RNA Transcription Flashcards
RNA overview
2’ OH group, uracil instead of thymine, and single stranded
macromolecule acts as genetic and catalytic molecule
3 classes: mRNA, tRNA, rRNA (and the miRNA)
act as genetic material in some viruses
no primer required (RNA pol binds to promoter regions of DNA)
transcriptome
sum of all RNA molecules produced in a cell under given set of conditions
DNA-dependent RNA polymerase requires
- DNA template
- all 4 ribonucleoside 5’ triphosphates (ATP, CTP, GTP, UTP)
- Magnesium
template vs. coding strand
serves as template for RNA synthesis
DNA strand that is identical in base sequence to RNA being synthesized
promoter found in template strand (differentiates between the 2)
always 5’ to 3’ synthesis (nucleotides added onto 3’ end)
How Mg2+ coordinates RNA synthesis
2 Mg2+ ions
1. First Mg2+ coordinates nucleophilic attack of 3’ OH on incoming alpha phosphate on new NTP
2. 2nd ion facilitates displacement of pyrophosphate
supercoils
RNA pol creates positive and negative supercoils to unwind bubble of DNA
topoisomerase relieves tension in the supercoils
negative coil at the exit side, positive on the DNA entry side
bubble formation
about 17 bp unwound at a time
8bp RNA-DNA hybrid region inside RNA pol (after which RNA peels away and DNA duplex reforms)
6 RNA pol in bacteria subunits and their functions
alpha - 2 subunits, assembly and binding to UP (upstream promoter) elements
beta - main catalytic subunit (top claw)
beta’ - DNA binding (bottom claw)
sigma - directs enzyme to promoter, each class of RNA pol holoenzymes have different σ subunit, binds transiently to core
omega - protects pol from denaturation
crab claw shape
UP elements
conserved promoter sequences
stimulate RNA synthesis at the promoter
-10 and -35 sequences are highly conserved, act as recognition sites for subunit σ70 in e coli
Transcription cycle in bacteria
AKA sigma cycle
1. directed by σ70 subunit bound to core enzyme, RNA Pol binds DNA promoter and forms closed complex
2. transcription bubble forms, forming open complex near -10 TATA box
3. Initiation causes conformational change which leads to elongation form and moving away from promoter (promoter clearance)
4. σ70 dissociates after elongation begins, replaced by NusA protein
5. transcription terminates and NusA dissociates
Transcription in eukaryotes requires
Typical eukaryotic Pol II promoter sequences
regulatory proteins
regulatory sequences affect pol II activity
Inr sequence - initiatory sequence at 1+
TATA box consensus sequence at -30
types of eukaryotic RNA polymerase
I: synthesis of rRNA
II: synthesis of mRNA and specialized RNA, can recognize 1000s of promoters, made up of 12 subunits in humans (overall conserved)
III: synthesis of tRNA, 5s rRNA and specialized RNA
CTD
carboxyl-terminal domain in Pol II in eukaryotes
long carboxyl terminal tail in RBP1 subunit of Pol II that consists of repeats of consensus heptad AA sequence
ex. yeast have 27 repeats, humans have 52 repeats
CTD is significant difference between bacterial and eukaryotic Pol II
Eukaryotic Pol II transcription mechanism
- TBP (TATA binding protein) binds to promoter, assembly of Pol II with all the TF which help Pol II bind to DNA (TFIIE and TFIIH last to bind)
TF II: A, B, D, E, F, H and CTD - Forms closed pre-initiation complex
- TFIIH is helicase and unwinds DNA to form bubble, forming open transcription complex
- TFIIE and TFIIH are released, CTD is phosphorylated by TFIIH
- Elongation complex, elongation factors help keep Pol II on
- Elongation factors dissociate, termination factors bind and CTD is dephosphorylated
- termination of RNA synthesis
TBP function
TATA binding protein - recognizes TATA box
