Eukaryotic Transcription Flashcards
What are the types of Eukaryotic RNA polymerases and where are they found?
- type I - 28S, 18S, 5.8 S rRNA
- type II - mRNAs most are snRNAs
- type III tRNAs, 5S rRNA
type I is in the nucleolus
Type II and III are in the nucleoplasm
There is only one in prokaryotes
Different types of RNA are generated in differenet locations
Give the characteristics of RNA polymerase II
- RNA pol II is made up of 10+ subunits
- the 3 subunits display similarities to bacterial RNA pol
- deletion analysis used to define promoter region
- promoter boundaries can be determined by making deletions that progressively remove more material from one side
- When one deletion fails to prevent RNA synthesis but the next stops transcription, the boundary of the promoter must lie between them
- define the minimal sequence requirment for promoter activity
- deletion of sequences that surround the TATA box and then removal of TATA box itself
Polymerase II Promoter region
- 90 bp(variable) upstream GC box
- 75 bp upstream CAAT box
- 25 bp upstream TATA box
- +1 is the start site
- presence of generic sequences, such as TATA box, CAAT box and some specific TF binding sites (GGGCGG for SP1)
Promoters have different combinations of TATA, CAAT and GC boxes
diversity of promoters suggests flexibility for pol II recruitment
multiple combinations are possible
TATA box is most common sequence that will help define promoter region
CAAT box is dispensable so are other sequences
TATA less promoter do exist and can support transcription
Initiation of transcription: interactions between TF, promoters, + RNA pol II
TF=Transcription faction
TBP=TATA Binding Protein
TAF=TATA binding protein Associated Factors
TFII D; consists of TBP or TATA-Binding Protein + TAFs (TATA-Binding Associated Factor)
Human TAFs and possible interactions with a transcription factor
The proteins binding to the enhancer sequence contain both a DBD or DNA Binding Domain and an AD: Activation Domain
Transcription factors that compose the various transcription complexes
Assembly of pre-initiation complex over the promoter region.
- TBP + TAFs = TFIID binds TATAA
- TFIIA binds upstream of TATA
- TFIIB binds downstream of TATA
- TFIIF bound to RNA pol II binds complex
- TFIIE bind to help initiate pol to start
- Addition of factors is not random but sequential.
- TFII D is first (containing the TATA binding activity).
- TFII A then binds upstream of TATA box.
- TFII B binds downstream of TATA box.
- RNA pol complexed with TFII F is then recruited, followed by TFII E downstream of the RNA pol plus TFs complex.
- TFIIH and H Bind
- TFIIH=kinase activity
- PC-terminal domain (CTD) of RNA pol II promotes promoter clearance
- activates RNA pol II + release of TFs
- Capping enzyme + splicing factors bind P-CTD
- TFII H possesses a Kinase activity that will be used to phosphorylate the C-Terminal Domain (or CTD) of the RNA polymerase II largest Sub-Unit.
- After phosphorylation of the CTD (changes in charge, more negative) the interactions with the DNA or mediator protein are reduced and the RNA polymerase can clear the promoter region.
- Partial release of TFs.
Note that the capping enzyme, necessary for mRNA modification and the release factors bind only to the phosphorylated form of the CTD.
RNA Polymerase II/Mediator Complexes transition from initiation to elongation
- Phosphorylation of CTD changes the electrostatic interactions between RNA pol II DNA and mediator(s) and favors the release of the ploymerase
- The CTD becomes accessible for elongation and processing factors
- Role of mediator may be to prevent the RNA pol from clearing the promoter region, through un-phosphorylated CTD-mediator interactions. The P-CTD becomes accessible for EFII to bind.
Model of transcription initiation complex assembly and promoter clearance for Pol II
- Binding of TBP of TFIID to minor groove of DNA (sequence-specific DNA binding)
- Assembly/Recruitment of Initiation Complex
- Pre-initiation Complex or minimal initiation complex
- Complete initiation Complex (closed)
RNA polymerase I promoter
RNA pol I uses different TFs: TFI
RNA promoter recognized only by RNA pol I
RNA pol I promoter has 2 elements
- upstream control element
- Core promoter
RNA pol I promoter are bi-partite, containg an upstream control element and often do not have a real TATA box
RNA pol I initiation complex
- upstream binding factor
- selectivity factor 1=TBP + 3 proteins
TFs: Upstream Binding Factor or UBF1 binds to G-C rich sequence
SL1 binds cooperatively with UBF1
Pol I
Promoters for RNA pol III can be upstream or downstream of start: (+55 to +80)
- 5S rRNA with Box A and Box C after startpoint
- tRNA where box A and box B are after start point
- U6 and snRNA with Oct PSE TATA before startpoint
Changes in location of binding sites results in difference in architecture of initiation complexes (note the location of RnA Pol III in U6snRNA)
All eukaryotic promoters rely upon TBP to place polymerase on promoter
- Pol I promoters - BP and SL1
- Pol II promoters TFIID and TBP
- Pol III promoters TFIIB and TBP
Eukaryotic Transcription Termination
Eukaryotic Transcription Termination:
- From Pol I transcription
- termination occurs >1000 bp past normal 3’ end of rRNA via cleavage
- 18 bp sequence recognized by outside factor
- From Pol III transcription
- unclear, possibly similar to bacterial
- rho-independent termination, minus the hairpin
Transcription termination specified by Poly A signal (Pol II transcript)
What additional Modifications to Pol II transcript
- Initiation
- Elongation
- 5’ cap applied
- cleavage where a complex of proteins that recognize and cleave after AAUAAA site
- Polyadenylation via polyA polymerase n= 20-200 A’s
poly-A regulate translation; aid in stablity
Cap: role in translation and prevents degradation
Cap structure is added during the synthesis of 1st 15-30 NTP
