Transcription 2

Question 1

What is involved in DNA sequencing?

Answer 1

Determining the sequence of nucleotide bases in a length of DNA

Question 2

What 4 components are needed for chain termination (Sanger sequencing)?

Answer 2

1. DNA polymerase
2. primer
3. Four DNA nucleotides
4. The template DNA to be sequenced

Question 3

Why is a primer needed in Sanger sequencing?

Answer 3

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

Question 4

What type of nucleotides are needed for Sanger sequencing?

Answer 4

Dideoxynucleotides (ddATP, ddCTP, ddTTP, ddGTP)

These are chain-terminating nucleotides and each is labelled with a different coloured dye

Question 5

What makes dideoxy nucleotides different from normal deoxynucleotides?

Answer 5

They lack a hydroxyl group on the 3rd carbon of the sugar ring

Question 6

What does the hydroxyl group on the 3rd carbon of a deoxynucleotide allow for?

Answer 6

The hydroxyl group acts as a hook allowing a new nucleotide to be added to an existing chain

Question 7

What happens once a dideoxynucleotide has been added to the chain?

Answer 7

No further nucleotides can be added

No hydrogen bonds can form as there is no hydroxyl available

Question 8

What is the first stage in Sanger sequencing?

Answer 8

DNA sample is placed in a tube with a primer, DNA polymerase, DNA nucleotides and dye-labelled dideoxynucleotides

Question 9

What happens in Sanger sequencing once the initial mixture has been prepared?

Answer 9

The mixture is heated to denature the template strand

This causes it to become 2 separate single starnds

Question 10

What happens once 2 single separate strands have been produced in Sanger sequencing?

Answer 10

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

Question 11

When will DNA polymerase stop adding nucleotides in Sanger sequencing?

Answer 11

When it reaches a dideoxynucleotide

Question 12

How can dideoxynucleotides allow the sequence of bases in a DNA strand to be indentified?

Answer 12

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

Question 13

What happens to proteins after translation?

Answer 13

They either remain in the cytosol or are fed into the endoplasmic reticulum

Question 14

What are the 2 types of ribosome found within a cell?

Answer 14

Cytosolic ribosomes

Membrane-bound ribosomes (rough endoplasmic reticulum)

Question 15

What do membrane-bound ribosomes start off as?

what initiates the transformation process?

Answer 15

Cytosolic ribosomes

They bind to a mRNA that encodes a secretory protein

Question 16

What happens when the ribosome begins to translate the mRNA that encodes for a secretory protein?

Answer 16

The first part of the protein produced is a string of hydrophobic amino acids

This is the signalling sequence

Question 17

what is the signalling sequence?

Answer 17

The initial stretch of 20 hydrophobic amino acids

Question 18

What happens after the signal sequence has been translated?

Answer 18

A signal recognition particle binds to the signal sequence

This causes the ribosome to dock to the endoplasmic reticulum

Question 19

What happens once the ribosome has docked to the ER?

Answer 19

The newly synthesised polypeptide is fed through the ER as it is translated

Question 20

What is the role of a signal peptidase?

Answer 20

It removes the signal sequence which releases the protein into the ER

Question 21

What happens if the signal sequence becomes embedded into the ER membrane?

Answer 21

This creates a transmembrane protein

Question 22

Which proteins are fed into the ER during translation?

Answer 22

Only proteins that have a signal sequence

e.g. proteins that are bound for organelles in the endomembrane system or the cell exterior

Question 23

what happens to proteins that do not have a signal sequence?

Answer 23

They remain in the cytosol for the rest of translation

If they have other address labels, they will be transported to relevant organelles

Question 24

What happens to proteins in the ER?

Answer 24

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

Question 25

What proteins are not transported to the Golgi apparatus?

Answer 25

Ones that have amino acid tags which state that they must stay in the ER

Question 26

What is prenylation?

Answer 26

The addition of a lipid molecule to a protein

Question 27

What causes a protein to become prenylated?

Answer 27

If it has been made in the cytosol and has a particular amino acid sequence at the C-terminus

Question 28

What does the amino acid sequence at the C-terminus lead to?

Answer 28

Part of the C-terminus is removed

A fatty acid chain is transferred to the sulphur atom of the cysteine residue

Question 29

What is the purpose of prenylation?

Answer 29

The fatty acid tail can bury into the lipid blayer

This helps to hold it in the correct position within the cell