L. 5 Copying DNA: Replication and Transcription

Question 1

L.O.

Answer 1

1. Outline the general mechanism for copying DNA to DNA before cell division - replication.
2. List the unique problems associated with replication and describe the strategies used by the cell to overcome these, including unwinding DNA, and leading versus lagging strand replication.
3. Describe the general functions of proteins that are required for DNA-replication
4. Outline the general mechanism for copying DNA to RNA - transcription.
5. List the unique problems associated with transcription (including unravelling DNA, and making multiple copies of small sections of the genome at different frequencies) and describe the strategies used by the cell to overcome these.
6. Describe the general functions of proteins that are required for RNA transcription
7. Compare and contrast the differences between making DNA and RNA

Question 2

Replication of DNA

Answer 2

• A complementary copy od DNA is created from a template strand od DNA
• Uses DNA Polymerase enzyme
• A Primer (short DNA/RNA piece) to start transcription
• Always builds in opposite direction of 5’ to 3’
• Nucleotide trophosphates are used as a substrate
• A monophosphate is added to the OH at the 3’ end.

Question 3

DNA Polymerase

Answer 3

• Making a DNA copy of a DNA template
• Needs a primer to start
• Works from 3’ to 5’, BUT builds from 5’ to 3’ direction
• Uses deoxynucleotide triphosphates as substrates
• Has a ‘proof read’ effect where it can tell if it made a mistake and remove a mismatched nucleotide

Question 4

Bacterial DNA replication

Answer 4

• Genomes are big and made up of long strands of dsDNA, which is circular
• Strands are complementary to one another

Question 5

Semi-conservative replication model

Answer 5

• Suggets that each newly generated dsDNA contains one original (the template) and one new strand
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Question 6

Helical DNA to be unwound…

Answer 6

• Pulling long helical strands apart leads to supercoiling
• Topoisomerase/ gyrase enzyme cuts the DNA strands allowing it to unwind and stick back together
• Only small sections are delt with at a time to avoid messes

Question 7

Biopolymer Synthase
- 3 steps

Answer 7

Initiation:
- Where, when & how to start

Chain Elongation:
- How the parts are added

Termination:
- Where, when & how to stop

Question 8

Initiation process

Answer 8

1. Origin is very AT-rich, it is easier to pull stands apart here
2. DNA binding proteins open the site to begin the process
3. DNA Helicase unwinds the DNA in small chunks (unzips), causing supercoiling
4. DNA topoisomerase/ gyrase stops the supercoiling
5. Replication forks form as Helicase and Gryase work in both directions of the DNA circle
6. Single-stranded binding proteins (ssbp) coat ssDNA to keep the strands apart and prevent base pairing

Question 9

Elongation

Answer 9

Both parental strands are copied at the same time and in both directions
- 2 Replication forks, 2 strands being copied at each fork, therefore a total of 4 strands are being synthesised during replication in circular DNA
- At each fork there is a leading and a lagging strand

Question 10

Leading Vs lagging strand solution in elongation

Answer 10

• DNA can only be synthesised from 5’ to 3’
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Question 11

Leading Vs Lagging strands

Answer 11

Leading:
- Primase making an RNA primer
- DNA polymerase 4 makes a DNA copy of the strand in 5’ to 3’
- Continuous copying

Lagging:
- Primase makes multiple RNA primers
- DNA polymerase 3 synthesises in 5’ to 3’ until it runs into next primer, creating Okozaki fragments
- DNA polymerase 1 replaces RNA primers with DNA
- DNA ligase joins the pieces of DNA
- Is discontinous copying

Question 12

Termination

Answer 12

• Joining up the new strands (4 strands become 2 new daughter strands)
• Roughly opposite the origin
• Strands complete circle and meet again
• Similar steps to lagging strand after Okozaki fragments

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Question 13

Transcription

Answer 13

DNA into RNA

Question 14

RNA Polymerase

Answer 14

• Makes an RNA copy from a DNA template
• Does NOT need a primer to start
• Uses Ribonucleotide triphosphates as a substrate (NTP’s)
• There is limited ‘proof reading’ as it works very fast

Question 15

Challenges of Transcription

Answer 15

Only small sections of genome need to be transcribed
- Where does inititiation occur?
- How does elongation occur?
- How does termination work?
- How is it regulated?

Question 16

Transcription: Initiation and Termination

Answer 16

• RNA polymerase binds to Promotor region of DNA and starts transcribing from 5’ to 3’
• Transcription stops at the Terminator
• Terminator and Promotor are in DNA sequence

Question 17

Directions of Transcription

Answer 17

• Only one strand of DNA is transcribed for each gene
• Uses ‘bottom’ strand of the DNA as a template, creating DNA complementary base pairs for the template, which is same as ‘top’ strand

Question 18

Transcription Inititation

Answer 18

1. Promotor region is -35 and -10
2. Transcription starts at +1
3. -10 is a TA rich site
4. Transcription proteins bind to DNA at specific sequence in the promotor region
5. Transcription proteins help RNA polymerase get to the transcription site
6. Transcription factors leave and RNA polymerase is ready
7. Elongation can now start

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Question 19

Transcription Elongation

Answer 19

• Unwinds the DNA in small parts in a ‘transcription bubble’
• working 5’ to 3’
• Works in a system of “Open, build chunk, Slide”
• RNA nucleotides are added until the termination

Question 20

Transcription termination

Answer 20

• RNA nucleotides are added until the termination site is reached

Method 1:
- G/C rich region causes the RNA to fold and pinch becoming double stranded, close to the polymerase
- This causes transcription to slow down and pause
- The RNA Polymerase and RNA are then released from the DNA strand

Method 2:
- Can have a protein bound (Rho Protein)
- Rho protein travels up RNA and binds to G/C rich site
- Rho protein then wedges itself between the polymerase and RNA, causing it to stop and terminate.

Question 21

Gene expression and regulation

Answer 21

Genes can be expressed at different frequencies by:
Promotor strength
- DNA sequence optimised for strong or weak sigma factors
- Strong binds = more RNA copies made
- Weak binds = less RNA copies made

Repressions
Accelerators/ Activators

Question 22

Transcription regulation: Repression

Answer 22

• Binds to the promotor region and blocks proteins from binding
• Can be modulated by small molecules, allowing the repressor to leave

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Question 23

Transcription Regulation: Accelerators

Answer 23

• Usually when there is a weak promotor
• Alters structures of the protein to bind more frequently.
• Can be modulated by small molecules, activating the accelerator.