DNA - Biochemistry
What is transcription
The way in which wew turn DNA inot RNA in the nucleus of the cell in order for protein synthesis to take place as DNA is too large too leave the cell
How is RNA different to DNA
It is single stranded so can form well defined secondary structures by folding
It has ribose sugar not deoxyribose
It hae Uracil not Thymine
What are the different types of RNA
mRNA-Messnger RNA used as a template for protein synthesis
rRNA- Ribsomal RNA is a mjaor component in the ribsosome structure
tRNA- Tranfer RNA that carries amino acids in the activated form to the Ribsome
snRNA- Small nuclear RNA used for RNA splicing
miRNA- Micro RNA bind to mRNA to promote degradation or inhibitation of translation
How is RNA made
By copying one of the DNA strands during transcription using normal base pair rules and with RNA polymerase but no need for a primer
Nucleotides are attached in the 5-->3 direction and 17 BP are undowund in the DNA to act as a template
What is needed for transcription in Prokayotes
RNA Polymerase, A holoenzyme = a2BB + Gamma enzyme to direct the enzyme to the initiation site
Transcription in Prokaryotic cells
Transcriptional units are marked by promoter sequences to detrmine the start point for transcription, which is located by initation site that has a lower affinity for non-promoter DNA region
Binding then to the template of the promoter sequence causes the starnd to seperate and RNA synthesis can happne anti-parrallel to the DNA strand 5-->3 direction
A transcription bubble is formed that move along the DNA with RNA polymerase in the bubble
What makes the promoter sequence
Pribnows box which is -10 BP upstream from the start of the RNA molecule
A consensus sequence that is 35 BP up stream from the RNA start point
They collectivly help the enzyme- RNA polymerase- locate RNA promoter sequence
How is RNA synthesis stopped
There is a termination sequence that tell RNA polymerase to stop. which is made of palindromic GC rich region followed by AT rich region.
The RAN transcript form hairpin formation so there can be complimentary pairing between the GC and AT regions to stop transcription
RHO factor may also help: Areas rich in Cytosine but low in Guanine so prevent hairpinn loop need RHO protein to bind to region causing the polymerise to destabilise and leave
Post transcription in Prokaryotes
Some RNA is further modified after:
tRNA- bases are methylated and CCA is added to 3-end for amino acid binding
rRNA- Cut into smaller chains that are more relevant for purpose
In eukaryotes what are 3 factors that influence gene expression
Nuclear membrane that seperate transcription and translation are seperate so dont happen at same time
RNA processing
Complex transcriptional regulation
What are the 3 types of RNA Polymerase
RNA polymerase 1 - Used to produce rRNA
RNA Polymerase 2- To produce mRNA and miRNA
RNA Polymerase 3 - tRNA production
How is mRNA futher modified in eukaryotic nuclei
Additon of poly a tail at the 3 primer end to increase stabilty and specify export to the cytoplasm
Addition of the cap to the 5 primer end to direct the protein translation that will take place
What is Splicing
Many eukarotic genes have intron in them that are spliced from pre mRNA to leave just the exons in the mRNA
This is performed by the spliceome
Why is Splicing useful
Fibroblast produce fibronectin mRNA that have amino acid EIIB and EIIA
These are important due to tightloy bound proteins in the fibroblast membrane
In liver cells they dont encode for EIIB and EIIA as they are spliced out so then allow fibronectin to be secreted to the blood
How can transcription be regulated
The interaction of RNA polymerase and promoter sequences can be enahnced by activators or blocked by repressors
Bacteria Genes in a common pathway are controlled by single unit called an Operon
- Lac Operon only on when lactose present and is controlled by upstream regulatory gene that is produced that can bind to the operator region and prevent RNA polymerase binding to some exons
What is Gene expression
Genes are expressed when they have been transcribed and tranlated to a protein
Genes expressed all the time = Consituitive expression
Genes expressed under certain condition = Regulated expression
Why is it hard to control processes in eukaroyotic cells
Tissue Specificty, cell type variation, developmental regulation, Gene to be transcribed may be inaccesible in condensed chromatin so may need remodelling to give RNA polymerase access
What are transcription factors
Basal ones - Bind to promoter and are common in most genes
Regulatory factors - Bind to promoter proximal element and enhancers, and are gene specific
What is miRNA
Helps regulate gene expression, due to small non-coding RNA it what it is made of
They are synthesized from precursor nuclear DNA
Cause silencing of mRNA/gene due to interfear with RNA binding to the DNA so then destabilize mRNA or prevent translation
What does DNA stand for
Deoxyribose nucleic acid
When and how as DNA discovered
Fpundin 1869 when it was found acid molecules rich in phsophate were isolated from tjhe cells in the pus
Found this by cell fractionization
How is DNA organised in eukaryotic cells
DNA is found in the nucleus and is packed into chromosomes that are a complex of DNA anjd a protein that are linear when organsized
How is DNA organsied in prokaryotic cells
DNA is not in the nucleus due to not having one but it is wrapped and super-coiled
It is not wrapped round a histone protein
The DNA is naked
What is a chromosome
They were named due to seen by dyes that were able to pick out areas of AT or GC rich of the genome to make a banding pattern
Each human cell have 46 chromosomes so 23 pairs and each one is made up of a single DNA molecule that are associated with a protein and wrapped to be compact
All cell have 2 copies of each chromosome = homologoius chromosome
Normal cells are diploid and gamate cells are haploid
What is the genome
The number of genes that an organsim can express this will varty depending on organsim type
The human genome project allows today to publish any human gene ever in less than a day
Gene number has a correlation to organsim complexity but no relation between complexity and number of chromosomes
What are genetic diseases
Down Syndrome- 3 copies of chromosome 21 with the extra 1 from the mother
Turner Syndrome - Women that have 1 x chromosome so only 1 parent contribute an x chromosome
Klinefelter syndrome - Males with only 1 y chromosome and 2 x chromosome
Philadelphia chromosome - Translocation of material between chromsome 9 and 22 so form an oncogenic fusion gene
Cystic fibrosis - CFTR gene found on chromosome 7 that generate a large protein that doesnt function due to deletion mutation
Sickle cell - Adenine changed to thymine at postion 7 of the gene in the Haemoglobin peptide
What is the Griffith experiment on DNA
Experimented on Mice, he found that Non virulant strands became virulant when mixed with heat killed virulatn strands
Thsi was a permanent transformation
Prpved DNA was the genetic material and that something from dead cel transformed living cells
What did Avery prove about griffith experiment
Transfromation was produced by tranfer of DNA from virulent cells
This was detsroyed by DNase but not by Protease or RNase
He utilsied fractionation methods to purify DNA and transforming activity was unaffected by protease and Rnase so mouse still dies but woth Dnase the mouse doesnt die as no tranformation
What was the Hershey and Chase experiment
They used bacteriophage T2 and e.coli and labelled the T2 with 35 s protein and 32p DNA
It then showed that only DNA from the T2 entered the e.coli so it could produce its own coat protein
What is DNA made of
It is made of deoxyribose + Phosphate group + Nitrogenous bases
What are the nitrgogenous bases
Adenine + Thymine are complimenrary to each other and are bonded by 2 hydrogen bonds
Guanine and Cytosine also bond together by 3 hydrogen bonds
Purines = Adenine and Guanine
Pyrimidine = Cytosine and Thymine
They complimnetary bond together in nucleotides to make the double helix of the DNA structure
What are the 2 bond of the DNA backbone
Glycosidic bond between the nutrogenous base and the deoxyribose sugar
Phosphodiester bond between the phosphate group and the dexoyribose on the next adjacent nucleotide
How did Watson and Crick come up with the DNA double helix structrure
Chargaffs rule: The 4 nucleotides are not present in the same abundace and ratios differ depending on species
However A=T and C=G so the purine number = prymidine number
What did Franklin and Wilkins do
They obtained x-ray diffraction patterns on DNA fibres that provided the fundamental repeating distances in the DNA
0.34nm gap between nucleotides
Can change conditions to turn A DNA that id dehydrated to B DNA that is hydrated and present in our bodies
What are some other structural features of DNA
The bases in the DNA are hydrophobic and are able to stack on top of each other in the core of the double helix
Seperation distances of staxkced bases is optimal for VDW forces
GC and AT bodnming are the same shape and size
The phosphates run on the outside of the helix due to being negative charged so outside of the DNA is vert hydrophillic
What direction does DNA run in
The 2 strands run anti-parrallel to each other so one will run 5 primer to 3 primer and the other will go 3 to 5 primer
The DNA has a right hanged helix due to curling on the clockwise direction
The base pairs are perpendicular to the helix axis and centre of helix runs through base pairs
What is B-DNA
The predominant form of DNA
It 10 BP per turn in the helix and 0.34nm gap between nucleotides
Has 2 grooves of enequal size due to bonding patterns of glycosidic bonding
What is A-DNA
The dehyrdrated form of B DNA
It is muich wider and the bases are tilted and offset from the helix axis
Bases dont stack up on each other
What is Z DNA
This if left handed DNA and if formed by some GC conjtainig sequences at a high salt concentration
It is symmetrical due to no major or minor groove
What are the properties of DNA
It will melt at high heat and wil be used to seperate the 2 strands and absorb light at 260
Single stranded DNA has a greater light absorbance atb 260 due to having no base stacking
GC rich DNA wil melt at higher temp than AT rich DNA duento being 3 Hydrogen bonds in GC bonding
How does the DNA pack into the nucleus
6 billion base pairs in each cell
In all Eukaryotic cells the DNA is condensed to chromatin
What is chromatin
Contain 5 proteins called histones = H1, H2A, H2B, H3 and H4
They are highly basic so have many charghed amino acids that interact with negativly charged DNA
2 copies of H2A H2B H3 and H4 make an octameric disk structure that make up the histone core that the DNA wrap around to form a nucleosome
What is the role of the H1 histone
It is a linker histone that bind to each of the produced nucleosome
They are in contact with both the DNA and the histone core
It will change the conformation of the DNA as it leave the nucleosome to help compact it by adding kink to the DNA chain
What is semi-conservative replication
Each new DNA doubkle strand is made up of an original parent strand along with a newly synthesized strand
What is the meselson and Stahl experiment for proving semi conservative replication
They grew bacteria on non-radioactive N15 and then cells with normal N14 DNA were tranferred to the gel
The DNA then underwent seperation after 1,2,3 generation of the DNA after replication took place
At 0 generation all DNA was N15
after 1 generation the DNA was density between N14 and N15
after 2 generations the DNA was 50% N14 and 50% N15
After 3 generations 75% was N14 and 25% was an intermediate DNA between N14 and N15
What are the types of centrifugation
Aside - Heavier particles will sedement faster but not at equilirbium method due to everyything go to bottom depending how fats you spin it
Sucrose Density - Sample put at top of tube with pre-existing sucrose gradient and it will seperate but not equilibrium as everything end up at bottom eventually
Cesium chloride density gradient - Sample are dispersed in cesium chloride, and is an equilibrium method under high centrifugal forces the cesium form concentration gradient
Samples then float at postion corresponig to buyoncy density
Where does DNA replication start
Start at a fixed point of bidirectional and there may be mnay replciation forks that are all bidirectional
What is the DNA replication fork
Parent starnd opened up by DNA helicase and produces new duaghter strands that act as template for new nucleoptides to bind to via complimentary base pairs
The synthesis of new DNA strand always go in the 5 to 3 primer direction so the new nucleotide added at the 3 primer end
DNA polymerase is the enzyme used to add a nucleotide but first a primer need to be added bt RNA primase that will lay down a small section of porimer for the DNA polymerase to be able to layw donw new nucleotides to the DNA chain being made
How do Okazaki fragemenst form
On the lagging strand the 3-5 primer there are short strand sthat are formed from RNA primase being laid down and DNA polymeras briefly extend before then breaking off
Okazaki fragments are joined together by DNA ligase and ATP is needed
Why is DNA Polymerase important
DNA PCR reaction
DNA sequencing
What are the types of gel used for DNA sequencing
Agarose gel - Physcial gel that is used to seperate double stranded DNA, and are able to visualise the DNA under UV light
Polyacrylamide gel - Chemical gel that is run vertically and uses denaturing gels with 8M urea to seperate single stranded DNA can be detected and seen by attachemnt of fluorescent dye
What is next generation sequencing
Short fragements of DNA is annealed to a slide using oligonucleotide adaptors
PCR to create multiple copies
Then use fluroscent nucleotides for sequencing and take image after each addtion
Reversible terminator is important due to allow many nucleotides to keep being added after an image is taken to get many different images + have modified nucleotide tha blocks 3 primer on the nucleotide so will strop reaction for short time and is reversible