Transcription in E. coli Flashcards
Highlight the details of an experiment used to prove that RNA synthesis occurs in 5’ to 3’
What are the pros of no nuclear membrane in prokaryotes?
What is the con?
Phosphorylation of 3’deoxyadenosine (RNA nucleoside where 3’ has H instead of OH) AND phosphorylation at 5’ HOCH2, but doesn’t produce RNA as can’t form the phosphodiester bond at 3’ H
Processes occur quickly & respond rapid change in environment
Transcription & translation are not separated in time & space - so going to live shorter
What is the RNA polymerase holoenzyme made up of? (subunits)
What is its core enzyme?
Which subunit binds to the promotor sequence?
What happens after this binds?
What is +1 on the DNA?
2a, ß, ß’, sigma
2a, ß, ß’
Sigma
Other subunits assembled
Site on DNA where first RNA nucleotide transcribed (promotor is upstream)
What two consensus sequences are found in sigma70 promotors?
What do they each do?
What is the amount of space between the two regions?
What is the more downstream region called?
-35 and -10 (TATA BOX)
-35: recognition site with bases specific for proteins/subunits to bind
-10: orientates the RNA polymerase to move 5’ to 3’
17bp
TATA box
The DNAse 1 Footprint experiment demonstrated that RNA polymerase binds to DNA. What reactants are in the two tests?
What were the results in both tests?
How was this shown after gel electrophoresis?
What method can be used to determine the DNA sequence?
- DNA & DNAse 1
- DNA, DNAse1 & RNA polymerase
- Phosphodiester bonds were cleaved resulting in DNA nucleotide fragments
- Phosphodiester bonds were only cleaved where RNA polymerase wasn’t bound
Footprint after running 2nd reaction
Chemical cleavage method of DNA sequencing
What do the different domains/subunits of the RNAP do?
Specifically, what does ß do?
How did experimental evidence prove this?
What was the result?
What are the 3 phases of transcription?
alpha = core enzyme/promotor recognition
sigma = promotor recognition
ß = catalytic domain
ß’ = template (DNA) binding domain
Binds ribonucleoside triphosphates & forms phosphodiester bond between them
Mixed RNA polymerase with radioactive uracil (exclusive to RNA) triphosphate which binds to the beta subunit & separate the subunits by cellulose acetate chromatography
Band corresponding to ß subunit is radioactive
Initiation - inserting nucleotide into correct position
Elongation - forming phosphodiester bond
Termination - recognising the end of the gene
Describe the closed promoter complex of RNAP before initiation
Describe the open promoter complex on initiation
What region on the promoter sequence aids melting?
What is formed?
Complex is bound to DNA & subunit assembled, both DNA strands still bonded
DNA unwinds at the front of the enzyme & re-winds behind, sigma factor dissociates (no longer needs to recognise promotor)
TATA box
Transcription bubble
What happens during the elongation phase of transcription?
What does the continuous melting (front) and movement form?
What are the 3 things that must happen in termination?
correct ribonucleoside triphosphates bind & form phosphodiester bond in ß domain
Transcription bubble
- RNA synthesis must end
- RNA needs to be released
- RNA polymerase needs to dissociate so DNA can reanneal
Give a basic description of rho independent intrinsic termination & rho dependent termination?
What are intrinsic terminators?
What is crucial at the end of the RNA sequence?
What more detailed occurs in rho independent termination?
Rho independent: forms a GC stem loop to release RNA
Rho dependent: rho binds & helps release RNA
Sequences of GC rich regions in the RNA
Uracil rich region
Stem loop forms in mRNA due tho GC complimentary regions, and weak uracil:adenine pairing from RNA:DNA helps release RNA polymerase & RNA
What kind of protein is rho?
What does it bind to?
How does rho terminate transcription?
Which termination type is more dominant in e coli and why?
Hexamer (6 identical subunits)
rut site 3’ of mRNA (c-rich)
Rho binds to rut & moves along RNA & releases it with ATPase activity - cleaves & releases RNA
rho independent - fewer materials & less ATP demanding
on the produce mRNA after transcription:
what is the 5’ leader sequence?
what is the 3’ trailer sequence?
what is the similarity in both of these sequences?
UTR untranslated region upstream of AUG/protein coding seq for ribosome binding & regulates mRNA production
rho independent terminator (U tail)
non-translated regions
