AOS1 Flashcards
Biomacromolecule
Proteins are biomacromolecules;
- A large organic molecule (in organisms) and are composed of smaller subunits.
Polymer
Proteins can also be defined as a polymer.
- A polymer is a large molecule composed of a chain of repeating similar smaller molecules (monomers)
Monomers
A simple molecule with 2 or more binding sites (to form a macromolecule).
Proteome
Is more diverse than a genome.
- It is the entire complement of proteins in a cell or organism.
Amino acid subgroups:
- Amino group (2 hydrogens + a nitrogen).
- Central carbon (H - C).
- R group (changes)
- Carboxyl group (C =O - O - H)
Polymerisation of amino acids:
- The hydroxyl group is broken off the carboxyl of one amino acid to form a covalent bond with the hydrogen from the other amino group to form a water molecule.
- Amino acid residues are joined together in a peptide bond. (condensation reaction).
( 100 or more chain = poly peptide )
( 20 or less = peptide )
4 levels of protein structure
- Primary.
- Secondary.
- Tertiary.
- Quaternary.
Primary
The sequence of amino acids in a protein.
Secondary
3D form of segments of a polypeptide chain; due to the interactions between nearby amino acids including the Alpha Helices and Beta Pleated Sheets.
Tertiary
The overall 3D shape of a protein (polypeptide).
(The shape of the protein determines its function).
- Folded into shape by chaperone proteins.
- When folded it is held by a hydrogen bond or a disulfide bond between cysteine amino acids.
Quaternary
- Only occurs if the protein is made of more than 1 polypeptide chain.
- When a polypeptide joins to another biomacromolecule.
Purines:
2 nitrogen-containing carbon ring.
- Adenine.
- Guanine.
Pyrimidines:
1 nitrogen-containing carbon ring.
- Cytosine.
- Thymine.
- Urical.
Proteins
Biomacromolecule made up of amino acid chains folded into a 3D shape.
Nucleic acids:
Information molecules that encode instructions for the synthesis of proteins.
- Pentose (5 carbon) sugar.
- Nitrogenous-containing base.
- Phosphate group.
3 main forms of RNA:
- Messenger RNA.
- Transfer RNA.
- Ribosomal RNA.
Messenger RNA
(mRNA).
Carries genetic code from DNA in the nucleus to the ribosome.
Transfer RNA
Carries specific amino acids from the cytoplasm to the ribosome and pairs with complementary code carried by mRNA.
Ribosomal RNA
The main structural component of ribosomes within cells.
RNA polymerisation.
RNA built by enzyme; RNA polymerase.
- Polymerase built on a 5’-3’ direction adding new nucleotides to 3’ end.
Structure of genes:
Exons, Introns, promoter and operator regions
Introns
Contain non-coding DNA.
- Transcribed regions of eukaryotic genes that are REMOVED from RNA before translation.
(Occurs in RNA processing).
- Not found in prokaryotic
Exons
Are EXPRESSED
- Transcribed regions of a gene that are translated.
- Coding DNA.
5’ UTR (untranslated region).
3’ UTR
“the leader”
- upstream; front of gene.
“the trailer”
- downstream.
Promoter
Ahead of gene
- the binding site for RNA polymerase.
- TATAAA
Activator/Repressor
Increase/Decrease
Enhancer/Silencer
Increases/Decreases the rate of transcription (rate of RNA subscribed).
Operator region
A binding site for repressor proteins which can inhibit gene expression.
- Interacts with a repressor to alter the transcription of an operon.
Termination sequence
Sequence of DNA that signals for the end of transcription.
Anticodon
Negative-sense sequence of 3 unpaired nucleotides in tRNA.
- Non-coding strand.
Codon
Positive-sense sequence of 3 nucleotides in mRNA and the coding strand of DNA.
Gene expression
The production of functional gene products.
Process of gene expression.
Transcription: copying of DNA –> pre mRNA.
Rna Processing: Modifies pre-mRNA to produce mRNA.
Translation: Decoding of mRNA strand into polypeptide chain.
Transcription:
Occurs in the NUCLEUS
- RNA polymerase enzyme runs along template (non-coding) strand, 3’-5’ unwinding and unzipping DNA, building a complementary strand of RNA, 5’-3’
RNA processing:
Occurs in the NUCLEUS
- Pre-mRNA; introns are cut out and exons join together (splicing).
- Methyl-g cap added to 5’
- Poly-A tail added to 3’
Translation
Occurs in the RIBOSOME
- Anticodon of tRNA pairs with a complementary codon in RNA.
- Amino acids carried to the ribosome this way are linked by peptide bonds.
Gene regulation
To save cells energy, by inhibiting and activating gene expression. (cells can be switched off)
–> In eukaryotic cells; makes it possible for cells to differentiate for particular functions.
Gene regulation by transcription repression; Low levels
Insufficient quantity of tryptophan causes the repressor protein to become inactive and detach from the operator region, allowing RNA polymerase to transcribe the trp structural genes so that the level of tryptophan can increase.
Gene regulation by transcription repression
- High levels of tryptophan
- Tryptophan binds to the repressor (conformational) changing its shape.
- Repressor protein changes into its active form.
- Binding to operator region and blocking path of RNA polymerase, stopping production of tryptophan
Gene regulation by attenuation
Attenuation: reduce the effect
mRNA transcript contains 4 domains capable of binding to the next.
- Domain 1; 2 tryptophan codons.
- Between domains 1 and 2 there is a STOP codon.
- Attenuator at 3’ of mRNA, containing U-A bonds; weak bonds.
Gene regulation by attenuation; tryptophan
When tryptophan is present, the ribosome runs pasts tryptophan codons and stops at the stop codon (1-2).
- Preventing domain 2 from pairing with 3, making 3 pair with 4, forming a hairpin.
- Putting tension on the attenuator, so mRNA pulls away from DNA ending transcription. (stopping production of tryptophan).
Gene regulation by attenuation; NO tryptophan
- Ribosome pauses at the 2 tryptophan codons waiting for a tRNA carrying.
- Allowing domain 2 to pair with 3, preventing 3 from pairing with 4, creating a hairpin.
- Due to being far away from the attenuator, mRNA does not pull away from DNA, and the ribosome continues along transcribing genes of the operon.
Trp Operon
series of genes within certain species of bacteria that encode for the production of the amino acid tryptophan.
High tryptophan levels: Attenuation.
1 The processes of transcription and translation of the trp operon begin and occur simultaneously.
2 The ribosome (in translation) arrives at the two tryptophan codons in a row.
The tRNA-bound tryptophan present in the cell travels to the ribosome and is added to the protein that is being made by the ribosome.
3 This causes the mRNA molecule being read by the ribosome to fold in a specific way via
hydrogen bonds and form a terminator hairpin loop.
4 The folding of the terminator hairpin loop causes the mRNA molecule to separate from
the template DNA at the attenuator sequence.
5 RNA polymerase detaches from the DNA, causing transcription to stop before any
structural genes are transcribed. Without these structural genes, new tryptophan
cannot be synthesised
Low levels of tryptophan: Attenuation.
1 The processes of transcription and translation of the trp operon begin and occur simultaneously.
2 The ribosome involved in translation arrives at the two tryptophan codons in a row. Due to there being no tRNA-bound tryptophan in the cell, when the ribosome involved in translation arrives at the attenuator sequence that codes for two tryptophan amino acids it pauses. Meanwhile, the RNA polymerase involved in transcription continues along the DNA.
3 Causing the mRNA molecule to fold in a specific way via hydrogen bonds and form an antiterminator hairpin loop.
4 The antiterminator hairpin loop does not cause the mRNA to separate from the template strand at the attenuator sequence.
5 RNA polymerase continues to read the DNA template strand, transcribing the
structural genes for proteins involved in the synthesis of tryptophan and translation
can continue
The protein secretory pathway (PSP)
Involves various different organelles that produce, fold, modify, and package proteins, eventually exporting them from the cell via the process of exocytosis.
- Rough endoplasmic reticulum, Golgi apparatus, transport and secretory vesicles, ribosome.
Ribosome PSP
Synthesises proteins; Assemble polypeptide chains from amino acids by translating mRNA.
Rough endoplasmic reticulum PSP
Folds and transports proteins; folding of newly formed polypeptide chain before passing to the Golgi apparatus.
Transport Vesicle
Transports proteins; buds off rough ER and travels to the Golgi body.
Golgi Apparatus
Modifies and packages proteins; Proteins have chemical groups that are either added or removed in the golgi body, where they are packaged into secretory vesicles for export into cytosol.
Secretory Vesicle
Transports proteins; Buds off Golgi (containing proteins), travels through cytoplasm and fuses with the plasma membrane, releasing protein contained within into the extracellular environment through exocytosis.
Proteome
The complete array of proteins produced by a single cell or an organism in a particular environment
Role of enzymes
Catalyse reactions by lowering the activation energy for a chemical reaction to start.
Catabolic
A chemical reaction that breaks a big molecule into smaller molecules. (Exergonic) No energy- releases energy
Anabolic
Reaction builds a bigger molecule from small molecules. (Endergonic) Absorbs Energy
Exergonic
Releases energy.
Endergonic
Reaction that absorbs energy.
Enzyme changes shape upon binding aligning substrates making it easier for bond to form, forming a product.
Activation energy
Minimum quality of energy which reacting molecules must possess in order to undergo a reaction.
A cell must couple…
A catabolic (exergonic reaction) with an anabolic (endergonic reaction), produces lots of energy.
Enzymes in catabolic reactions:
- Substrate binds to active site, due to almost perfect complementary shape.
- Upon binding the enzyme changes shape by lowering activation energy stressing the bonds in the substrate helping it catabolise 2 products.
The active site:
Part of the enzyme that the substrate binds to with a complementary shape.
Almost perfect fit
An induced fit occurs, changing the enzyme shape.
How enzymes work
- Speed up chemical reactions.
- Not permanently changed/used up in reaction.
- Re-usable.
- Specific to one type of substrate
Reaction rate
How fast a chemical reaction is processing.
- How much product is being made.
- How much energy is being absorbed/produced.
- How much substrate is being used up.
Variables affecting reaction rate
- Temperature.
- pH level.
- Inhibitors.
Temperature affecting enzyme catalysed reaction.
Too cold:
- Reaction rate SLOWS due to the movement of molecules (kinetic energy) lowering as well.
Too hot:
- Enzymes can be denatured.
Denatured
Permanent change in the shape of a protein due to the breaking of hydrogen bonds between non-adjacent amino acid residue side chains.
Tolerance range
A range where enzyme works best.
A little above or below; they still work but not as well.
pH levels affecting enzyme-catalysed reactions
pH high or lower than optimal:
- Reaction will slow down because pH can change the shape of the enzyme and substrate.
- Enzymes MAY be denatured by extreme pH (high or low).
Enzyme concentration and reaction rate
High enzyme concentration = faster reaction rate.
Substrate concentration and reaction rate.
Substrate concentration higher = rate of reaction faster.
(Increasing substrate concentration beyond the point of saturation will not increase the rate of reaction further).
Saturation
A point which active sites of all enzymes in solution are occupied by a substrate molecule.
Inhibitors
Any molecule that binds to an enzyme and prevents the enzyme from binding with it’s normal substrate.
Different types:
- Competitive inhibitors.
- Non-competitive inhibitors.
- Irreversible inhibitor
- Reversible inhibitor.
Competitive inhibitor
Bind to active site of an enzyme and prevents the substrate from binding to the same active site.
Non-competitive inhibitor
Binds the allosteric site (any part of the enzyme other than the active site), changing the shape of the enzyme so that the substrate cannot bind to the active site.
Irreversible inhibitor
Forms a covalent (strong) bond with part of the enzyme causing a permanent change.
Reversible inhibitor.
Binds to enzymes non-covalently (weak).
Co-enzymes
Assist in catalysing reactions; releases energy and is recycled during reaction.
Co-factor
Some enzymes require assistance from a co-factor to catalyse reactions.
- Assists enzyme function.
In co-enzyme assisted reactions:
The enzyme remains unchanged; but the structure of the co-enzyme changes.
- co-enzyme binds to the active site donating energy/molecules (can’t be reused).
- After the reaction; the co-enzyme leaves the enzyme and is recycled by accepting more energy assisting with more reactions
Endonucleases
A broad range of enzymes are responsible for cutting strands of DNA.
Restriction endonucleases
When enzymes target specific recognition sites.
- Sourced from bacteria; produced naturally as a defence mechanism against invading viral DNA that could harm bacteria.
To cut DNA…
Endonuclease enzymes cleave the phosphodiester bond of the sugar-phosphate backbone that holds DNA nucleotides together
Recognition site
In restriction, endonucleases are specific to each enzyme.
Palindromes
the 5’ to 3’ sequence of the template strand is the same as the 5’ to 3’ sequence of the non-template strand.
Blunt ends
(Endonucleases) cut DNA in the middle of the recognition site.
- A straight cut with no over-handing nucleotides.
Sticky ends
A staggered cut, with overhanging unpair nucleotides.
“Sticky” because unpaired nucleotides will be attracted to a complementary set of unpaired nucleotides.
Advantage of sticky end endonucleases
Ensures that an inserted gene is orientated correctly when manipulating DNA.
Ligases
Join fragments of DNA or RNA together.
- Catalysing formation of phosphodiester bonds between the 2 fragments to merge together.
2 types:
- DNA ligase.
- RNA ligase.
Ligases reverse role of endonuclease
Ligases stick; endonuclease cut.
Ligases lack restriction endonucleases; therefore can join together any blunt or sticky end.
- Due to the substrate being sugar or phosphate groups of DNA and RNA, rather than nitrogenous bases (restriction enzymes).
Polymerase
Synthesise polymer chains from monomer building blocks.
- RNA polymerase. Monomer: RNA nucleotide. Polymer: RNA strand.
- DNA polymerase. Monomer: DNA nucleotide.
Polymer: RNA strand.
RNA polymerase
Used in the transcription of genes.
DNA polymerase
The replication or amplification of DNA.
- Can be used to synthesise more strands of DNA, therefore amplifying DNA.
Primer
A short, single strand of nucleotides that act as a starting point of the template strand for polymerase enzymes to attach.
Once polymerase is attached to primer…
It can start to read and synthesise a complementary strand to the template strand in a 5’ to 3’ direction.
Function of CRISPR Cas9 in bacteria:
- When bacterium infected with virus, it uses cas nuclease to create a double stranded break on the viral piece of DNA known as a protospacer (PAM)
- CRISPR spaces are transcribed and converted into gRNA.
- gRNA binds to Cas9 creating a CRISPR Cas9 complex, directed to complementary DNA inside the cell.
- gRNA forms a hairpin loop from the transcribed palindromic repeats either side of the spacer.
CRISPR Cas9 when infected again with another virus.
- The Cas9-gRNA complex scans for matching viral DNA sequences.
- When a match is found, Cas9 cleaves phosphate sugar backbone to inactivate the virus.
- Cas 9 contains 2 active sites to cut both strands of DNA creating blunt ends.
- This cut disables the virus from replicating
- The bacteria retain a copy of the viral DNA in their CRISPR array for future protection
Proctospacer
Short sequence of DNA extracted from bacteriophage by Cas1 and 2, which is yet to be incorporated into the CRISPR gene.
guide RNA
(gRNA).
The specific sequence determined by CRIPSR to guide Cas9 to a specific site.
CRISPR Cas9 in gene editing.
- The CRISPR Cas-9 identifies a PAM region (NGG) on the DNA and then unwinds the DNA
- The Cas9 checks if the sgRNA is complementary to the DNA sequence, upstream from the PAM region on the opposite stand (the opposite strand is checked for the complementary nucleotides)
- If the sgRNA is complementary to the DNA, Cas9 cleaves both stands of DNA upstream from the PAM
- The cell detects and attempts to repair the broken stand of DNA, knocking out a gene
or:
- CRISPR Cas9 identifies PAM sequence in target DNA.
- Creates sgRNA complementary to target sequence 5’ of PAM sequence.
- Combines sgRNA with Cas9 to form a complex.
4.Cas9/sgRNA guided to target sequence by sgRNA.
- PAM sequence in target DNA is complementary DNA; cutting of DNA.
- DNA repair mechanisms change original sequence.
Limitations of CRISPR Cas9.
Ethical implications:
- Treat Embryo before cells differentiate.
–> doesn’t respect sanctity of human life.
–> It’s illegal to genetically modify human embryos.
- Safety.
- Informed consent.
- Inequality.
- Discrimination.
The polymerase chain reaction (PCR)
A DNA manipulation technique that amplifies DNA by making multiple identical copies.
Scientists can analyse:
- Paternity testing.
- Forensic testing; samples of bodily fluids.
- Analysing gene fragments for genetic diseases.
Restriction endonucleases
Any enzyme that acts like molecular scissors to cut nucleic acid strands at specific recognition sites; also known as restriction enzymes
After each cycle on the PCR…
The amount of DNA present is doubled.
x=2^n
Taq polymerase
Heat-resistant DNA polymerase enzyme (from bacteria) that amplifies a single-stranded DNA molecule by attaching complementary nucleotides.
Elongation
Synthesise a longer polynucleotide.
Process of PCR
- Denaturation.
- DNA heated = 90-95 degrees,
- break hydrogen bonds between bases, and separate strands. - Annealing.
- Separate strands cooled to 50-55degrees -
- Primers bind to complementary sequences on separate strands. - Elongation.
- DNA heated to 72 degrees,
- Taq polymerase can work optimally, binding to primer (starting point) and beginning to synthesise a new complementary strand of DNA. - Repeat multiple times to make more copies of DNA.
Forward primer
Binds to START codon on 3’ of template strand, taq polmerase reading DNA in the same direction as RNA polymerase, synthesising a new strand of DNA.
Reverse primer
Binds to STOP codon on 3’ on coding strand, causing taq polymerase to synthesise new DNA strand in REVERSE direction than RNA polymerase would function.
Gel Electrophoresis
Used to measure the size of DNA fragments.
Gel Electrophoresis Process:
- DNA samples with fragments of varying sizes is combined with DNA loading dye.
- Mixture placed in a well at one end of the agarose gel using a micropipette.
- Agarose gel immersed in a buffer solution.
- at top, + at bottom.
4. Electric current is passed through, causing DNA to move towards the positive electrode.
- Smaller fragments move through the agarose gel faster than larger fragments.
- Fragments appear as bands and can be observed under UV light.
Variations in gels movement:
Voltage: Stronger force = further DNA travel.
Gel composition: Great density + agarose concentration = increase difficulty for larger fragments to pass through.
Buffer concentration; greater concentration = more electric current conducted; DNA moves further.
Time; longer = further travel.
DNA profiling
Analyse Short Tandem Repeats (STRs) in a piece of DNA.
Aids in discovering how 2 people are related.
STRs
Small sections of repeated nucleotides vary in length between people and are found in the non-coding areas of autosomal chromosomes.
- Only 2-5 base pairs long.
Tandem because repeats occur one after the other.
Plasmid
Small circular loop of DNA separate from chromosomes found in bacteria.
Making recombinant plasmid
Introducing DNA to an organism where it doesn’t naturally occur.
- Insert foreign DNA into a plasmid, to be taken up by bacteria.
- Bacteria then expresses protein encoded by foreign DNA.
Recombinant plasmid
Circular DNA vector that is ligated to incorporate gene of interest.
Bacterial transformation
A process where bacteria take up foreign DNA from their environment.
Vectors
Help introduce foreign DNA into an organism, used in bacterial transformation.
Gene of interest
A sequence of DNA encoding proteins we wish to produce.
Plasmid vectors
Is selected into which the gene of interest will be inserted.
4 DNA sequences in Vectors
- Restriction endonuclease sites.
- Antibiotic resistance genes.
- Origin of replication.
- Reporter gene.
Restriction endonuclease sites.
in gene of interest.
gene of interest + plasmid,
cut with the same restriction endonuclease generating identical sticky ends on both ends of a DNA sequence.
Hanging genes of interest nucleotides will be complementary to overhanging nucleotides of plasmid vectors allowing them to form hydrogen bonds.
DNA ligase in the gene of interest.
Joins plasmid to a gene of interest, forming phosphodiester bonds in the sugar-phosphate backbone.
Creating a circular piece of DNA; the recombinant plasmid.
2 methods to promote recombinant plasmid uptake
Heat shock
Electroporation
Heat shock
- Bacteria+plasmid into calcium ion solution on ice.
- + calcium ions; make plasma mem more permeable to - charged plasmid DNA. - Solution heated to 37-42 for 25-45 secs before being put back into ice.
(Change in temp=more permeable plasma mem)
Electroporation
- Electrical current passes through solution containing bacteria and plasmid vectors.
- Current causes plasma membrane to become more permeable.
Process of producing recombinant human insulin.
- Creating the recombinant plasmid.
- Creating transformed bacteria.
- Protein production and Extraction.
Creating the recombinant plasmid
insulin
- Plasmid vector produced with ampR and tet and restriction sites.
- Insulin A and B subunit genes (without introns) are cut and ligated to form recombinant plasmids.
Creating transformed bacteria
insulin
- Plasmids are added to bacteria to create transformed bacteria.
- Bacteria colonies tested to find bacteria that successfully took up recombinant plasmids.
- lacZ gene is inserted into the plasmids.
- Plasmids containing lacZ added to bacteria to create transformed bacteria.
Protein production and extraction.
insulin
- Bacteria colonies tested to find bacteria that successfully took up recombinant plasmids containing lacZ.
- Insulin sub-units genes expressed attached to large b- b-galactosidase proteins.
- Insulin A and B subunit proteins are isolated, purified and combined to make human protein.
Genetic engineering
Alternation of an organism’s genome.
2 types of GMO (genetically modified organisms)
Cisgenic organism - genes from the same species.
Transgenic organism - genes from different species.
GMO in agriculture
- Increase crop productivity.
- Increase disease resistance of crop.
Producing transgenic plants
- Gene identification; gene of interest identified and isolated.
- Gene delivery; The gene of interest is delivered to cells of the host organism.
- Gene expression; the transformed gene is grown repeatedly before being applied in the field.
Transgene
The gene that is artificially introduced into the genome of a separate organism.
Increasing crop productivity
- Quality of crops can be improved; increasing the nutritional value and ability to grow in different countries
Growth of population = more food needed.
Increasing disease resistance
To become less impacted by plant pathogens.
Issues surrounding GMOs
- Potentially unsafe or unnatural.
- Range of biological, social and ethical implications that are hotly debated.
