1) Transcription Flashcards
What is the central dogma? Why are the series of steps needed?
DNA –> RNA –> Protein
- DNA is too stable to be an intermediate to determine whether proteins are made= Need something that is more unstable
- Originally thought it was unidirectional BUT it is now shown that RNA can be reverse transcribed to DNA using reverse transcriptase
- RNA are able to be produced only when needed, then broken down which makes them the appropriate intermediate
- Double stranded DNA molecules are transcribed to produce single stranded RNA molecules
What is transcription?
- Occurs in prokaryotes and eukaryotes using the enzyme RNA polymerase
- DNA —> RNA
- Involves creating a single stranded complementary RNA copy of a DNA template
- Sense strand= 5’ to 3’
- RNA made so that it is complementary to the template which runs from 3’ to 5’= RNA copy now also runs from 5’ to 3’
- Transcription may occur at many different positions along a bacterial chromosome
What are the differences between DNA and RNA?
1) DNA has DEOXYRIBOSE while RNA is made of RIBOSE sugar
2) DNA has THYMINE as there is a methyl group on the carbon while RNA has URACIL as there is just a CH group
But bases in RNA are also joined by phosphodiester bonds
What are some examples of other RNA molecules and what do they do?
Not all RNA molecules are transcripts
Examples: rRNA and tRNA (involved in translation)
What is the structure and function of RNA polymerase?
Function: Enzyme which transcribes DNA
1) Clamp: Keeps polymerase anchored to DNA (made up of ‘jaws’)
2) Flap: Ensurers mRNA is retained and does not detach from the enzyme
3) Active site: Middle of enzyme, where newly synthesised RNA strand is made
DNA does not enter the mouth of the polymerase directly= Usually held sideways with sharp bend to help force the two DNA strands apart
mRNA= Leaves from back of polymerase
Also have ribnucleotide triphosphate which enter the active site on the same side that DNA is pulled through but through a secondary channel
What are the different subunits and states which bacterial RNA polymerase can exist in?
States:
1) Holoenzyme= whole, complete= 5 subunits
2) Core enzyme= 4 subunits, loses sigma subunit , carries out bulk of transcription during elongation
Subunits: separate proteins
2x α (alpha)= Promotor binding= Helps polymerase bind to the promotor
1x β (beta)= Nucleotide binding= Controls the movement of the chain through the system, base by base
1x β’ (beta prime)= Template binding
1x σ (sigma )= Initiation= Anchors the polymerase to the right place to transcribe the gene + helps to separate the two strands to make the bible. This subunit dissociates once it has completed its functions
What are the 3 stages of transcription? (Names only)
1) Initiation
2) Elongation
3) Termination
RNA polymerase needs to first find the correct section of DNA to start transcribing, as the genes are arraigned in very long DNA molecules (chromosomes) so it cannot just start transcribing at the beginning of a chromosome
What is a promotor?
Initiation:
Very time and energy consuming if RNA polymerase transcribed junk DNA= needs to know where to actually transcribe
Promotor: Upstream (to the left or the 5’ end) of the coding sequence of a gene (on sense strand)
3’ end= Transcription terminator
Promotes are also called consensus sequences= Similar in all genes as they are hexametric (6 bases) sequences at -35 and -10 bases upstream of the start of transcription
What happens during the first stage of transcription?
INITIATION:
1) RNA polymerase in holoenzyme state recognises the consensus sequences at -35 end= start of transcription
2) Sigma factor binds tightly to the promotor of the gene by the polymerase and DNA undergoing a series of conformational changes- Open up DNA, position it to active site and tighten grip of enzyme around it
3) RNA polymerase start to unwind (melt) the DNA at Pribnow box (-10)
4) Melting continues forward to cover the start site
5) mRNA begins to be synthesised (few bases) and sigma factor is released from holoenzyme
What happens during the second stage of transcription?
ELONGATION: lengthening of new RNA strand
1) RNA polymerase continues to unwind DNA as it goes= makes transcription bubble
2) Free ribonucleotide triphosphate bind to form new chain as RNA pol catalyses the formation of phosphodiester bonds to link the nucleotides together= 3’OH group attaches to 5’ phosphate of a new nucleotide
3) Polymerase makes sure they are the correct matches by using proof reading function
What happens during the third stage of transcription?
TERMINATION
1) Inverted repeat sequence of the terminator sequence= Synthesised RNA molecule will pair to itself= Form hairpin structure
2) Makes it very difficult for Pol flap to hold onto the RNA= pulled away form active site
3) RNA chain is released
Bacteria: 2 types of termination signals and both involve inverted repeats
How does a stem and loop secondary structure form?
What are the two different types of terminators? (Names only)
Through hydrogen bonding between Guanines and Cytosines
Terminators:
1) Intrinsic terminators
2) Rho-dependent terminators
What are intrinsic terminators?
Have a G-C rich stem followed by a run of As in the template strand= produces run of U residues in transcript
They have more A-U bonds than G-Cs which makes it easier to release RNA chain as they are weaker bonds as they have less
Hairpin structure is also very stable
More thermodynamically stable than DNA-RNA hybrid formed= More difficult to continue transcription
Also: RNA hairpin may come into contract with flap structure on outer surface of the beta subunit= Movement of flap= Breakage of DNA-RNA hybrid may be increased
What are Rho-dependent terminators?
Usually inverted repeats but NO RUN OF As in the transcribed strand
Rho protein is needed for the termination= Rho attaches to the transcript and moves along the RNA towards the polymerase
Rho= Purses the polymerase but is always behind it, until the Pol stalls at termination site the Rho uses helices enzyme to break base pairs between DNA and RNA
