Lecture 1 Flashcards
Originally people through that DNA directly encoded proteins. Why is this wrong?
DNA is too stable to be the intermediate. The unstable intermediate is RNA. DsDNA is converted to RNA- bidirectional.
Death cap mushroom
Amanita phalloides- causes death by inhibiting the process of transcription. Produces alpha amanitin- transcription inhibitor.
Why is transcription and translation simpler in prokaryotes?
Everything occurs in one compartment.
What 3 things do you need for transcription to occur?
DNA template for complementary base pairing
4 ribonuclease triphosphates (ATP, CTP, GTP, UTP)
RNA polymerase
What is one advantage of using RNA polymerase?
It does not need a primer
Describe DNA
Deoxyribose no OH on 2' AGCT Nucleotides joined by phosphodiester bonds double stranded secondary structure is a helix stable
Describe RNA
Ribose presence of OH on 2' AGCU Nucleotides joined by phosphodiester bonds single stranded Many secondary structures Easily degraded
Ribosomal RNA
In the cytoplasm.
Role: binding of mRNA & tRNA and protein synthesis.
Messenger RNA
In the cytoplasm.
Role: carrier of gene sequence
Transfer RNA
In the cytoplasm.
Role: adaptor between mRNA and protein sequences- involved in translation
Mircro RNA
In the cytoplasm and nucleus.
Role: regulates transcription and translation
Primary and secondary structure of RNA
RNA has a primary structure (A) and a secondary structure (B)
Common RNA secondary structures
Tetraloops
Pseudoknots
Stem-loops
RNA polymerase clamp
Keeps the polymerase anchored to the DNA. Clamp made up of jaws.
DNA is held sideways with a sharp bed to its left as it exits the polymerase. Bending helps to force the two strands apart.
rNTPs (ribonucleotide triphosphates) enter the active site at the same side that DNA is pulled through, but through a secondary channel. mRNA leaves from the back of the polymerase and the flap ensures that mRNA is retained.
5 Subunits of bacterial RNA polymerase
two copies of alpha
single copy of Beta
Single copy of Beta prime
Single copy of omega
What does the core enzyme of Bacterial RNA polymerase catalyse?
the elongation of the RNA molecule by the addition of RNA nucleotides
How is the holoenzyme formed
The sigma factor joins the core to form the holoenzyme which is capable of binding to a promoter and initiating transcription.
Omega subunit function in bacterial RNA polymerase
stabilises the enzyme
Three stages of transcription
Initiation
Elongation
Termination
INITIATION
What region does RNA polymerase bind?
To the promoter region as directed by the protein-sigma factor.
INITIATION
What does the promoter region determine?
Where to start transcription
Which strand of DNA to transcribe
INITIATION
Which strand is the template and where does transcription begin?
Either strands can be templates
Part of each promoter region is the initiation site where transcription begins. DNA polymerase will open a bubble to start transcribing genes.
INITIATION
Describe promoter regions in bacterial DNA.
Promoters of all bacterial genes have regions of similarity. These consensus sequences are similar in all genes studied. They are hexameric (6 bases) sequences at -35 and -10 bases upstream of the start of transcription.
INITIATION
What is the commonly first transcribed base?
Adenine
INITIATION
Where does RNA polymerase holoenzyme bind?
The RNA polymerase holoenzyme (including the sigma factor) recognises the consensus sequences at -35 and -10 and binds.
The sigma factor recognises and binds to the -35 consensus to anchor the polymerase
INITIATION
What is the advantage of the sigma factor?
Allows RNA polymerase to bind more tightly with specific connections.
INITIATION
How do the sigma and beta prime factor work together?
They separate the two strands to make a bubble.
INITIATION
Where does RNA polymerase start to unwind the DNA?
At the Pribnow box (-10) and melting continues forward to cover the start site.
INITIATION
RNA polymerase holoenzyme slides along DNA but what happens when it recognises the promoter of a gene?
Promoter of gene is recognised through a sigma factor and the polymerase binds tightly to the DNA
INITIATION
What happens once the holoenzyme has bound to the active site?
The RNA polymerase holoenzyme and DNA undergo a series of conformational changes that consist of opening the DNA up and positioning it in the enzyme’s active site and then tightening the grip of the enzyme around it so it does not become detached before transcription is finished.
ELONGATION
How does synthesis start?
Opening of the double helix. DNA unwinds 10 base pairs at a time.
ELONGATION
What direction does RNA polymerase read the template strand?
3’ to 5’ direction
ELONGATION
What direction are the nucleotides added?
5’ to 3’ direction
ELONGATION
What molecules are used as substrates?
Ribonucleoside triphosphates
Two phosphate froups are removed from each substrate molecule to release energy to drive the polymerase reaction
ELONGATION
What links the nucleotides together in a chain?
Phosphodiester bonds
ELONGATION
What happens once the mRNA has begun to be synthesised?
The sigma factor is released from the holoenzyme and RNA polymerase moves down the chain in a more efficient manner.
ELONGATION
When does RNA polymerase unwind DNA?
As it moves along the DNA it makes a transcription bubble.
ELONGATION
How does polymerase make sure the mRNA are correct matches?
Proof reading function
ELONGATION
How quickly do mutations occur?
Mutations occur in mRNA at a rate of 10^4 to 10^5 bases, this is not detrimental as many copies of RNA are made and RNA has a short lifespan so is not as harmful as DNA mutations
TERMINATION
What specifies termination of transcription?
Particular base sequences
TERMINATION
When must transcription stop?
Once the coding sequence has been transcribed into mRNA
TERMINATION
When must the RNA polymerase be released?
The RNA polymerase releases the growing chain when it encounters a termination signal. The terminator is transcribed and all of the sequence appears in the mRNA.
TERMINATION
What can termination signals occur as?
Formation of a stem and loop secondary structure through H bonding between Gs and Cs
Protein binding - Rho dependent terminators
TERMINATION
How does the hairpin structure form?
The inverted repeat sequence of the terminator sequence means that the synthesised RNA molecule will pair to itself and form a hairpin.
TERMINATION
What is the purpose of the hairpin structure?
Formation of the hairpin helps to pull the transcript away from the RNA polymerase active site. The RNA chain is released. The hairpin is stable so its formation is thermodynamically favoured
TERMINATION
Intrinsic terminators
G-C rich stem followed by a run of A’s in the template strand. This produced a run of U residues in the transcript.
TERMINATION
Run of U’s
A=U pairing has only 2 hydrogen bonds which is weaker than the bonds in the stem loop so it becomes increasingly difficult for RNA-DNA hybrid to stay attached and the RNA gets released.
TERMINATION
Flap structure
The stem loop structure comes into contact with the flap, movement of the flap contributes towards the breakage of the RNA-DNA hybrid and the ultimate termination of the chain.
TERMINATION
Protein binding- Rho dependent termination
The Rho protein will attach to the transcript and move along the RNA towards the polymerase, but will never catch the polymerase. Polymerase stalls at the termination site and Rho catches up and breaks the base pairs between the DNA and RNA (helicase enzyme) which stops transcription
Why must transcription be controlled?
Not all proteins are needed all the time
Controlling transcription avoids energy waste
