T7 genetics

DNA

origins

hershey and chase: determined that DNA was the carrier of genetic information.
they grew virsues in radioactive sulphur or phosphorous as sulphur only appeared in proteins and phosphorous only appeared in DNA
then they took the viruses and placed them in a centrifuge, the spinning causes all the heavy stuff to sink to the bottom, therefore the nucleus (contatining the genetic info) should also sink to the bottom.
as a result they found that only those viruses grown in phosphate had a radioavitve pellet (sink to bottom) and those grown in sulphur were only radioactive in the supernatant (liquid)


franklin rosevelt and x-ray crystallography: used to deduce the structure of DNA:

  • took DNA and laid them vertically within a glass tube
  • shot x rays at it and prokected the diffractions on photographic film
    from this they deduced that:
  1. must be double helix, double stranded
  2. phospahte outside, nucleotide inside

when building the model they found that there were equal number of purines and purimidines , as well as AT was double bonded and CG was triple bonded, leading the conclusion that during DNA replication:

  • the molecules run by complentary base pairing
  • the direction is bidirectional

DNA replication

there are a few important enzymes that are involved in dna replication:

  • helicase - the unwinding enzyme that splits the helix into 2 strands, occurs at specific regions (called origins of replication) and creates replication fork
  • DNA gyrase - relieves torsional strain on the DNA as it is uncoiling by negative supercoiling
  • SSB proteinS (single stranded binding) - bonds to DNA strands after they have been seprated to stop re-annealling, and protect the nucleotides from being idgested by nucleases will depart once DNA polymerase III arrives
    DNA primase - adds RNA primers to singal the begining of replication
    DNA polymerase III: extends chain by adding nucleotide (does this as free nucleotides exist as triphosphates, DNA pol III cleaves the 2 phosphates and uses the energy to generate a phosphodiester bond) that are lined up agaisnt DNA strand via complementary base pairing, bonds to 3' end of primer and moves in a 5->3"
    -- leads to a lagging strand, away from fork
    -- and leading strand, toward the fork

if lagging strand, need 2 more enzymes:

  • DNA polymerase I, replaces the primers with nucleotide bases
  • DNA ligase - joins the okazaki fragments of DNA together - by creating phosphodiester bonds

origin of replication: sequences where dna replication is inititated within a genome,

  • in bacteria as they are circular DNA, they have only 1 origin
  • as eukaryotes are linear and have more dna in general, they will have multiple points
  • DNA synthesis may occur bi-directionally, which forms a replication bubble
    -- the replication bubble will eventually fuse as the two forks meet, however its ability to go both ways drasticlaly decreases the time for DNA replication (i.e. withough replication for 1 chrom would take 1 month)
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okazaki fragments : sequences of dna created during dna replication on the lagging strand
as dna strands are anti-parrallel the DNA pol II must move in opposite directions, therefore on the lagging strand the Pol would need to always return and discontinoulsy make fragements
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telomeres: regions of repetitive dna on the ends of chromosomes that help prevent chromosomal deterioration.

  • as with each replication, the terminal RNA primer is unable to be replaced by DNA pol I at the extreme ends of the chromosome, therefore it will inevitably get shorter
  • this is also why the shortening of telomeres is associated with ageing (usulaly 40-60 replications - hayflick limit) as cells may stop dividing when too much telomere dna is lost
  • however telomere can be lengthed by an enzyme called telomerase, yet too much = cancer, so cancer researchers are finding inhibition drugs for telomerase to treat cancer
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DNA sequencing: a method used to present the DNA seqeucnce of something, and is done through using dideoxynucleotides (ddNTP)

  • ddNTP are unable to form phosphodiester bonds due to a lack of the hydroxyl group, this means that they will terminate the dna replication seqeucen when meeting is complementary pair, i.e. if using ddGTP, then the place where it terminates must have a cytosine

appication:

  • need 4 PCR set ups, each tube contating the DNA, free nucleotides and 1 type of ddNTP,
  • then do PCR to generate a billion copies and place solution in side a gel electrophoresis machine

  • as each PCR contains a difference ddNTP, the overall gel electrophoresis should be enough to piece together what the DNA sequece looks like



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noncoding dna: only 1.5% of the human genome consists of coding genes, the rest, before thought to be useless are actually pretty functional (e.g. STRs in dna profiling)


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not exaclty non-coding DNA but coding DNA vs repetitive dna


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nucleosomes: are when dna is wrapped around an ocatmer of histones to help with condensing DNA during supercoiling:

  • process:
  1. dna is wrapped around an octamer of histones
  2. histone tails link to form a chromatasome
  3. chromatasome coil to form a solenoid
  4. solenoid coils to form a 30nm fibre
  5. the fibre then folds to form chromatin
  6. chromatin supercoils to from chromosomes

histones are used to be bound to dna, as the negative dna is attracted to the positive on the amino acids

  • histones also have N-terminal tails which extrude outwards from the nucleosome and during condesation, they link up with each other to form a chromatasome



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supercoiling: the additional twist of DNA strands which create strain on that strand

  • there are two types of supercoiling: positive (oerwound) and negative (underwound), most DNA is underwound
  • supercoiling works to be more efficient when packing dna into the body

  • nucleosomes help with super coiling and during dna replication, positive supercoiling occurs, putting strain, therefore dna gyrase will negativly coil the dna strand in order to reduce the strand



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transcription: the act of using DNA to create a mRNA strand

process:

sections of a gene:
a gene is a seuqence of dna that can be transcribed and consits of 3 main parts:

  • promoter - the non-coding sequence that inittiations transciprtion, it allows RNA polymerase to bind and thus locates the begining of transcription
  • the core promotoer sequece is found near the coding sequence, however, transcriptional factors that mediate RNA pol to bind to the promoter can be either proximal control elements (close) or distal control elements (far away)
  • coding sequence - is the section of DNA that is being transcribed after the RNA pol binds to the promoter sequence
  • terminator - a sequence of dna that tells the RNA polymerase to stop and detach


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terms:

  • antisense strand aka template strand is the strand being transcribed,

  • sense strand aka the coding strand is not being transcribed and is the DNA copy of the mRNA sequence


    and as either polynucletide strand may contain a gene, you can never know which one is sense and antisense



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transcription: is the process by which genes are copied into mRNA via RNA pol

  • this happens as free nucleotides exist as triphosates (NTPs)and in order to bond them togethr RNA pol will cleave two phosphates off to join the NTPs together
  • transcritption happens in a 5'->3' direction

there are 3 steps:

  • initiation: the RNA pol binds to the promoter and unwinds the helix
  • elongation: the RNA pol moves along the strand synthesising the mRNA strand
    -termination: the RNA pol reaches a termination sequence and detaches from the strand, helix rewinds
  • and in eukaryotes the mRNA must go through post-transctiptional editing before becoming mature mRNA


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post transcriptional events

mRNA: mRNA needs to be editied before being used in translation and they are done through 3 events:

  • capping: placing a methyl- cap on the 5' end of the mRNA


    -- protects agaisnt degredation by exonucleases and helps transcriptional machinery recognize it (e,g, ribosome)


  • polyadenylation: the addition of a chain of Adenine on the 3' end of the mRNA strand


    -- improves stability of mRNA strand and helps with exporting out of nucleus


  • splicing: the act of removing introns from mRNA strand


    -- introns are non-docing sequences within genes, whereas exons are expressed sequences


    -- introns are cut out whilst exons stay


    however you can also cut out various exons in order to create diff proteins, this is called alternative splicing



the capping and polyadelylation are all to transport the mRNA out of the nucleus into the cytoplasm, however as proaryotes do not have compartments, they do not need to seperate transcription and translation, thus the ribosome can begin to work when mRNA is still transcribing, this is possible as both processes occur in a 5'->3' direction


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regulating transcription:
transcription is controlled by two groups of proteins:

  • transcritional factors, - bind to RNA pol to form a complex that initiates transcription
  • regulatory protiens that bind to DNA sequences outside of promoter and affect transcritional factors:
    -- i.e. activators bind to enhancer sequences which help mediate transcription (through complex formation) and repressor sequences bind to silencer sequences which help prevent complex formation

these proteins can bind to control elements, which are regions of DNA that are either close (proximal) or far (distal) from the gene
-regulatory proteins usually bind to the distal control elements, oppo for the transctitional elements

  • most genes have many control elements surrounding it therefore showing that transcription is highly regulated

moreover the presence of certain transcritional factors may be tissue specific or may be regulated by chemical signals (e.g. hormomes)
plus the environmenet can also affect gene expression, for example, hydrangeas change color based on the PH of water they are in (blue for acidic red for basic)

epigenetics:the study of phenotypic changes that do not involve alterations in DNA sequence (changes in heritable traits that do not involve the alteration of base sequences


it all starts with histones:

  • histones have positive N1 tails that are attracted to the negative charge of DNA
  • when adding a acetyl group(negative) (acetylation) to it , i will neutralie the charge and loosen the coil
  • if adding a methyl group (methlation) it will increase the positive charge causing it to be tighter

DNA can exist as heterochromatin or euchromatin:

  • hetero means tightly coiled and thus unable to transctipt


    -euchromatin means loosely coiled and available for transciption


    different genes will have different areas of hetero and eu, and some segements may be permanently supercoild while others change with time



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direct methylation of DNA can also decrease expression, (just increases coiling), which is why genes with higher methylation are less transcribed
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epigenetics shows how our environment can affect our mmehtlyation patterns and thus gene expression, identical twins look less alike as they grow older, different cells in our boday can also have differing methylation patterns (influeneced by heritability but not genetically pre-determined)

3 main types to express genes into proteins:

  • mRNA - transcription
  • tRNA - brining aminoacids for translation
  • rRNA - makes up a large part of ribosomes used for translation

the body alos has other RNA to regulate expression:

  • small nuclear RNA snRNA - component of splicsosome
  • short interfereing RNA siRNA - moderates gene expression
    -- splits into two, the guiding strand will bing to mRNA and create a complex and destroy the targeted mRNA strand

operons: a DNA sequence that includes a cluster of genes meaning they are all expressed together or not at all

  • made up of promoter, operator, structural genes

  • related to stimulons and regulons



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reverse transcription: retroviruses are able to create DNA sequences from RNA sequences, due to the presence of an enzyme called reverse transceiptionase
they are commonly used to create gene expression files as they only transcribe active genes (actively transcribed)


the DNA strand synthesized is composed of complementary DNA or cDNA and because they represent active genes they are commonly used for gene profiles and gene transfer experiments (no introns)

translation: the process of taking mRNA and synthesizing protiens out of it

ribosomes and tRNA:

  • ribosomes are built of two sub units, a large one containing 3 tRNA binding sites (EPA) and a small one contaning one bnding site for mRNA,
  • they can be found either freely floating in cytoplasm or boud to RER (determins where the protein synthesized willl go afterwards)
  • ribosomes are smaller in pro (70s) than in euk (80s)




tRNA are responsibile for carring the amino acid to the ribosome

  • they consist of an acceptor stem (for amino acird)
  • d arm - for tRNA activating enzyme
  • t arm for ribosome

  • anticodon, to match the codon area on mRNA



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tRNA activation:

  • tRNA needs to be activated by adding energy and an amino acid group, this is done through an amino acid specific to its antidocod and thus aminoacid


  • there is one enzyme for one amino acid


    process:

  • first an ATP molecule comes and bonds to the enzyme and an amino acid bonds to the other side of the enzyme, creating an amino acid -AMP complex as a PP bond is released
  • the tRNA comes and binds to the amino acid, AMP is released and tRNA is released

the ATP -> AMP charges the tRNA to be able to form polypeptide bonds during translation


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proteins

protein structure;
protein structure can be divided into four levels, primary, seondary, tertiary and quartenary


primary - the sequence of amino acid which comprise the polypeptide chain

  • determines all types of bonding afterwards
  • made by the peptide bonds between amine and carboxyl groups

secondayr - the way in which the pp chain folds in repeating arrangements to form alpha helces and beta pleated sheets

  • folding is a result of hydrogen bonds forming inbetween them
  • if not alpha or beta, pp chian exists as a random coil
  • folding gives rise to mechanical stability due to H bonds

tertiary - the way the pp chain coils in order to form 3D shapes

  • demonstares enzyme specificty, e.g. shape is very important, determines function
  • formed by many bonding
  • can be either globular or fiborous

globular tend to have more function purpose (i.e. hameglobin, insulin, enzymes), more reactive to changes in environement and generally more soluable inwater
fiborous tend to have more structural purpose (i.e. collagen, keratin), less reactive to changes in environement and generally insoluable inwater


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quatnery - when protein includes more than 1 pp chain or a prosthetic group (inorganic group)

  • example of this is hameglobin and the heme group (conjugted protein)

  • held togetehr by a variety of bonds



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protein destinations: can be seen by where the ribosome is

  • if ribosome is free , then protein is used intracelluarly and within the cytosol
  • if bound to ER, the protein is targeted for secretion, mebrane or lysosomes

what determines the ribosomes position is the presence of an initial signal seuqence on the pp chain, this signal recruits a signal recognition particle (SRP), which halts translation,

  • it then moves the ribosome to a receptor on the ER membrane
  • translation is restrted and the pp chain continues to grow but this time via a transport channel into the lumen of the ER
  • this allows for the protein to the transported via a vescile in the golgi complex for outside or embedded in membrane via ER

  • the signal sequence is cleaved and teh SRP is recycled for another time



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polysomes: a group of two or more ribosomes translating an mRNA sequence simolutaneoulsy

  • they will appear as beads on a string
  • in prokaryotes the polysomes may form as transcription is still ocurring

  • ribosomes at the 3" end will be longer than those at the 5" (3 is end and 5 is begining)



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protein modification:

  • can happen by adding new functional groups, via phosphorylation, methylation etc. thus affecting properties and function
  • also by removing exisitng elements,
  • reacemization - converting proteins from arragement to another, changng intermolecular interactions between amino acids

summary of protein expression:

  • transcriptional control - through controlling transcriptional factors and regularoty proteins
  • rna processing - through preventing mature mRNA from being produced
  • rna transportation - preventing them from leaving nucleus in eukaryotes
  • translational control - preventing ribosome from forming assembly
  • protein activity control - destroying proteins via modifications


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amino acid polarity:

  • the polarity of amino acids have different functions in different situations: in membrane bound proteins, as enzymes, water soluable proteins

membrane bound:

  • non polar on the outside interacting with membrane
  • polar on inside, hydrophillic china

water soluable:

  • non- polar nside for stability
  • polar outside for solubility

enzymes:

  • diversely spread out as plairty plays a role in binding for the active site

visualising proteins:

  • haemoglobin - made up of 4 sub units 2 alpha 2 beta, quartenary structure with heme prosthetic groups
    aquaporins - intergral membrane proteins allowing the passage of water
  • keratin - fibrous protein, gives structure in hair nails skin, formed by long twisted strands

  • green flourescnet protein - produced by jellyfish, a barrel like shape made of 12 pp chains



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process: translation occurs in 3 main steps:

  • initiation
  • elongation + translocation
  • termination

initiation:

  • small unit of ribo moves along mRNA till finding start codon, then calls in large unit, assembles ribosome, tRNA comes in and binds to P site

elongation:

  • second tRNA comes and binds to A site
  • first tRNA transfers amino acid to A site tRNA via peptide bond

translocation:

  • ribosome moves by 1 codon position
  • p tRNA become e tRNA and leaves, the tRNA carrying pp chain moves to p
  • another t RNA comes and the cycle is repeated

termination:

  • ribosomes reaches a stop codon
  • this recruits a release factor
  • pp chain is release, tRNA leaves and ribosome disassembles