copies

• each cluster is a nucleolar organizer region (NOR)

• size of nucleolus depends on the metabolic activity of the cell

  • size means number of clusters involved in rRNA synthesis

RNAP III

tRNAs, other small RNAs including 5S rRNA

has 3 types of promoters recognized in different ways by different groups of transcription factors:

• 2 types of internal: transcribe 5S rRNA and tRNA genes

• upstream: transcribes snRNAs, similar to RNAP II TATA promoters

Type 1 RNAP III Promoters

internal type found only in genes for 5S rRNA

• TFIIIA to BoxA then TFIIIC binds to BoxC

• subsequent binding of TFIIIC displaces TFIIIA and allows TFIIIB (positioning factor) to bind upstream from startpoint

• TBP can bind to TFIIIB

• RNAP III can be recruited and TFIIIC is displaced

efficiency of transcription is altered by changes in region upstream from the startpoint (spacing or sequence)

Type 2 RNAP III Promoters

transcribe tRNA genes

• TFIIIC binds to BoxA AND BoxB downstream of startpoint

• allows TFIIIB (positioning factor) to bind near the startpoint and TBP can be positioned

• RNAP III recruited

• TFIIIA and TFIIIC are assembly factors to help find TFIIIB

• have to be removed without affecting transcription

• TFIIIB is the only true initiation factor required by RNAP III

Type 3 RNAP III Promoters

transcribes some snRNAs 

more conventional arrangement (like RNAP II) with upstream elements to regulate transcription initiation

• TATA element is immediately upstream of startpoint: efficiency increased by other elements - factors binding here act cooperatively

• TATA element is bound by TFIIIB that actually recognizes DNA promoter sequence

• TFIIIB/TBP for RNAP III has the same role as TFIID/TBP for RNAP II

• in all 3 promoter types, TFIIIB binds to promoter to form preinitiation complex → directs binding of RNAP III

RNAP I, RNAP II, and RNAP III

TATA binding protein is a component of the POSITIONING factor no matter which RNAP is transcribing → allows each type of polymerase to bind to its promoter

• involved in coordination of activities of all 3 polymerases through binding to other polymerase-specific factors

• TBP = #1 commitment factor that coordinates transcription factors and pulls in the right RNAP

  • within SL1 complex (RNAP I)

  • within TFIID (RNAP II) 

  • within TFIIIB (RNAP III)

  • also in TATA-less II promoters

Structure of RNAP II

x-ray crystallography of 3D yeast RNAP II crystals and posed on synthetic DNA (no promoter)

25 angstrom channel in the face of the RNAP II which can accommodate 20 bp of DNA; channel formed by Rbp1 and Rbp2

• at the opening of the channel “jaws” role: grabbing of dsDNA 

• sliding clamp - composed of parts of Rbp1, 2, 6

• active site is on Rbp2 includes Mg2+ and conserved aspartate motif - RNA synthesis (facilitates nucleophilic attack) 

• two pores - one should be the exit for the growing RNA 

basically large subunit structure that make a clamshell that allows DNA and RNA to be transcribed

Core Subunits of RNAP II Structure

rpb1, 2, and 3, are absolutely necessary

rbp1 binds DNA, responsible for a-amanitin sensitivity

2 forms of large subunit: phosphorylation on carboxyl-terminal domain (CTD)

• alpha is non-phosphorylated

• o is phosphorylated

II alpha containing enzyme binds to promoter; II o-containing enzyme is in elongation phase (functionally different)

rpb2 is the polymerization active site; rpb3 is a 20 aa region of similarity with bacterial subunit a, 2 monomers in holoenzyme

CTD Tail

unique to pol II (not in I or III)

stretch of 7 aas that are repeated multiple times (at least 10) on rpb1 subunit

• tyr-ser-pro-thr-ser-pro-ser

5 of these 7 have -OH so this is a hydrophilic, phosphorylation site (mostly polar amino acid residues) 

• critical for viability 

• un-phosphorylated CTD tail used to initiate transcription

• phosphorylated CTS is present only for high levels of transcription

• critical for methyl cap addition and polyadenylation; splicing 

Promoters Recognized by RNAP II

class II promoters (promoter regions) have 2 parts:

1. core elements (aka core promoter or just promoter)

2. regulatory elements (one is the enhancer)

Core Promoter of Class II Promoters

minimal set of elements required for accurate in vitro transcription initiation by RNAP II

necessary for recruitment, binding, and proper positioning of RNAP II

• TATA box at ~-30 TBP binding site

• initiator on TSS

• downstream promoter element

• TFIIB, upstream

but lots of promoters don’t have initiator and downstream element and there are TATA-less promoters too

Regulatory Elements of Class II Promoters

bind regulatory proteins

classification of the elements (and the regulatory proteins that bind to them) is not always straightforward

• presence or absence of regulatory elements (sequences) can influence transcription rates through binding of regulatory proteins

• sometimes the regulatory elements are necessary for initiation of transcription

generally classified based on the distance from a core promoter and activity

• upstream elements (upstream and relatively close to, but not in, the promoter)

• enhancers and silencers (everywhere in any orientation)

• boundary elements and insulators

Core Promoter

TATA Box:

• highly conserved sequence 25-35 bp upstream from start site

• similar in action to e. coli TATA box but is further upstream

TATA-less promoters: found in housekeeping genes - absolutely necessary genes or in specialized genes (made in only certain cells)

• those genes must have either initiator core element or GC boxes (upstream element) to start transcription

Functions of TATA Box

is an element of the core promoter

SV40 early promoter (viral promoter): WT has 3 possible transcription start sites (all Gs)

• deletions of the sequences between TATA and WT start sites indicate transcription will start somewhere downstream of the TATA site → sequence doesn’t matter; DISTANCE from TATA to start is most important

1. TATA box finds start of transcription ~30 bp downstream

2. tata box sometimes important for the efficiency of transcription

3. TBP binds to TATA box and starts assembly of general transcription factors and RNAP

Enhancers/silencers can also be present in ______ ___ or ______.

structural gene; downstream

Initiator Element

part of core promoter; could be part of TATA-less promoters

• majority have C at -1 position and A at +1 position

• strength of promoter determined by surrounding nucleotide sequence

• transcription with initiator elements or TATA boxes begins at precise site

Downstream Elements

part of core promoter; could be part of TATA-less promoters

binds TFIID (general transcription factor)

TFIIB Recognition Element (BRE)

binds TFIIB (General transcription factor)

considered part of a group of regulatory elements

Regulatory Elements

may be present individually or in “sets”

upstream elements:

1. GC boxes: GC rich stretch, orientation independent (can be flipped 180 degrees) but must be close to TATA box - 20-50 bp upstream of start site, usually in housekeeping genes

   1. could be a part of TATA-less promoter and transcription could be initiated at any one of multiple possible sites over 20-200 bp resulting in mRNAs with multiple alternative 5’ ends (UTRs_

2. CCAAT boxes: enhancer element 30-75 bp upstream with CTF (CCAAT transcription factor)

   1. no prok. equivalent; may be necessary in euks.

3. promoter-proximal elements: control regions 100-200 bp upstream from start site with various numbers of sequences and effects

   1. cell type specific - specific set of elements dictates expression

   2. identified through 5’ deletion series

Enhancers

part of regulatory elements

• control elements that typically stimulate transcription

• could be a multiple binding site for different transcription factors

• transcription factors that recognize enhancer = enhancer binding proteins = activators → interact with general transcription factors

• sometimes the same element could be both enhancer and silencer based on the protein that is bound

• can be thousands of bp away

• orientation doesn’t matter

• can occur downstream in an intron or in the exon

• transvection: enhancers on adjacent chromosomes (local concentration of factors)

Insulators and Boundary Elements

insulators: form chromatin boundary between euchromatic and heterochromatic regions in an individual chromosome

• loops made by CTCF and cohesin, access in under locus control regions’ control

insulated neighbourhoods: regions of DNA within extruded ‘loops’ of eukaryotic DNA; chromosome territories are respected

Review of RNAPII Promoters

class II promoters have two parts: core elements and regulatory elements

core elements include:

• TATA box, initiator, downstream element, TFIIB recognition element

regulatory elements include:

• upstream elements (GC boxes, CCAAT boxes, promoter proximal elements)

• enhancers and silencers

• boundary elements and insulators

How do we recognize the RNAP II promoter?

• position RNAP II at transcription initiation sites and bend DNA (promote tight binding) to prevent nucleosome reformation

• required by transcription of most genes that are transcribed by RNAP II

• transcription initiation complex

• TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH where D is the largest and is made of TATA binding protein and TBP-associated factors

  • has 2 basic roles: foundation for transcriptional complex and prevent nucleosome stabilization in the promoter region

The transcription initiation complex is made of _____ and _____.

RNAP II; GTF bound to promoter region

Importance of TBP

it is the first protein to bind to the TATA box → is a positioning factor

• it is highly conserved with similar C-terminal domains

• monomer that makes a saddle shape and bends minor groove

• alternative TBP in small # of cells (TRF1 is very rare and cell-specific)

• positioning factor so helps RNAP find its promoter, no matter which is transcribing

  • within SL1 complex (RNAP I)

  • within TFIID (RNAP II)

  • within TFIIIB (RNAP III)

  • Also in TATA-less II promoters

Which factor determined whether or not TFIID stays at the promoter?

TAFs - TATA-associated factors

• some promoters are more difficult to transcribe from than others

Steps of Eukaryotic Transcription

1. TFIID binds at TATA via TBP and TAFs

2. once TBP/TFIID is bound, TFIIB (monomeric protein) binds - rate limiting step

3. TFIIF binds to mediator RNAP II → make a preformed complex and direct mediator RNAP II to GTFS assembled on the promoter

4. 2 large RNAP II subunits interact with TFIIB which is already positioned on DNA

   1. unphosphorylated CTD tail of RNAP II is in direct contact with TFIID

5. TFIIE and TFIIH bind to the complex (TFIIE = DNA-dependent ATPase, TFIIH = helicase and protein kinase activity)

6. TFIIH helicase activity unwinds DNA with energy from TFIIE, kinase activity phosphorylates CTD tail of RNAP II

7. phosphorylation of CTD tail detaches RNAP II from TFIID and promoter

8. transcription pause released by more phosphorylation of CTD and other factors by CDK9

9. promoter clearance (mediator and GTFs released)

10. RNAP II goes to elongation

Which TF is critical for RNAPII orientation on the promoter?

TFIIB; near RNA exit site and active site

Promoters Lacking TATA

in majority, TBP needs to be placed at the right spot first with respect to existing core element and transcription start site

initiator element:

• TBP can bind to promoter itself with initiator minus TATA through TBP-associated factors (TAFs) → TFIID binds

GC boxes:

• TBP can’t bind itself to promoter with GC but no TATA through TAFs → TFIID binds

after TFIID binding, events are the same as in TATA promoters

Proteins that Affect Transcription

basal transcription is seen in vitro; in vivo, euk promoters have to be activated by upstream bound transcription factors 

GTFs and TFs directly influence RNAP binding:

• GTFs bind to core promoter or RNAP II or each other to anchor RNAP II

• TF (activators and repressors) bind to DNA at proximal elements, enhancers/silencers, even core promoter elements

co-activators and mediators bind to both activators and basal apparatus as a bridge

chromatin remodelling enzymes and other regulators act on local chromatin structure

In order for a protein to be a transcription factor it:

1. has to be able to bind DNA

2. has to be able to activate or repress transcription

transcription factors are modular proteins → different domains have different functions

• DNA-binding domains (DBD) interacts with DNA sequences to bind DNA

• transcription activation domain (AD) interacts with other proteins to stimulate or impair transcription from a nearby promoter

• some have dimerization domains for homo or hetero dimers

• ligand-binding domains bind small molecules to regulate TF activity

Domains vs. Motifs

domains: tertiary structure of large proteins organized in distinct regions of the proteins; can be active and stable on its own when removed from the rest of the protein

motifs: specific combinations of secondary structures which are organized into specific 3D structure inside the domains (but refer to protein secondary structure)

DNA Binding Domain Overview

• Zn fingers (C2H2, C4, C6)

• one alpha-helix in homeodomain

• basic alpha-helix part in bZip

• basic alpha-helix in bHLH

DNA Binding Domain

structural motif present in euk transcription factors responsible for its interaction with specific DNA sequences

has a variety of structural motifs for the classification of transcription factors based on interactions and how they bind with DNA

Structural Motifs in DNA Binding Domain

1. zinc finger: proteins with regions around a zinc ion also seen in protein that do not bind to DNA, 3 types; only one that can bind as a monomer

2. homeodomain protein: one of the oldest forms of DBD and are important in development of organisms, a highly conserved 60 aa sequence organized into 3 alpha-helices, two of which are organized into helix-turn-helix and third fits into major groove with N-terminal into minor groove

3. leucine-zipper proteins (bZIP) have hydrophobic leucines in every 7th position of the C-terminal end of the protein (every second turn) for dimerization

4. helix-loop-helix (HLH) is similar to zipper motif with hydrophobic residues on one side of C-terminal and aloha helix dimerization; tend to form dimers with the second alpha helix responsible for DNA binding to make homo or heterodimers

Types of Zinc Fingers

1. C2H2 holds zinc with 2 Cys and 2 His residues, alpha helix inserts into major groove of DNA - usually 3 or 4 finger domains, can bind by self as a monomer

2. C4 has 2 groups of 4 Cys residues binding 2 zinc ions binding as a homo or heterodimer (homo needs 2 copies, hetero needs another copy of a different protein to bind)

3. C6 zinc finger in yeast uses 6 Cys residues to bind 2 zinc ions into a globular domain and small recognition helix; requires alpha helix dimerization domain (homo or hetero doesn’t matter just needs dimerization)

Transcription Activation Domain Overview

• acidic

• glutamine

• proline rich

Transcription Activation Domains

allows for proteins to do protein-protein interactions for activation/repression of transcription

protein-protein interactions allow for conformational changes in the protein for it to activate or repress

• acidic activation domains interact with co-activator

  • some yeast activators, mammalian glucocorticoid receptor, herpes virus activator

  • lots of aspartic and glutamic acid

• glutamine-rich domains

• proline-rich domains

DNA Binding and Transcription Activation Domains

experiments with chimeric hybrid proteins show activating domain and DNA binding domain are separate and independent

Gal4 is an activator with DNA binding and activation domain and Lex A is a repressor with DNA binding and activating domain that acts as a repressor → cut off repressing activating domain of Lex A and hook up to activation domain of Gal4 → whole protein acts as an activator when bound to lexA sites in DNA

DNA binding domain from repressor + activation domain from activator = acting as activator even though DBD is from a repressor

therefore, activation domain is important ONLY for activation and DNA binding domain is ONLY for binding; they are independent

Dimerization Domain Overview

• one of the Zn fingers (alpha helix in zinc finger C6)

• leucine zipper part in bZip

• second helix in bHLH

Ligand Binding Domain Overview

• in Zn finger - C4 factors where the steroid hormone is the ligand

Dimerization Domain

allows for greater diversity and complexity of factors (more control of gene expression)

• combinatorial control of different activation domains into a third activation domain

• recognition of different binding sites

• heterodimerization can alter DNA binding specificity of transcription factor for combinatorial control → control by combination of different proteins

• dimerization does robust activation compared to monomer itself since it can recognize more sites

Ligand Binding Domain

a ligand is any type of effector molecule or covalent attachment

binding/modification of this domain will cause conformational change and modulate the native active of the regulatory molecule

is typically reversible and there are a variety of ligands (e.g. end products like tryptophan, carbon sources like lactose, covalent modification like phosphorylation)

example: CCAAT Enhancer Binding Protein B is a transcription factor for endometrial cells that can be phosphorylated to become active and open up → big structural change

Enhancers

any TFs that bind can interact

• have multiple binding sites as landing pads for transcription factors

cooperative binding makes an enhanceosome, a nucleoprotein complex by bending or looping DNA

• binding certain proteins makes assembly of other proteins → combinatorial control

• gene is only expressed with the correct combination of TFs in the cell

Co-activtors

make direct contact with activators or repressors through transcription activation domain

are not transcription factors themselves so need to bind something else to regulate transcription

have specific functions, such as histone modification of tails to affect the degree of compaction of nucleosome → allow for greater accessibility of TFs to the transcribed gene

Mediator Complex

protein complex needed for assembly of the pre-initiation complex (RNAP II and GTFs) and successful initiation

• allows for fine-tuning and cooperation of proteins (Remodeling of local region of transcription) → does this through activator domains contacting different mediator components

function is to coordinate a combination of activators/elements and can do conformational changes of the complex → changes rate of RNAP II initiation (engages more quickly or more slowly)

helps RNAP II coordinate the signals it’s getting from all the TFs

Tissue Specificity of Cis Regulatory Elements

• DNA is the same in every cell/tissue therefore the cis elements are the same everywhere but we don’t want all the same proteins expressed everywhere

• tissue specificity comes from the action of tissue-specific TFs 

• regulatory proteins are tissue specific and this is what dictates the tissue specificity of cis elements (crucial for transcription initiation)

Transcription Initiation by RNAP II

highly regulated in proks and euks because it has to respond to developmental timing and environmental changes

• one gene can be transcribed in multiple condiitons

• different conditions are associated with different cis elements which bind different transcription factors

• combinatorial control since different factors at each site will interact and that determines if the gene is turned on and expressed

no consistent location for where cis regulatory elements will be found - a factor’s presence/absence could increase/decrease transcription

• different genes that respond to the same signal in the same way all have the same cis element

promoters are modular - variety of elements can contribute to promoter function meaning none are essential or common to all promoters

• elements can differ in number, location, and orientation

Influences on Transcription Initiation

• direct influence on TFs on assembly of initiation complexes

  • recruitment of GTFs

  • co-activators

  • interactions

  • enhanceosomes

  • architectural changes

• TFs regulate changes in chromatin structure (remodeling) and control histone acetyl and deacetylation

• concentration and activities of TFs

• protection of active gene promoters from methylation

Recruitment of GTFs

RNAP in euks is not directly recruited to DNA unlike proks that can scan and find

• TFs help recruit GTFs (which associate with RNAP)

• holoenzyme model: RNAP II and GTFs are recruited as a preassembled whole that lands on the transcription unit

• enhanceosome model: happens stepwise where TFIID complex binds first due to interactions of TAFs with activators bound to the enhancers (opens up region to get access, TFs assemble) but steps may happen in parallel

Co-activators

• interact with TFs with modulator complex, co-activators, each other, ligands → promote or prevent GTF binding

• e.g. CREB protein that binds cAMP response element and activate associated genes by binding to CREB-binding protein (CBP) → CREB is activated by phosphorylation to allow association with cAMP response element (CRE)

Interactions

TFs interact with each other or ligands, those interactions promote or prevent binding of GTFs

Interactions Involving Repressor Proteins:

• repressor inhibits gene activation by binding to site that overlaps activator site

• repressor binds adjacent to activator and interferes with AD (activation domain) of activator

• repressor binds upstream and interacts with mediator through GTFs to inhibit initiation

• co-repressors recruited that alter nucleosomes to inhibit transcription

Interactions Involving Ligand Binding Domains

both activation and inhibition switch on and off depending on if ligand is bound or not due to conformational changes in the TF

retinoic acid

Interactions Involving Enhanceosomes

• TFs make these at any distance from GTF binding site for specific gene expression, usually for tissue-specific gene expression

  • multiple enhancers and modular arrangements give fine control through different combinations and concentrations of TF → combinatorial control

Interactions Involving Architectural Changes

TFs bind to specific binding sites to change the shape of the DNA in a control region → changing interactions transcription factors, and/or GTFs, stimulating transcription

difficult for DNA to bend or loop if the distance between core promoter and enhancer is too short, may need more architectural TFs

Interactions Involving Insulators

sets up boundaries between DNA domains - prevents activation or repression of genes by close but unrelated activators and repressors

Histone Acetylation and Deacetylation

TFs regulate this

• core nucleosome has histones with a flexible tail where modifications occur to compact or loosen the chromatin

• 20-40 aa N-terminal tails have positive lysine residues that can be acetylated or deacetylated

  • acetylated = positive charge is neutralized and eliminates interaction with DNA → DNA less condensed, promoter regions available (euchromatin)

  • catalyzed by histone acetyltransferases (HAT) associated with activation of gene expression

  • deacetylation → repression of gene activity where deacetylases are in co-repressor complexes

Histone acetylation is ____ but not ____ for activation.

essential; sufficient

acetylation of histone tail makes the chromatin less compact but we still have nucleosomes intact which need to be repositioned to expose promoter elements - chromatin remodeling protein complexes

• use ATP energy to remodel and move nucleosomes out of the way (displace histone or slide it along DNA) 

• swi/snf proteins are important for altering structure of nucleosome core, swi proteins move/remove/replace nucleosomes on DNA 

• remodeling complexes don’t bind DNA sequences themselves → are recruited by activators or repressors

Concentrations and Activities of TFs

regulated during cell differentiation and in response to hormones and signals from other cells

as we have differences in concentration, we make a gradient that affects which genes will be expressed (think Drosophila local concentration of TFs in different parts of the organism that affects different genes

• critical point: transcription of genes which are transcription factors themselves

  • transcription activation based on concentration of effected cell important during development

• accessible binding sites and respective binding proteins are necessary for transcription

Protection of Active Gene Promoters

protecting from methylation

• some promoters have CpG islands are preserved as unmethylated to keep genes active

  • islands are stretches of 200-500 nucleotides with GC content more than 50% that act as core promoters or upstream elements

• mechanism of protection is still unclear but could be prevention of DNA methyltransferase binding, demethylase existing, GTFs and RNAP II and H3 tail mods excluding DNA methyltransferase (DNMT) from sites of transcription initiation

• could also reduce amount of GC pairs but we can’t control the DNA so we just don’t have CpG islands in some areas

• deamination of normal C to U or methylated C to T which gets recognized by repair system and get fixed as a mutation (getting rid of methylated C through conversion to T)

Which enzyme recognizes single strand methylation and repairs the new strand?

maintenance methylase; there are no methyl groups on newly made DNA strand, the parent strand is methylated

involved in protecting active gene promoters from methylation

CpG methylation is known as _____ _____.

epigenetic modification; means on top of the gene

the DNA sequence is not changed, just methylated which changes gene expression

if methyl groups are removed from CpGs in promoter regions, transcription is able to occur but not necessarily will

no methylation = necessary but not sufficient for transcription to occur but this is not a universal method for regulation of gene expression (e.g. drosophila)