BoC genes in action L1 and 2

Question 1

Draw the central dogma diagram for genes (good to put in intro)

Answer 1

See notes

Question 2

What are the 6 levels of DNA packing?

Answer 2

DNA double helix –> expanded chromatin ‘beads on a string’ –> condensed chromatin (30nm diameter fibre) –> scaffold associated chromatin –> condensed scaffold associated chromatin –> metaphase chromosome

Question 3

What amino acids do histones have a lot of?

Answer 3

Arginine and Lysine giving strong positive charge

Question 4

What is nucleosome structure?

Answer 4

octameric, 8 histones (2A,2B,3,4) x2

Question 5

How can you see the amount of DNA per nucleosome and how much is it?

Answer 5

Using partial nuclease digestions, 200bp

Question 6

What does histone H1 do?

Answer 6

Its not part of the octameric core but functions as a clamp to keep the nucleosme closed

Question 7

What happens at histone tails?

Answer 7

Post-translational modifications eg. methylation of lysines and arginines, acetylation
Influences DNA packaging and accessibility for translation

Question 8

What is else can influence epigenetic control?

Answer 8

Nucleotide-driven actions to condense DNA

Question 9

What did an amphibian oocyte study show?

Answer 9

That stability of condensed chromatin relies on the proteins cohesin 1 and cohesin 2

Question 10

What are a series of 4 landmark experiments looking at DNA replication? What are names of scientists who did them?

Answer 10

1) Chargaff, saw in DNA proportion of A=T etc.
2) Watson and Crick resolved DNA double helix
3) Mendelson and Stahl proved semi-conservative replication
4) Cairns identified the replication fork

Question 11

What was Mendehlson and Stahl’s experiment?

Answer 11

They used density centrifugation to separate DNA strands grown in the presence of different N isotopes
First grown in 15-N labelled then moved to 14-N labelled growth medium and allowed to replicate
DNA extracted and analysed by density centrifugation
(See notes for diagram)

Question 12

What is minimum length of E Coli origin and what is it called?

Answer 12

Ori C, 245 bp

Question 13

What is found in this region

Answer 13

1) DNA Unwinding Element (DUE) with 3x AT-rich sequences (because only 2 H bonds)
2) 5x 9bp regions which act as recognition sites for Dna a
3) Several GATC sequences, which are targets for Dam methylase mediated methylation (Ori C needs to be methylated before Dna a binds)

Question 14

Dna a function?

Answer 14

Initiator, forms complex with ATP and binds the 9x 5bp binding sites
Partially unwinds DNA in the DNA unwinding region

Question 15

Dna b function?

Answer 15

Helicase, it is a hexamer

Question 16

Dna c function?

Answer 16

Loader, loads Dna b onto partially unwound DNA, also a hexamer

Question 17

DnaG function?

Answer 17

binds DnaB making primosome, makes RNA primer

Question 18

Draw DNA pol 111

Answer 18

See notes for diagram

Question 19

What are functions of beta, alpha, gamma, tau and eta and theta subunits?

Answer 19

beta- sliding clamp to keep DNA pol 111 bound
alpha- synthesises new DNA 5’ to 3’ (ie new DNA runs this direction), one for leading one for lagging
g- clamp loading/unloading
t- mediates dimerisation of the 2 alpha subunits
e and th- proves 3’-5’ (Backwards) exonuclease ie removing necleotides and proof-reading activity

Question 20

How often is there an error in replication?

Answer 20

1/10^9 nucleotides replicated

Question 21

What is the site of active synthesis called?

Answer 21

The replication fork

Question 22

How is movement of the replication fork helped?

Answer 22

By unwinding of DNA by DnaB and by the beta subunit of DNA pol 111
NB: DnaB active all of replication

Question 23

What was Okazaki’s pulse chase experiment?

Answer 23

3H-tritium (radioactive thymidine) was added to replicating E.Coli (the pulse)
Cells were either harvested immediately or 3H-Tritium was removed and they were allowed to replicate (the chase)
DNA was denatured and separated by size via ultracentrifuge
See notes for diagram of results

Question 24

What is the Trombone model and who was it suggested by?

Answer 24

Alberts
Suggests template for lagging strand is looped around to allow RNA primer to be placed on exposed DNA which can be pulled through by advancing polymerase
When RNA primer of previous sequence is reached synthesis stops so a nick exists between RNA and DNA sequences, lagging strand produced as alternating sequence of RNA primers and newly sequenced DNA
(see notes for diagram)

Question 25

How is nick between RNA and DNA sequences healed?

Answer 25

DNA pol 1 removes and replaces the RNA primer using a 5’ to 3’ (forward) exonuclease activity
DNA ligase joins nicks, glues the 3’ -OH of the DNA strand to the 5’ phosphate of the adjacent strand to form continuous DNA
(See notes for diagram)

Question 26

What primer does DNA pol 111 use?

Answer 26

RNA primer that is 10 bases long made by DnaG

Question 27

How does termination happen?

Answer 27

Roughly opposite OriC are ter sites that interact with the replication site making it pause
separation of replicated chromosome carried out by topoisomerase 4 and recombinase enzymes