BoC genes in action L1 and 2 Flashcards
Draw the central dogma diagram for genes (good to put in intro)
See notes
What are the 6 levels of DNA packing?
DNA double helix –> expanded chromatin ‘beads on a string’ –> condensed chromatin (30nm diameter fibre) –> scaffold associated chromatin –> condensed scaffold associated chromatin –> metaphase chromosome
What amino acids do histones have a lot of?
Arginine and Lysine giving strong positive charge
What is nucleosome structure?
octameric, 8 histones (2A,2B,3,4) x2
How can you see the amount of DNA per nucleosome and how much is it?
Using partial nuclease digestions, 200bp
What does histone H1 do?
Its not part of the octameric core but functions as a clamp to keep the nucleosme closed
What happens at histone tails?
Post-translational modifications eg. methylation of lysines and arginines, acetylation
Influences DNA packaging and accessibility for translation
What is else can influence epigenetic control?
Nucleotide-driven actions to condense DNA
What did an amphibian oocyte study show?
That stability of condensed chromatin relies on the proteins cohesin 1 and cohesin 2
What are a series of 4 landmark experiments looking at DNA replication? What are names of scientists who did them?
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
What was Mendehlson and Stahl’s experiment?
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)
What is minimum length of E Coli origin and what is it called?
Ori C, 245 bp
What is found in this region
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)
Dna a function?
Initiator, forms complex with ATP and binds the 9x 5bp binding sites
Partially unwinds DNA in the DNA unwinding region
Dna b function?
Helicase, it is a hexamer
Dna c function?
Loader, loads Dna b onto partially unwound DNA, also a hexamer
DnaG function?
binds DnaB making primosome, makes RNA primer
Draw DNA pol 111
See notes for diagram
What are functions of beta, alpha, gamma, tau and eta and theta subunits?
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
How often is there an error in replication?
1/10^9 nucleotides replicated
What is the site of active synthesis called?
The replication fork
How is movement of the replication fork helped?
By unwinding of DNA by DnaB and by the beta subunit of DNA pol 111
NB: DnaB active all of replication
What was Okazaki’s pulse chase experiment?
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
What is the Trombone model and who was it suggested by?
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)
How is nick between RNA and DNA sequences healed?
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)
What primer does DNA pol 111 use?
RNA primer that is 10 bases long made by DnaG
How does termination happen?
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
