islamic university of gaza faculty of...

Islamic University of GazaFaculty of Medicine

Molecular Biology, 2012‐20132

• The two cells extremely different but contain the same genome!!

• Differentiation = synthesizing and accumulating different sets of RNA and protein molecules.– The DNA sequence is generally not altered– All the differences are achieved by changes in gene expression

3 Molecular Biology, 2012‐2013

• Mediated by1. Gene regulatory elements ‐‐ specific cis‐acting DNA

sequences2. Sequence‐specific DNA‐binding proteins ‐‐ trans‐

acting transcription factors.• Interpret the information present in gene promoters and other regulatory elements

• transmit the appropriate response to the RNA pol II transcriptional machinery

3. The complexity and diversity of the multiprotein complexes that regulate gene expression


• The unique combination of regulatory elements and the transcription factors that bind them.

• Regulatory elements – Promoter elements

• The core promoter • Proximal promoter elements

– Long‐range regulatory elements• Enhancers and silencers• Insulators• locus control regions (LCRs)• Matrix attachment regions (MARs)

• Transcription factors– General– Gene specific


• Gene promoter: is loosely defined as:– The collection of cis‐regulatory elements, positioned near the transcriptional start site• required for initiation of transcription• or increase the frequency of initiation

– These include:• The core promoter• The proximal promoter elements “upstream regulatory elements”


• Approximately 60 bp DNA sequence overlapping the transcription start site (+1)– Serves as the recognition site for RNA pol II and general transcription factors


• The position and orientation of promoter elements are essential to their operation

• The general transcription factor TFIID is responsible for the recognition of all known core promoter elements– Exception: TFIIB recognizes the BRE (TFIIB recognition element).

• A particular core promoter may contain some, all, or none of these elements.


• The vast majority of yeast genes contain a single upstream activating sequence (UAS)– Regulate TFIID binding to the core promoter element– usually composed of two or three closely linked binding sites for one or two different transcription factors.


• A typical multicellular eukaryote gene is likely to contain several proximal promoter elements– located just 5′ of the core promoter – usually within 70–200 bp upstream of the start of transcription.


• Tend to be located in clusters.• Increase the frequency of transcription initiation

– only when positioned near the transcriptional start site.

• Bound by transcription factors that– May directly activate or repress transcription. – May recruit long‐range regulatory elements, such as enhancers, to the core promoter


• Examples:– The CAAT box is a binding site for

• CAAT‐binding protein (CBF) • CAAT/enhancer‐binding protein (C/EBP).

– The GC box is a binding site for the transcription factor Sp1.


• Can work over distances of 100 kb or more from the gene promoter.

• Mediate the complex patterns of gene expression in different cell types during development.

• Not generally observed in yeast (with few exceptions)

• In multicellular eukaryotes include– enhancers and silencers– Insulators– locus control regions (LCRs)– matrix attachment regions (MARs)


• Contains in the order of 10 binding sites for several different TFs.

• Can be tissue‐specific or developmental stage‐specific

• Elements that repress gene activity are called silencers.


• Typically 0.3‐2 kb in length with two distinct functions• Chromatin boundary marker:

– Marks the border between regions of heterochromatin (gene‐poor) and euchromatin (gene‐rich) in eukaryotes.

– Natural barriers to heterochromatin spreading into neighboring DNA particularly when active genes are nearby.

• Enhancer blocking activity: – Prevents inappropriate cross‐activation or repression of neighboring genes by blocking the action of enhancers and silencers.


• Organize and maintain a functional domain of active chromatin

• Enhance the transcription of downstream genes. • Operate in an orientation‐dependent manner.• Present in many loci, including gene clusters encoding

– α and β‐globins– visual pigments– major histocompatibility proteins– human growth hormones– serpins (a family of structurally related proteins that inhibit proteases)

– T‐helper type 2


• The genes of the β‐like globin gene cluster are expressed in erythrocytes in a tissue‐ and developmental stage specific manner: – ε‐globin gene is activated in the embryonic stage, – γ‐globin is activated in the fetal stage– β‐globin gene is expressed in adults.

• LCR ‐ contain clusters of transcription factor‐binding sites– TFs interact via extensive protein–DNA and protein–proteininteractions.


• The LCR is a minimum requirement for globin gene expression.– Physiological levels of expression– high‐level transcription

• The regulation of chromatin “loop” formation is the main mechanism controlling developmental expression of the β‐globin genes.– developmental regulation of the β‐like globin genes is mediated by the regulatory elements within their individual promoters.

– LCR interacts with only one gene promoter at any one time.


• RNA pol II is first recruited to LCR in vivo.

• RNA pol II is then transferred from the LCR to the β‐globin gene promoter– Stimulated by the erythroid transcription factor NF‐E2

• Other transcription factors are required for the physical interaction between the β‐globin LCR and the β‐globin promoter


• Scattered throughout the genome at 5–200 kb intervals

• Responsible for formation of chromatin loops in eukaryotic genomes

• Plays a central role in transcriptional control.• Thought to organize the genome into ≈ 60,000 chromatin loops with (on average 70 kb/loop).– Active genes tend to be part of small looped domains ≈ 4 kb

– Inactive regions of chromatin larger domains of up to 200 kb.


• >70% of characterized MARs are AT rich.• Some MARs are GT rich and have the potential to form Z‐DNA.

• MARs are typically located near enhancers in 5′ and 3′ flanking sequences.

• They are thought to confer tissue specificity and developmental control of gene expression by– recruiting transcription factors– providing a “landing platform” for several chromatin‐remodeling enzymes.

– Some MARs include recognition sites for topoisomerase II.


• Two types of nuclear matrix-binding sites are proposed to exist within the loops

• Structural (constitutive) MARs serve as anchors• Functional (facultative) MARs are more dynamic and help to bring genes onto the nuclear matrix.


• 5‐10% of the total coding capacity of the genome of multicellular eukaryotes transcription regulatory proteins .

• These proteins fall into three major classes:– The general (basal) transcription machinery– Transcription factors– Transcriptional coactivators and corepressors


• Three major components of the general (basal) transcription machinery – RNA pol II– General transcription factors– Mediator

• The polymerase + other factors (general TFs) form a preinitiation complex on core promoters transcription initiation


• RNA pol II from the budding yeast Saccharomyces cerevisiae:– 12 subunits (Rpb1 to 12), numbered according to size – highly conserved among eukaryotes.– Contains a 10‐subunit catalytically active core

• Contain a positively charged “cleft.” • The nucleic acids occupy this deep cleft• 9 bp of RNA–DNA hybrid forms at the center


• A unique tail‐like feature = up to 52 heptapeptide repeats (aa consensus sequence: Tyr‐Ser‐Pro‐Thr‐Ser‐Pro‐Ser )

• Unphosphorylated CTD Transcription initiation • Phosphorylated CTD Elongation.

– Phosphorylation is catalyzed by TFIIH and other kinases (at positions 2 and 5 in the repeat during transcription)

– When phosphorylated, the CTD can bind mRNA processing factors

• CTD dephosphorylated again recycling of RNA pol II and reinitiation of transcription


• RNA pol II is absolutely dependent on them– promoter recognition – unwinding the promoter DNA – initiation of transcription

• Binding of TFIID recruits other general TFs and RNA pol II to the promoter.



• In vitro: FIIB TFIIF + RNA pol II + more proteins such as Mediator TFIIEand TFIIH which bind downstream of RNA pol II.

• In some cases, TFIIA is recruited prior to TFIIBand contributes to complex stability.

• TFIID = TATA‐binding protein (TBP) + 14 TBP‐associated factors (TAFs).– TBP

• Specifically recognizes the TATA box, as well as some other core promoter elements.

• highly conserved and appears to be required for transcription by all three eukaryotic RNA polymerases.

– Binding of TFIID to the core promoter is a critical rate limiting step

• Allows activators and/or chromatin remodeling factors to control transcription

• TAFs interact with the transactivation domains of specific transcription factors


• TFIIH contains at least nine polypeptide subunits with diverse functions.– a cyclin‐dependent protein kinase (CDK7)– cyclin H– a 5′ → 3′ helicase– a 3′ → 5′ helicase– others.

• TFIIH is the protein kinase that phosphorylates the CTD of RNA pol II,

• ATP‐dependent helicase activity was demonstrated for yeast TFIIH

• TFIIH functions not only in transcription but also in DNA repair


• The complex of core promoter, general TFs, and RNA pol II (in association with Mediator) is called the preinitiation complex (PIC).

• Promoter melting is mediated by the ATPase/helicase subunit of TFIIH, with the help of TFIIE.

• Unwinding is followed by “capture” of the nontemplate strand by TFIIF.

• The template strand descends to the active site of RNA pol II


• Transduces regulatory information from activator/repressor proteins to RNA pol II

• In vitro transcription assays – RNA pol II and associated factors can stimulate low levels of transcription (basal transcription).

– Core RNA pol II is not responsive to transcriptional activators that work in vivo.

– An additional factor is required for activator‐responsive transcription (Mediator)



• Connects transcriptional activators bound at enhancers, or other long-range regulatory elements, with RNA pol II

• Promotes preinitiation complex assembly

• Stimulates the kinase activity of TFIIH.

• Unique to eukaryotes ‐ expressed ubiquitously in eukaryotes from yeast to mammals.

• The majority of Mediator complexes act as transcriptional coactivators – there is a Mediator complex that represses transcription

– Mediator T/S can act as both a repressor and an activator


• Sequence‐specific DNA‐binding proteins – bind to gene promoters – Bind long‐range regulatory elements

• Changes in the amounts or activities of TFs regulation of gene activity

• Transcription factors themselves may be regulated by– transcriptionally induced or repressed expression by other regulatory proteins

– Proteolysis– covalent modification– ligand binding


• By:– specific interactions with DNA regulatory elements – interaction with other proteins

• Several mechanisms:– Stimulation of the formation of a preinitiation complex.

– Stimulation of the enzymatic activity of the general transcription machinery (induction of a conformational change, phosphorylation).

– Interaction with chromatin remodeling and modification complexes to permit enhanced accessibility


• Consist of three major domains– DNA‐binding domain– Transactivation domain– Dimerization domain

• Typically have a nuclear localization sequence (NLS)

• Some also have a nuclear export sequence (NES) • Some also have ligand‐binding (regulatory) domains, such as hormone‐binding domains, which are essential for controlling their activity.


• Proteins that increase or decrease transcriptional activity through protein–protein interactions without binding DNA directly.

• serve as scaffolds for the recruitment of proteins containing enzymatic activities– Or have enzymatic activities themselves for altering chromatin structure.

• Include chromatin remodeling and modification complexes


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