Transcription in Eukaryotes
Atif Hassan KhirelsiedAtif Hassan Khirelsied, , B.Sc., M.Sc., Ph.DB.Sc., M.Sc., Ph.D. .
Department of BiochemistryDepartment of Biochemistry
Faculty of Faculty of MedicineMedicine
International University of International University of Africa,Africa, KhartoumKhartoum, Sudan., Sudan.
Eukaryotic gene transcription
E k ti i iti ti i lEukaryotic initiation is more complex.
About 50 different proteins (transcription factors) bind to promoter .
RNA polymerases bind several transcription factorsRNA polymerases bind several transcription factors.
The Eukaryotic Transcription Factors
Th th l t ( l t )• The are many other regulatory sequences (elements) in eukaryotic genes .
• The number and type of these elements varies with each gene.
The Eukaryotic Transcription Factors
• Eukaryotes have three types of RNA polymerases, I, II,Eukaryotes have three types of RNA polymerases, I, II, and III, and prokaryotes only have one type.
• Eukaryotes contain many different promoter elements: TATA b i iti t l t d tTATA box, initiator elements, downstream core promoter element, CAAT box, and the GC box
The basal promoter elements
Th b l t l t th CCAAT b ( t)The basal promoter elements are the CCAAT‐boxes (cat)
and TATA‐boxes .
The TATA‐box resides 20 to 30 bases upstream of the
l d l h ktranscriptional start site and is similar to the prokaryotic
Pribnow‐box .
Eukaryotic Transcription UnitEukaryotic Transcription Unit
The Eukaryotic PromoterThe Eukaryotic Promoter
The Eukaryotic RNA Polymerases
Eukaryotic cells have three distinct, RNA polymerases (pol) I, II and III.
Each polymerase synthesize a different class of RNA.Each polymerase synthesize a different class of RNA.
Classes of RNA pol merases ere identified b theirClasses of RNA polymerases were identified by their sensitivity to α‐amanitin.
The Eukaryotic RNA Polymerases
Diff t t f RNA th i d b diff t RNA P lDifferent types of RNA are synthesized by different RNA‐Polymerases.
The Eukaryotic RNA Polymerases
α‐amanitin inhibits RNA polymerases
The inhibitor of transcription
i h ll idAmanita phalloides
The Eukaryotic Transcription Factors
• Numerous Transcription Factors interact with the TATA• Numerous Transcription Factors interact with the TATA‐
box.
• TF interacting with are RNA polymerase II include:
– TFIIA
– TFIIB
– TFIID
– TFIIEReview their structural and functional features at the end of this presentationTFIIE
– TFIIF
– TFIIH
features at the end of this presentation
– TFIIH
Generalized scheme of binding of transcription factors to the promoter regions of eukaryotic cells.p g y
Following the binding of the transcription factors IID, IIA, IIB, IIE and IIF a pre‐initiation complex is formed RNA polymerase II then binds to thisa pre‐initiation complex is formed. RNA polymerase II then binds to this complex and begins transcription from the start point 1.
The Eukaryotic Transcription Factors
The basal transcription factors (BTF)
BTF th t i f t• BTFs are the proteins necessary for accurate
transcription.p
• They are distinct from the regulatory transcription
factors, which bind to sequences far away from the
start and serve tomodulate levels of transcriptionstart and serve to modulate levels of transcription.,
Eukaryotic transcription factors
• The protein C/EBP (for CCAAT‐box/ Enhancer BindingThe protein C/EBP (for CCAAT box/ Enhancer Binding
Protein) binds to the CCAAT‐box.
• Other transcrpition factors include:
1. CTF (CAAT Transcrition Factor).
2 Sp1 (specificity protein 1)2. Sp1 (specificity protein 1).
Eukaryotic transcription factors
Eukaryotic transcription factors
Diff t bi ti f t i ti f t t• Different combinations of transcription factors exert
differential regulatory effects upon transcriptional
initiation.
ll d ff b f• Various cells express different combinations of
transcription factors leading to tissue specific regulation of p g p g
gene expression.
Eukaryotic transcription factors
Eukaryotic transcription regulatory elements
Structural Motifs in DNA Binding Proteins
The helix‐turn‐helixThe helix turn helix
I t i th h li t h li (HTH) i j• In proteins, the helix‐turn‐helix (HTH) is a major structural motif capable of binding DNA.
• It is composed of two α helices joined by a short strand of amino acids
• It is found in many proteins that regulate gene expressionexpression.
Structural Motifs in DNA Binding Proteins
Structural Motifs in DNA Binding Proteins
Th L i ZiThe Leucine Zipper
• A leucine zipper, leucine scissors, is a super‐secondary structural motif with two parallel α ‐helices.p
• It is a common dimerization domain found in some proteins involved in regulating gene expression
Structural Motifs in DNA Binding Proteins
Structural Motifs in DNA Binding Proteins
The Zinc Finger
• Zinc fingers are small protein structural motifs that can coordinate one or more zinc ions to stabilize theircan coordinate one or more zinc ions to stabilize their folds.
• They function as interaction modecules that bind DNA RNA iDNA, RNA, proteins.
Structural Motifs in DNA Binding Proteins
Structural Motifs in DNA Binding Proteins
The helix‐loop‐helixThe helix‐loop‐helix
• This motif is characterized by two α‐helicesconnected by a loop.
• In general, transcription factors including this domain In general, transcription factors including this domainare dimeric, each with one helix containing basic amino acid residues that facilitate DNA binding.a o ac d g
Structural Motifs in DNA Binding Proteins
TFIIA
• Consists of three protein subunits in humans and
drosophila
• TFIIA binds directly to TBP (TATA binding protein) and
stabilizes its binding to DNA,
• TFIIA acts as an anti‐repressor, stabilizing TFIID binding byTFIIA acts as an anti repressor, stabilizing TFIID binding by
blocking repressors of transcription
TFIIBTFIIB
• Binds directly to TBP, recruits RNA polymerase II, through an interaction with the small subunit of TFIIF.
• Stabilizes TBP binding to TATA element• Stabilizes TBP binding to TATA element.
• Required for association of RNA polymerase II to the initiation complex.
TFIID = (TBP + TAFs)TFIID = (TBP + TAFs)
• Is multimeric (>5 subunits) transcription factor that• Is multimeric (>5 subunits) transcription factor that recognizes and binds to the promoter DNA.
• Consists of TATA‐binding protein (TBP), and several TBP‐i t d f t (TAF )associated factors (TAFs).
• It initiates transcription and recruits other transcription factors through a direct interaction with TFIIB.
TBPTBP
• The TBP protein resembles a saddle, with the inner
f t ti DNA d th t f ksurface contacting DNA and the outer surface make
protein‐protein contacts.p p
TFIIE
• Two subunits. Forming a tetramer of two molecules of
h b i h l b i h i fieach subunit, the large subunit has a zinc finger
domain.domain.
• Recruits TFIIH to the initiation complex and modulates
TFIIH kinase and helicase activities.
TFIIFTFIIF
TFIIF i f RNA l II t t bl• TFIIF is necessary for RNA polymerase II to stably
associate with the TFIIF‐TFIIB‐promoter complex.p p
• Helps recruit RNA polymerase II to the initiation
complex in collaboration with TFIIB.
TFIIHTFIIH
• TFIIHs have at least six subunits.
• The two largest TFIIH subunits are ATP‐dependent helicases.
• TFIIH is essential for promoter melting.