Transcription 2 Flashcards
(29 cards)
What is involved in DNA sequencing?
Determining the sequence of nucleotide bases in a length of DNA
What 4 components are needed for chain termination (Sanger sequencing)?
- DNA polymerase
- primer
- Four DNA nucleotides
- The template DNA to be sequenced
Why is a primer needed in Sanger sequencing?
The primer is a short piece of single-stranded DNA
It binds to the template DNA and acts as a starting point for the polymerase
What type of nucleotides are needed for Sanger sequencing?
Dideoxynucleotides (ddATP, ddCTP, ddTTP, ddGTP)
These are chain-terminating nucleotides and each is labelled with a different coloured dye
What makes dideoxy nucleotides different from normal deoxynucleotides?
They lack a hydroxyl group on the 3rd carbon of the sugar ring
What does the hydroxyl group on the 3rd carbon of a deoxynucleotide allow for?
The hydroxyl group acts as a hook allowing a new nucleotide to be added to an existing chain
What happens once a dideoxynucleotide has been added to the chain?
No further nucleotides can be added
No hydrogen bonds can form as there is no hydroxyl available
What is the first stage in Sanger sequencing?
DNA sample is placed in a tube with a primer, DNA polymerase, DNA nucleotides and dye-labelled dideoxynucleotides
What happens in Sanger sequencing once the initial mixture has been prepared?
The mixture is heated to denature the template strand
This causes it to become 2 separate single starnds
What happens once 2 single separate strands have been produced in Sanger sequencing?
The mixture is cooled so that the primer can anneal to the single-stranded template
The temperature is raised again so DNA polymerase can synthesise new DNA in the 5’ to 3’ direction
When will DNA polymerase stop adding nucleotides in Sanger sequencing?
When it reaches a dideoxynucleotide
How can dideoxynucleotides allow the sequence of bases in a DNA strand to be indentified?
The process is repeated until a dideoxynucleotide is incorporated at almost every single position of the target DNA
The DNA fragments are arranged in order of increasing length
The terminating nucleotide can be identified by the colour of the dye
What happens to proteins after translation?
They either remain in the cytosol or are fed into the endoplasmic reticulum
What are the 2 types of ribosome found within a cell?
Cytosolic ribosomes
Membrane-bound ribosomes (rough endoplasmic reticulum)
What do membrane-bound ribosomes start off as?
what initiates the transformation process?
Cytosolic ribosomes
They bind to a mRNA that encodes a secretory protein
What happens when the ribosome begins to translate the mRNA that encodes for a secretory protein?
The first part of the protein produced is a string of hydrophobic amino acids
This is the signalling sequence
what is the signalling sequence?
The initial stretch of 20 hydrophobic amino acids
What happens after the signal sequence has been translated?
A signal recognition particle binds to the signal sequence
This causes the ribosome to dock to the endoplasmic reticulum
What happens once the ribosome has docked to the ER?
The newly synthesised polypeptide is fed through the ER as it is translated
What is the role of a signal peptidase?
It removes the signal sequence which releases the protein into the ER
What happens if the signal sequence becomes embedded into the ER membrane?
This creates a transmembrane protein
Which proteins are fed into the ER during translation?
Only proteins that have a signal sequence
e.g. proteins that are bound for organelles in the endomembrane system or the cell exterior
what happens to proteins that do not have a signal sequence?
They remain in the cytosol for the rest of translation
If they have other address labels, they will be transported to relevant organelles
What happens to proteins in the ER?
They are folded into their correct shapes and may have sugar groups attached to them
They are then transported to the Golgi apparatus in membrane vesicles
