Topic 5: Copying DNA and RNA

Question 1

What is used as substrate in copying DNA and how is it used in making new DNA strand?
What type of bond is formed between the nucleotides?
What is released during the bonding of new nucleotide?

Answer 1

Deoxynucleotide triphosphates

As the new strand is made, nucleotide monophosphate makes a phosphodiester bond with the sugar group at 3’ OH end of the previous nucleotide.
Pyrophosphate (the 2 phosphate groups left) is broken down into 2 phosphates to provide energy for reaction.

Question 2

In which direction is DNA being made?

Answer 2

Always 5’ to 3’

Question 3

What does DNA need to start replication?

Answer 3

A primer/short piece of RNA to let DNA polymerase start adding nucleotides

Question 4

Why is DNA replication less likely to make mistakes?

Answer 4

Often have 3’ to 5’ exonuclease activity to remove mismatch nucleotides

Question 5

Which replication model does DNA replication follow?

Answer 5

Semi-conservative: each newly formed DNA double strand has 1 original strand and 1 new one.

Question 6

In general, biopolymer synthesis follows how many steps?

Answer 6

3 steps: initiation, chain elongation, termination

Question 7

Describe the steps in the first stage of DNA replication

Answer 7

There is an “origin” of replication - starting point
Usually AT-rich –> easier to melt/be pulled apart

• DNA helicase: unwinds/ open up part of the DNA
• DNA topoisomerase/ gyrase: stops supercoiling by cutting the strands, allowing them to unwide and then religate
• ssDNA (binding proteins): coat the single stranded DNA to keep the strands apart and prevent small sections of base pairings
• Form replication forks

Question 8

How many strands are being made at one time in DNA replication?
2 types of making strands?

Answer 8

At 2 replication forks, both original strands are copied making 4 strands/ 2 double stranded DNA

Each replication fork, 1 leading and 1 lagging strand

Question 9

Describe steps in the second stage of DNA replication

Answer 9

• DNA primase: bind to the original strand and make primer or short stretch of nucleotides to initiate the replication
• DNA polymerase III: wraps around the original strands and use the nucleotides floating around to add in, making new strand
• Leading strand: original strand runs 3’ to 5’ so the new strand is continuously made as the helicase unwinds the strand
• Lagging strand: on original strand running 5’ to 3’
  + multiple primers are added
  + DNA polymerase III makes lots of Okazaki fragments until it reaches the next primer
  + DNA polymerase I replaces the RNA primers with DNA
  + DNA ligase joins the DNA fragments together
  + slower

Question 10

What is the Replication Fork Complexes and since lagging strand is made slower than leading strand, how does it help to manage this?

Answer 10

Replication Fork Complexes incorporates the DNA polymerase III, primase and many other components.

As lagging strand takes longer to replicate, the complex helps to pull out a big loop of ssDNA waiting to be replicated without slowing down the leading one.

Question 11

Describe the steps in the last stage of DNA replication

Answer 11

• New strands join up to make 2 new daughter strands
• Termination point roughly opposite to the origin

Question 12

What are some differences in eukaryotic DNA replication compared to prokaryotic?

Answer 12

• Many origins of replication
• Strip off nucleosomes before replication and then reform right after
• Telomerase especially for the ends (telomeres) of chromosomes

Question 13

Describe characterstics of RNA transcription that are different from DNA replication

Answer 13

• DNA template but RNA copy
• Don’t need a primer
• Use ribonucleotide triphosphate as substrate
• Limited proofreading, no 3’ to 5’ exonuclease activity

Question 14

What are some challenges in transcription, eg. regarding sections being copied and number of copying times?

Answer 14

• Only small sections of genomes need to be transcribed and these sections need to be copied for thousands of times
• Some are rarely copied in 1 cell and copied many times in another cells

Question 15

What parts of the DNA are RNA transcription initiation and termination associated with?

Answer 15

Initiation: RNA polymerase binds to promoter region
Termination: transcription stops at terminator

Question 16

In which direction does transcription occur and what is special about number of strand being made compared to DNA replication?

Answer 16

Can happen in both directions, but only 1 strand of DNA is transcribed for each gene. Which strand is copied can vary even in the same region of chromosomes

Question 17

Describe the steps of transcription initiation

Answer 17

• Sigma factors bind to RNA polymerase to change its affinity to a certain promoter sequence. When the RNA polymerase has already bind and start transcribing, the transcription factor leaves.
• Whereever the RNA polymerase moves to, it melts the DNA strands and creates a local “transcription bubble” and one of the components in its complex unwinds the DNA.
• When RNA has passed by, the 2 strands of DNA reanneal.
• Very fast process, the transcription bubble is quite small

Question 18

The promoter region incorporates which sequence of the DNA and what is the feature that makes it easy for the strand to be pulled apart and be transcribed?

Answer 18

Promoter region sitting right upstream has 2 consensus sequence, at -10 and -35. The -10 region often has sequence TATA –> easy to melt

Question 19

What is transcription factor?

Answer 19

Proteins capable of recognising a specific base sequence

Question 20

Describe the steps in transcription termination (2 ways)

Answer 20

• Way 1:
  + Use a signal encoded in the transcribed RNA to stop + G/C rich sequences followed by the A/T rich sequences
  + G/C rich sequence form double stranded haripin structure on the RNA –> transcription pauses and then stops
• Way 2: Rho protein binds to RNA, acts like helicase to travel up the RNA and dissociate the DNA/RNA hybrid complex

Question 21

Gene expression frequency is regulated by what factors?

Answer 21

• Promotor strength:
  + consessus sequence optimised for strong or weak binding between transcription factor/sigma factor and RNA pol
  + strong binding = more RNA copies and vice versa
• Repressors
• Accelerators/ Activators

Question 22

How do repressors affect RNA transcription?

Answer 22

Repressor - a protein - blocks the binding between RNA pol and sigma factor –> No RNA pol binding –> no transcription –> no gene expression

Question 23

How do accelerators affect RNA transcription?

Answer 23

Help to modulate weak promoters

Transcriptional activator - a protein - binds to DNA at specific region to modify the structure –> encourage the sigma factor to bind more frequently and tightly

Also interact directly with the RNA polymerase to recruit it to weak promoters.

Question 24

Repressors and activators can be modulated by what?

Answer 24

Small molecule binding (metabolites) to either activate activator or relieve repressor

Question 25

What is special/different in eukaryotic transcription?

Answer 25

• More complex regulation
• More transcription factors -unique sets depending on the cell type
• Many layers of condensation so chromatin accessibility is important

Question 26

What are the similarities in nucleic acid synthesis (DNA replication and RNA transcription)?

Answer 26

• Utilize base-pairing for correct pairs of complementary bases
• Use polymerases and dNTPs or NTPs to add new nucleotides
• Hydrolysis of pyrophosphate to get energy
• DNA strands need to be unwinded
• Recognition of start sites (primer for DNA and transcription factor for RNA)