Lecture 7: Mechanisms of Eukaryotic Transcription Flashcards
- What is transcription? - RNA polymerases - Mechanism of basal transcription
What is transcription?
- Transcription is 5′ to 3′ on a template that is 3′ to 5′
- Coding (nontemplate) strand – The DNA strand that has the same sequence as the mRNA and is related by the genetic code to the protein sequence that it represents
- RNA polymerase: An enzyme that synthesizes RNA using a DNA template
- > formally described as a DNA-dependent RNA polymerase
Describe and explain the structure of a gene
- Promoter: A region of DNA where RNAP binds to initiate transcription
- Starpoint: The position on DNA corresponding to the first base incorporated into RNA (begins at +1)
- Terminator: A sequence of DNA that causes RNAP to terminate transcription
- Transcription unit: The sequence between sites of initiation and termination by RNAP; it may include more than one gene
Explain RNA polymerisation
The 3’-OH group in ribose of the last ribonucleotide reacts with an incoming ribonucleoside 5’ triphosphate-> to be added to chain
State how many subunits does each RNAP have
I= 14 II= 12 III= 17
State where each RNAP is located and their RNA products
I-> nucleolus-> pre-rRNA
II-> nuceloplasm-> pre-mRNA= some snRNAs
III-> nuceloplasm-> t-RNA, 5s rRNA, U6 snRNA + 7SL RNA
List the RNA subunits that are shared
Rpb5, Rpb6, Rpb8, Rpb10, Rpb12
Explain what the transcription bubble is
- RNAP separates the 2 strands of DNA in a transient “bubble.”
- Uses 1 strand as a template to direct synthesis of a complementary sequence of RNA
- The length of the bubble is ∼12 to 14 bp
- The length of RNA-DNA hybrid within it is ∼8 to 9 bp
Explain the overview of transcription
- RNAP binds to a promoter site on DNA to form a closed complex
- RNAP initiates transcription after opening the DNA duplex to form a transcription bubble =the open complex
- RNAP the escapes the promoter from the initiation site
- Elongation-> transcription bubble moves along DNA
- The RNA chain is extended in the 5′–3′ direction by adding nucleotides to the 3’ end
- When transcription terminates or stops-> the DNA duplex reforms + RNAP dissociates at a terminator site
Explain the importance of TFs
- RNAP cannot initiate transcription from specific start sites without the assistance of other proteins-> transcription factors
- Each RNAP has its own specific set of transcription factors (TFIX, TFIIX, TFIIIX)
- > assist in locating Pol I, II or III transcriptional start sites
- Around each transcription start site is a core promoter
Explain what focused initiation is
- > starts from a single nucleotide or within a cluster of several nucleotides
- > Predominant means of transcription in simpler organisms
- > Regulated genes tend to have focused promoters
Explain what dispersed initiation is
- > there are several weak transcription start sites over a broad region of about 50 to 100 nucleotides
- > observed in approximately two-thirds of vertebrate genes
- > constitutive genes typically have dispersed promoters
Describe and explain the Eukaryotic core Pol II promoter motifs
- TATA box: TATAXAX consensus sequence (X is A or T) located 25-30 bp upstream of the transcription start site
- Initiator element (Inr): overlaps the transcription initiation site
- Downstream promoter element (DPE): extends from about +28 to +34
- TFIIB recognition element (BRE)
- XCPE1: present in TATA-less human core promoters (1% human genes)
Explain the transcription initiation process
- TBP region of TFIID recognises TATA box with aid of TFIIA
- TFIID + TFIIA recruit TFIIB
- TFIIB binds to complex
- TBP bends the TATA box around the C terminal domain of TFIIB
- The N-terminal domain of TFIIB brings the complex to RNAPII to join+ positions the transcription initiation site in the active site of RNAP
- The RNAPII-TFIIB- promoter complex recruits TFIIE and binds
- TFIIE recruits TFIIH and bind-> PIC formed + closed complex
- Helicase activity of TFIIH unwinds DNA in the area of the initiation site
- TFIIF captures the non-template strand after unwinding
- The template strand descends to the active site-> open complex
Explain the function of each TFIID in the PIC
-TBP is the TATA binding subunit of TFIID
Binds TATA element+ deforms promotor
-Platform for the assembly of TFIIB + TAFs
-Bind Inr, MTE, DPE + DCE promotor elements
Explain the function of each TFIIA in the PIC
-Stabilises TBP +TFIID binding –Blocks inhibitory effects of TAF1 + other proteins
Explain the function of each TFIIB in the PIC
- Stabilises TFIID- promotor binding
- Contributes to transcription start site selection
- Helps to recruit RNAPII-TFIIF to core promotor
Explain the function of each TFIIF in the PIC
- Binds RNAPII
- involved in recruiting polymerase for PIC
- Required to recruit EFIIE+EFIIH to PIC
Explain the function of each TFIIE in the PIC
- Helps to recruit TFIIH to core promotor
- required for promotor melting
Explain the function of each TFIIH in the PIC
- Functions in transcription + DNA repair
- has kinase+ helicase activities
- is essential for open complex formation
Explain what TBP is and what it does
- TATA-binding protein (TBP) is saddle shaped and bends DNA by 80°
- Interaction between TBP and the TATA box involves conformational changes in both TBP and the TATA box
- TBP interacts with TATA in the minor groove
Explain what TAFs are and what they do
- TBP-associated factors (TAFs) bind promoter elements other than TATA
- Additional proteins present in TFIID are called TBP-associated factors (TAFs)
- TFIID contains a core set of 13 TAFs
- TAFs are required for high levels of transcription & to transcribe genes that lack a TATA box
- TATA-less promoters ->the TAFs that bind to regulatory elements
Explain the overview of transition from initiation to elongation
- Open complex formation: initiation of transcriptional bubble
- RNA chain initiation: first 2 ribonucleotides line up on template strand in the transcription bubble + RNAP catalyses phosphodiester bond formation
- Abortive transcription: formation of short transcripts which are released
- Promoter escape: chain extension beyond 7 ribonucleotides-> triggers TFIIB release
=formation of the transcriptional elongation complex
State what part of RNAP is phosphorylation for elongation
Carboxyl terminal domain (CTD) of the largest subunit in RNAP 2 (Rpb1)
List the properties of TFIIH
- 10 subunits
- ring shaped
- 5’3’ and 3’ 5’ helicase activities
- cyclin-dependent protein kinase (CDK) activities
- ATPase
Explain the phosphorylation process of CTD for promoter clearance
- Phosphorylation of Ser5 by TFIIH-> recruits 5’ end capping enzymes on primary transcript
- Phosphorylation of Ser 2 by P-TEFb-> activates elongation, splicing + poly adenylation
- Phosphorylation on Ser7 by unknown kinase-> ?
- Dephosphorylation of Ser 5 by Ser5 phosphatase
- Polyadenylation factors bind to Ser2 phosphorylated repeats w/ peptidyl-prolyl bonds in trans configuration
- Dephosphorylation of Ser2 by Ser2 phosphatase
- Dephosphorylation of Ser7 by potential Ser7 phosphatase
- RNAPII able to be recycled + bind to another promotor
Explain the importance of phosphorylation of CTD
- Phosphorylation of CTD is involved in processing mRNA transcript
- > provides binding/recognition sites for mRNA processing
Describe the overview of elongation
- More stable than the initiation complex
- Approx. 14 bp are melted to form the transcription bubble
- First eight nucleotides within bubble are paired with the RNA chain
- Double stranded DNA opens up in front of the bubble and closes up behind the bubble as RNAP moves along
Explain why RNAP doesn’t have a steady pace during elongation
- elongation is temporarily delayed at pause sites
- pausing -> arrest and termination
- > arrested by Mg ion at active sit or RNAPII
- > arrested is an important step in proofreading
- TFIIS role -> reactivates arrested RNA polymerase II
Describe what makes up the promoter for RNAP I
- RNA polymerase I promoters consist of:
- core promoter
- upstream promoter element (UPE)
Explain how RNAP I binds to the promoter
- The factor UBF1 binds to UPE -> wraps DNA around a protein structure
= brings core and UPE close together - RNAP I holoenzyme= core binding factor SL1 + RNAP I
->binds to core promotor - SL1 includes TBP factor-> involved in initiation by all 3 RNAPs
- RNAP I binds to the UBF1-SL1 complex at the core promoter
Describe what makes up the promoter for RNAP III
- RNAP III has 3 types of promoter- 2 of which are internal promoters
- Internal promoters: have short consensus sequences located within the transcription unit
- > cause initiation to occur at a fixed distance upstream
- Upstream promoters: contain three short consensus sequences upstream of the start point that are bound by transcription factors
Explain how RNAP III binds to the promoter
- TFIIIA + TFIIIC bind to the consensus sequences
= enable TFIIIB to bind at the startpoint
- TFIIIB has TBP as one subunit -> enables RNA polymerase to bind
= PIC formed
state the difference between Internal type 1 pol III promoter +Internal type 2 pol III promoter
- Internal type 2 pol III promotor-> 2 TFIIIC bind to short consensus sequences
- Internal type 1 pol III promotor -> TFIIIA + TFIIIC bind to short consensus sequences
