VL 22 (Michael Lenhard) Flashcards
Important definitions
RNA polyermase
(DNA-dependent RNA polymerase)
* E
* DNA template→RNA
Promoter:
DNA region where RNA Pol binds to initiate transcription
Start point:
DNA position corresponding to first base incorporated into RNA
Terminator:
DNA sequence that causes RNA Pol to terminate transcription
Transcription unit:
sequence between sites of initiation and termination by RNA Pol;
may include > 1 gene
Transcription Occurs by Base Pairing in a “Bubble” of Unpaired DNA
- DNA melted→transcription bubble
- template strand used for complementary RNA-strand synthesis
- coding strand (nontemplate) around RNA Pol
Transcription reaction has 3 stages:
1. Initiation
–> Template recognition: RNA polymerase binds to duplex DNA
–> DNA is unwound at promoter
–> Very short chains are synthesized and released
2. Elongation
–> Polymerase synthesized RNA
3. Termination
–> RNA polymerase and RNA are released
Bacterial RNA Polymerase
Consists of Multiple Subunits
Holoenzyme:
* RNA Polymerase form that is competent to initiate transcription
* consists of 5 SU of the core E + σ factor
RNA Polymerase Holoenzyme Consists of the Core Enzyme and Sigma Factor
- Holoenzyme
–> α2ββ ́ω core E: catalyzes transcription
–> σ SU: only required for initiation
–> sigma factor changes DNA-binding properties of RNA Polymerase
→affinity for general DNA is reduced + for promoters increased - Sigma factor drives RNA Pol away from non-promoter DNA→RNA Pol binds more tightly to promoter DNA
Sigma factor controls DNA-binding by recognizing specific sequences in promoters:
conserved sequence:
sequences in which many examples of particular nucleic acid/protein are compared and same individual bases/aa are always found at particular locations
A promoter with short consensus sequences at specific locations
- purine at start point
- hexamer with sequence close to TATAAT at -10 (-10 element/TATA box), * hexamer with sequence similar to TTGACA at -35 (-35 element)
–> Individual promoters usually differ from consensus at on/more positions
RNA Polymerase–Promoter and DNA–
Protein Interactions Are the Same for Promoter Recognition and DNA
Melting
- purified protein of interest
- DNA substrate bound by protein of interest; label DNA (e.g. radioactive) on
one end of one ss
–> DNA flanked by 2 restriction E sites (blunt cut + 5 ́ overhang)
–> DNA Pol to fill in the resist 3 ́ end with labelled nucleotides → dsDNA - DNAase A (nonspecific DNA cleavage)
- DNA-bound protein protects DNA from digestion
- isolate DNA
- denature → ss
- ss → gel electrophoresis with high-precision, high-res gels
- control gel: no DNA-bound protein present
- missing bands, because DNase couldn ́t get DNA-access
- → define DNA-binding sites of specific proteins at single base resolution
Termination – Bacterial RNA Pol terminates at discrete sites:
- Two classes of terminators
–> Intrinsic terminators (don ́t need additional proteins to act)
–> Rho-dependent terminators (require Rho factor) - The DNA sequences required for
termination are located upstream
of the terminator sequence - Terminator sequence in DNA → hairpin structure in RNA
Pictures
Left:
* sequences required for termination are located in transcribed region → stops transcription after it has transcribed through it
Right:
* RNA can fold up by bp
–> stem region: RNA ss paired up with itself
–> loop region → stem-loop region
–> ss-U-run → no stable bp → RNA Pol gets destabilized;
backtracking prevented by stem-loop region → RNA Pol released
How Does Rho Factor Work?
- rut (rho utilization site) sequence present in nascent RNA
- hairpin structure/paused transcription
→ rho catches up
→ unwind DNA/RNA hybrid + release nascent RNA + RNA Poldissociates from template - Rho, core E → recycled
Supercoiling Is an Important
Feature of Transcription
- Neg. supercoiling increases efficiency of some promoters by assisting the melting reaction
- Transcription generates positive supercoils ahead of the E + neg. supercoils behind it → must be removed by gyrase + topoisomerase
- topoisomerase, gyrase cut one ss → wind one ss around the other → reseal
Competition for sigma factors can regulate initiation
- different σ factors can compete for core E-association → direct holoenzyme to different promoter sets → switch on different genes
- anti-sigma factor: protein that binds to σ factor to inhibit its ability to utilize specific promoters
- example: heat shock
Antitermination Can Be a Regulatory Event
- antitermination complex allows RNA Pol to read through terminators
- N utilization site (nut):
–> DNA sequence that is recognised, bound by N (N-protein) antitermination factor - Phage lambda uses antitermination systems (different mechanism) for regulation of both (early/late transcripts)
Eukaryotic transcription
- chromatin must be open → RNA Pol binds promoter
- basal TFs: TFs required by RNA Pol II to from the initiation complex at all RNA Pol II promoters
–> factors are identified as: TFIIX, X: letter - core promoter: shortest sequence at which RNA Pol initiates transcription (lower level than displayed by a promoter containing additional elements)
–> core promoter doesn ́t sustain high levels of transcription
–> for RNA Pol II: min. sequence; basal transcription apparatus assemble; includes three sequence elements (Inr, TATA box, DPE)
–> 40bp - enhancer
–> cis-acting sequence (affect activities only on the same molecule on which they themselves are located; specific binding site for certain protein)
–> increases the utilization of (most) eukaryotic promoters
–> can function in either orientation + any location (up-/downstream) relative to promoter
There are Three Eukaryotic RNA Polymerases.
- RNA polymerase I synthesizes rRNA in the nucleolus.
- RNA polymerase II synthesizes mRNA in the nucleoplasm.
- RNA polymerase III synthesizes small RNAs in the nucleoplasm.
- heterogeneous nuclear RNA (hnRNA) – RNA that comprises transcripts of nuclear genes made primarily by RNA polymerase II; it has a wide size distribution and variable stability
The start point for RNA Pol II:
RNA Pol II
–> requires general TFs (TFIIX; basal transcription factor for RNA Pol II) to initiate transcription
–> promoters frequently with short conserved sequence Py2CAPy2 (initiator Inr) at start point
TATA box
–> component of RNA Pol II promoters
–> AT-rich oxtamer; 25 bp upstream of start point
Downstream promoter element (DPE)
–> component of RNA Pol II promoters
–> don ́t contain TATA box (TATA-less promoter)
core promoter for RNA Pol II includes Inr + either TATA box/DPE + eventually other minor elements
Initiation is followed by promoter clearance and elongation:
- CTD coordinates RNA processing with transcription
- histone octamers must be temporarily modified during the transit of RNA Pol
- Inr = initiator/+1-site
Picture
* 7 aa stretch repeated in c-terminal domain + different post-translational CTD-modification states→coordinate transcription by RNA Pol II by other activites that are needed to process DNA
Enhancers contain bidirectional elements that assist initiation:
enhancer
* activates promoter
* any distance up-/downstream of promoter
upstream activating sequence (UAS)
* yeast
* behaves like enhancer o upstream of promoter
Picture
* mediator complex bridges between enhancer- bound TFs + basal transcription machinery at core promoter by bending / looping back
→ physical proximity enhancer sequence – core promoter
