U3 AOS 1 | DNA manipulation

Nucleic acids, proteins, enzymes, genes, DNA manipulation

proteins are…

group of molecules which have many functions in a cell

types of proteins

carrier proteins = move molecules through by adapting to shape.

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channel proteins = molecule cross straight through the membrane.

polypeptide

one of the four types of biomacromolecules

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structural word describing a chain

proteome

all the proteins that are expressed by a cell or organism at a given time

protein functions (CHEATS + D)

* contractile - actin and myosin
* hormones - insulin to send msgs
* enzymes - speed reactions


* transport - move things
* structure - keratin
* defence - antibodies

what is an amino acid

monomers of proteins

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* there are 20 amino acids

amino acid structure

* central carbon bonded to hydrogen
* carboxyl group (COOH)
* NH2 (amine group)
* R - group (varies for each amino acid)

chemical properties of R groups

non polar making them hydrophobic

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polar making them hydrophillic

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(+) or (-) charged

describe how amino acids polymerise

* hydrogen and oxygen from the carboxyl of one amino acid and the hydrogen from another amino acid break to form a water molecule and form a covalent bond (peptide bond)

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* this creates a polypeptide chain

4 levels of protein structure

1. primary - sequence of amino acids which affects overall 3D shape due to the interactions which impacts its function.

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2. secondary - folding of primary structure into beta pleated sheets, alpha helixes and random coils. alpha helix is flexible and beta pleated sheets provides more structure.

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3. tertiary - overall 3D shape due to interaction between amino acids and R groups. enables proteins to complete a function

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4. quarternary - not all go up to this stage. when many polypeptide chains join. creates larger proteins with more structure, complex functions

functional diversity of proteins

* can create so many combinations of amino acids that fold into polypeptides of varying shapes and sizes

nucleic acids

polymers of nucleotides which store genetic info to help produce proteins

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* DNA - deoxyribonucleic acid
* RNA - ribonucleic acid

basic nucleotide structure

1. phosphate group
2. pentose (5-carbon) sugar
3. nitrogenous base

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the pentose sugar is assigned a number in clockwise direction.

* 1’ which attaches to the nitrogenous base
* 3’ which attaches to the phosphate of the following nucleotide
* 5’ which attaches the five-carbon sugar to the phosphate group of the nucleotide.

what two categories are nucleotides categorised into..

* purine: two rings. eg: guanine, adenine
* pyrimidine: single ring. eg: cytosine, uracil, thymine

what end are nucleic acids added to?

3’ end

DNA

* double helix shape, antiparallel
* carries the instructions for proteins which are required for cell and organism survival

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DNA is found in other places besides nucleus; mitochondria and chloroplast have their own DNA.

prokaryotes dont have a nucleus and have a circular chromosome containing DNA.

structure of DNA

A double bonds with T (adenine = thymine)

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G triple bonds with C (guanine and cytosine)

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always equal numbers of A and T. and C and G nucleotides

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DNA polymerase is the enzyme which polymerises DNA

why does nucleic acid (DNA) have 5’ and 3’ ends

* two strands antiparallel
* DNA can be transcribed in one direction (specific)
* enzymes build new strands along template strand by adding nucleotides to 3’ end

RNA

single stranded. includes mRNA, rRNA, tRNA

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has uracil which pairs with adenine instead

mRNA

carries a copy of genetic info from nucleus (DNA) to ribosomes for protein synthesis.

tRNA

delivers specific amino acids from the cell cytoplasm to the ribosome and pairs with the complementary code carried by mRNA

rRNA

main structural component of ribosomes (around 60%). the rest is protein

DNA vs RNA

deoxyribose sugar has an absent oxygen on 2’ position of the sugar.

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DNA is found in nucleus, mitichondria and chloroplast, RNA found in cytoplasm

exocytosis

a type of bulk transport which exports things out of a cell

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requires energy

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* occurs due to the fluid nature of the membrane which allows fusion.

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1. vesicle contains teh secretory prodycts and is transported to membrane
2. vesicle and plasme membrane fuse
3. the secretory products released from cell into extracellular environment

endocytosis

involves the engulfment of molecules by extensions of the plasma membrane, importing them into the cell.

protein secretory pathway

* nucleus - has DNA which contains code for genes

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* ribosomes - site of protein synthesis which assemble polypeptide changes by translating mRNA.
* free floating ribosomes make protein for the cell
* ribosomes attached to rough ER make protein for export
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* rough ER - if a protein is to be exported, the ribosome creating it is usually attached to the rough ER which allows for folding of the chain before transporting it to the golgi.

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* transport vesicle - a transport vesicle containing the protein buds off the rough ER and travels to golgi which fuses with golgi membrane and releases it.

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* Golgi apparatus - proteins can have chemical groups added/removed thus modified where they are packed into secretory vesicles for export or released into cytosol

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* secretory vesicle - undergoes exocytosis to transport proteins.

other organelles involved in the protein secretory pathway

* mitochondria which is the site of ATP synthesis which helps with transportation.

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* plasma membrane which fuses with vesicles and facilitates release of proteins

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* nucleus which stores DNA which contain the instructions for mRNA.

what is a gene?

section of DNA that carries the code to make a protein

genome

the complete set of DNA housed within an organism

three adjacent nucleotodes in DNA

triplet

triplet to mRNA

codons

tRNA bases

anticodons

universal

almost all living organisms use same codons to code for specific amino acids

degenerate

the same amino acid can be coded by different codons

unambiguos

each codon can only code for specific amino acid

non overlapping

each triplet/codon read independently of adjacent triplets or codons

parts of a gene + function (eukaryotes)

promoter (5’) - RNA polymerase binds

introns - non coding sections of DNA

exons - coding sections of DNA

termination sequences - signals end of transcription

gene structure + function (prokaryotes)

introns not found in prokaryotes

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operator region - binding site for repressor proteins

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leader region - one mode of regulating gene expression

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^^ in additions to promoter, exons ad termination sequence

stages of gene expression

1. transcription

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2. RNA processing

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3. translation

transcription

* copies DNA into pre-mRNA as DNA is too big to leave nucleus
* occurs in nucleus

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* DNA unwinds/unzips
* RNA polymerase catalyses transcription through the joining of complementary RNA nucleotides in a 5’ to 3’ end
* transcription of the DNA template strand into pre-mRNA occurs
* pre-mRNA is complementary to the DNA template strand
* in the pre-mRNA, adenine (A) pairs with uracil (U), not with thymine (T).

RNA processing

after transcription to make pre-mRNA into mRNA.

* in nucleus

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* addition of methyl G-cap - so bind to ribosome
* adition of poly-A tail - so it cant degrade. adds stability
* introns removed
* exons spliced

alternative splicing

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* rearranges the order of exons so single gene can give rise to many mRNA strands.

translation

reading and converting the information in mRNA into a polypeptide chain

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* mRNA exits nucleus and binds to ribosome (either in cytosol or rough ER)

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* ribosome binds to and reads the mRNA molecule
* tRNA anticodons are complementary to the mRNA codons
* tRNA brings the corresponding amino acids to the ribosome


* adjacent amino acids are joined together into a polypeptide chain via a condensation reaction.

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→ every polypetide chain starts with Met

→ once mRNA reaches STOP codon, no amino acids added

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* ATP needed
* after translation, polypeptide folds into the protein which can be used in cell or exported out

what is gene regulation

inhibiting or activating gene expression. switchin on or off

regulatory genes

code for proteisn which affects gene expression

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eg: repressor proteins

structural genes

codes for proteins that are involved in structure and function in a cell

benefits of gene regulation

* prevents unnecesary production of gene products (proteins).
* conserves energy
* also ensures cells produce approapriate protein

operon

linked genes that share a common promoter and operator

trp operon

series of genes involved in production of trp.

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* found in prokaryotic cells

trp operon structure

* has two promoters because one for each gene type
* the regulatory gene is always expressed

function of the promoter region

The promoter region is the site where RNA polymerase binds to DNA to enable the transcription of the structural genes.

the two methods of regulating trp operon

* repression

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* attenuation

repression - high levels of trp

1. the regulatory gene codes for repressor protein

2. the tryptophan binds to repressor protein which causes it change shape

3. the repressor protein is activated and binds to operator which stops RNA polymerase from binding to promoter and transcribing. → inhibits the structural gene

repression - low levels of trp

• there is no trp to bind to repressor proteins which makes the repressor protein to becom inactive

• this results in RNA polymerase being able to bind to promoter and transcribe the structural genes of trp operon to create enzymes needed for tryptophan production.

leader region

* Have complementary sections which enable it to make hairpins
* Region 1 has two trp codons.
* The end of the leader region, have attenuator
* Hairpin forms at attenuator

attenuation - high levels of trp

• translation and transcription occur simultaneously

• ribosme comes up to attenuator sequence

• tRNA with trp amino acid travels to ribosome

• causes terminator hairpin loop to form

• mRNA seperates from template strand and RNA polymerase and ribosome detaches

attenuation - low levels of trp

* transcription and translation occur simultaneously
* ribsome pauses while RNA polymerase continues transcribing as no tRNA has trp to bring to ribosome
* causes mRNA to fold and make antiterminator hairpin loop
* this loop enables RNA polymerase to contine transcribing the structural genes.

what are enzymes?

organic biological catalysts which speed up reaction by lowering activation energy

→ enzymes are proteins thus made of amino acids

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* without enzymes, reactions would occur really slowly

catabolic

big into small

* exergonic (releases energy)

anabolic

small into big

* endergonic (absorb energy)

features of an enzyme

* reusable - not used up in reaction
* specific shape - due to interactions between amino acids
* active sit is complementary to substrate

enzyme models

lock and key: substrate fits perfectly iton active site. this isnt true as proteins are flexible and can change shape

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induced fit model: active sit changes slghtly to better fit substrate

how to enzymes catalyse a reaction

* active site and substrate have complemetary shape.
* active site and substrate bind = enzyme susbtrate complex
* react to form products

activation energy

• min amount fo energy needed for reaction to begin

dding more enzymes doesnt lower activation energy further but the reaction DOES speed up as there are more active sites for substrate to bind to.

denaturation

active site of enzyme changes → no longer function

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irreversible

factors affecting enzymes (temp)

low temp = slow rate of reaction and slow movement

optimal temp = fastest rate of reaction

high temp = enzymes denature

factors affecting enzymes (pH)

* if too high or low then denatures
* only functions at optimal pH

factors affecting enzymes (substrate conc)

* rate of reaction increases if susbtrate conc increases
* saturation point is reached when too many substrates and all active sites are reached = reaction rate os constant after this

factors affecting enzymes (enzyme conc)

* if enzyme conc is high than the rate of reaction will be high as lots of active sites for substrate to bind to.
* once all substrates are binded, reaction rate stays the same

competitive inhibition

• block active site so substrate cant bind

• inhibitor has complementary shape to active site

• no reaction

• increasing substrate can minimise effect of copetitive inhibitors

The more competitive inhibitors = inhibit enzyme activity more = less likely for substrate to bind

non competitive inhibition

* bind to allosteric site changing active site shape thus substrate cant bind

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reversible inhibition

* bind to active site weakly → temporary, bonds can be broken
* they slow rate of reaction but dont stop forever
* can be washed out by dialysis

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* inhibitor can have its effects overcome by increasing the amount of substrate present
* non competitive, reversible inhibitors arent impacted by substrate conc.

irreversible inhibition

* form strong bonds between R-groups = not broken
* enzymes will be unable to bind with substrate or catalyse reactions

cofactor

small inorganic substance (not made of carbon and hydrogen. Eg: Zn, Mg ions → body needs this for enzymes to work better). Bound within the enzyme but not active site

coenzymes

these assist enzymes in catalysing reactions be enabling substrate to better fit.

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* not proteins but are organic molecules (lipid, carbohydrate).
* reversibly loaded and unloaded
* subset of cofactors

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during reaction, conenzyme binds to active site and donates energy. substrate also binds.

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after reaction, coenzymes leave the enzyme and is recycled by accepting more energy to assist other reactions

biochemical pathways

* series of reactions to create an end product
* each enzyme catalyses a different product which used in the series of reaction

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* if something goes wrong (eg: enzyme 2 couldn’t function; inhibitor, denaturation, gene mutation) then effects the end product as well as the rest of the reaction)

three types on enzymes used for DNA manipulation

* endonucleases
* ligases
* polymerases

(restriction) endonucleases

* cut at specific recognition sites
* active site of an endonuclease is complementary to recognition site
* create sticky ends (overhanging nucleotides) or blunt ends (straight thru, no overhangs)

how many fragments are formed?

circular DNA - n.o of cuts = n.o of fragments

linear DNA - n.o of cuts = fragments (cuts + 1)

ligases

* join the fragments by forming phosphodiester bonds

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polymerase

* add nucleotides to DNA or RNA which can lead to copying entire genes
* RNA polymerase is generally used in transcription
* DNA polymerase is generally used in replication and amplication of DNA

Primers

* a short, single strand of nucleic acids that act as starting points for polymerase enzymes to attach.
* add to 5’ end
* a primer is required for a polymerase to attach to template strand
* once polymerase attaches to a primer, it reads and synthesises a complementary strand to template in 5’ to 3’ end.

what is PCR

* amplifies sample of DNA creating additional copies

what is PCR used for? (purpose)

* paternity testing
* forensics
* testong for genetic diseases

materials needed for PCR

* DNA sample
* taq polymerase (type of DNA polymerase) → used as it has higher optimal temp
* nucleotide bases
* specific DNA primers to join to 3’ end

how is a target region of DNA isolated

* primers must be attached to DNA section to be copied (PCR)
* restriction enzymes used to cut

process of PCR

1. denaturation - heated to 95 to break H bonds and seperate strands
2. annealing - the strands cooled to 50 so primers can bind
3. extension/elongation - DNA heated to 72 so taq polymerase bonds to primers and synthesises new strand
4. steps 1-3 are repeated to create many DNA strands

why are two genetically different primers used during PCR?

* DNA is anti-parallel double stranded therefore two different needed

why is taq polymerase used instead of eukaryotic DNA poly

* Taq poly can withstand high temps of PCR compared to DNA poly which would denature at such high temps

gel electrophoresis process

1. DNA samples loaded into wells using micropipette.

   • gel is made of agarose - has tiny pres to enable DNA fragments to move

   • gel is immersed in a buffer to carry the current and maintain temp

2. electric current passes through the gel using two electrodes

   • negative electrode is near the wells. - DNA is negatively charged due to its phosphate backbone

   • when current is applied, the DNA fragments will move from negative to positive

3. smaller fragments move faster and further than large fragments

   • large fragments closer to negative electrode

   • use a UV light or dye to see fragments

standard ladder/molecular marker

* known lengths
* used for comparing sizes

variations/errors in gel results occur due to

* voltage applied
* gel composition
* concentration of buffer
* time

what is gel electro used for?

* genetic testing - using PCR to amplify DNA and see if it matches infividuals geneotype
* DNA pofiling, paternity testing

factors that affect migration of DNA fragments through agarose during gel electro

• the size of the molecules, as the larger molecule will move more slowly

• the charge of the molecule, as the negative charge means that DNA moves towards the positive electrode

• the length of time the voltage is applied, as there may not be enough time for the DNA to migrate through the gel

• the concentration of the agarose, as denser agarose results in the molecules moving more slowly

bacterial transformation refers to

the process by which bacteria take up foreign DNA

plasmids

small circular chromosomes. look like circles

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* replicate independently

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role of vectors

• transport material into another thing

what is needed for bacterial transformation

* gene of interest - shouldnt have introns
* plasmid vector
* restriction endonuclease
* DNA ligase

steps involved in bacterial transformation

1. Plasmid extracted from bacterial cell
2. Same restriction enzyme used to cut the plasmid and gene of interest
3. DNA ligase (creates phosphodiester bonds to join sugar phosphate backbone) used to bind the foreign DNA into plasmid DNA. After binding, the DNA fragment permanent of recombinant plasmid
4. Recombinant plasmid added to bacterial culture via heat shock/electroporation (makes membrane permeable).
5. antibiotic selection to determine which bacteria has been transformed successfully
* Spread antibiotic on petri dish
* The ones with antibiotic resistance gene will survive (recombinant plasmid) .
* The one without the antibiotic resistance will die.
* The ones that die don’t have the recombinant plasmid

why does bacterial transformation work?

this works because DNA is ‘universal’ - same genetic code codes for the same amino acids