Biology U3AOS1

What are the four biomacromolecules?

Carbohydrates (monosaccharides, CHO)
Proteins (amino acids, CHON(S))
Lipids (fatty acids, glycerol, CHOP)
Nucleic Acids (Nucleotide)

What does proteome mean?

The proteome of a cell is all the proteins that at found within a cell, or can be expressed by the cell

What is the TATAAA box

- A type of promotor region for eukaryotes
- determines the start point for transcription.
- Where RNA polymerase binds for transcription
-Located in the promoter region of the upstream flanking region
- This DNA sequence found in the promoter indicates where a genetic sequence can be read and decoded

What is Post-transcriptional modification?

- Changes to newly transcribed pre-mRNA (hnRNA) after transcription has occurred and prior to translation into a protein product
- A methyl cap is added at the 5' prime end which is essential for efficient gene expression and cell viability
- A poly A tail is added at the 3' which makes the RNA molecule more stable and prevents its degradation
- Splicing also occurs in which introns are spliced out and exons are left and still are present in the mRNA unlike the introns
- After this process it is considered mRNA

Transgenic

- That one or more DNA sequences from another species have been introduced by artificial means

Transcription

There are three stages
1. Initiation
- Transcription factors bind to the promotor region to - initiate transcription
- RNA then binds to the promotor region
- This signals hydrogen bonds to break between the strands of DNA, exposing each strand and the DNA unwinding.
2. Elongation
- RNA polymerase moves along the template strand in 3'-5' direction reading the DNA nucleotide sequence and using free-floating complementary RNA nucleotides to synthesise a pre-mRNA single strand in 5' to 3' direction.
3. Termination
- Ends when RNA polymerase reaches a termination sequence.
- RNA polymerase detaches, releasing the pre-mRNA and the DNA winds up again

Translation

- Reading and converting the information carried in the mRNA molecule into a polypeptide chain
1. Initiation
- 5' binds to ribosome, read until start codon
- tRNA with complimentary anticodon binds to ribosome and delivers the specific amino acid to indicate the start of translation
2. Elongation
- mRNA is read through ribosome so that the next codon can be matched with its complimentary tRNA anticodon
- Then, the complementary tRNA molecules deliver specific amino acids to the to the ribosome
- tRNA will bring in specific amino acids according to the mRNA by anticodons which are complimentary to the codons of the mRNA
- Amino acids joined together through condensation reactions creating peptide bonds between amino acids
3. Termination
- Ribosome reaches a stop codon on the mRNA molecule
- Signals to end translation as there is no corresponding tRNA
- Ribosome detaches and the polypeptide chain is released.

Coding strand

- The coding strand has the nucleotide bases in which is wanted in the mRNA
- But, when copying DNA to mRNA during transcription, it uses complimentary base paring
- So if the wanted strand, the code strand is
TAC GAT TTA CAG
- If you were to copy it into mRNA, it would give you the complimentary of it. But that is not what is wanted, the exact nucleotides are wanted of the coding strand but in mRNA
- So the template strand, which is complimentary to the coding strand is used instead.

Coding Strand: TAC GAT TTA CAG
Template Strand: ATG CTA AAT GTC
mRNA: UAC GAU UUA CAG

- See how by copying the template strand you get the copy of the coding strand but in mRNA?

- The coding strand (or informational strand) is the DNA strand whose base sequence is identical to the base sequence of the RNA transcript produced

Template strand

- The sequence that copied during the synthesis of mRNA during transcription
- Is complimentary to the coding strand
- 3' to 5'

Initiation factors

- Proteins that bind to the small subunit of the ribosome during initiation of translation and have the ability to interact with activator to increase the rate of translation

Transcription factors

- Proteins that bind to the promotor region or TATAA box and control the functioning of RNA polymerase
- Gene activity determines whether is transcribed
- Affects transcription

Alternative splicing

- Process where different exons may be spliced, resulting in a single gene producing multiple different mRNA strands.
- Exons can be removed during the splicing process. Only in alternative splicing
- This means that single gene can produce many different mRNA molecules depending on which exons are spliced out or kept
- This allows for a single gene to give rise to many different mRNA strands and code for many different proteins
- Exons are joined in different combinations, leading to different (alternative) mRNA strands
- For example exons 1 2 3 4 5
- But 2 and 4 removed so, the exons (coding DNA) will be 1 3 5
- This allows for different combinations of mRNA
- Affects gene expression, affects what information a gene is synthesised into a product

Inhibition of expression

- Silencing a gene
- Prevents the gene from expressing at a post-transcriptional level (when gene splicing occurs, so after transcription)
- Preventing the gene from expressing itself
- Used in gene therapy to silence mutated genes

Activators

- A protein that promotes gene expression
- Positive control over gene expression and promote gene transcription
- A transcriptional factor

Repressors

- A protein that turns off the expression of one or more genes
- Negative control over gene expression
- Binds to the promotor region, preventing the production of messenger RNA since RNA polymerase can not attach
- A transcriptional factor

Enhancers

- Enhancers are regions in a gene
- DNA sequence that can be bound by activators to increase the likelihood of that gene being transcribed

Silencers

- Silencer refers to a region of a gene
- DNA sequence capable of binding transcription regulation factors, repressors
- DNA sequence where repressors bind

Structural Genes

- A gene that codes for any RNA or protein that will be a structural protein
- Structural protein is a structural element which helps with cell shape and contribute in psychical structures

Regulatory Genes

- A gene that regulates the expression of structural or other genes
- Creates a regulatory protein which controls other proteins or gene other genes
- These proteins can affected the rate of transcription of other genes

Trp Operon

- Trp operon is a series of genes that are involved in the production amino acid, tryptophan
- It is a repressible system because it is essentially always being expressed
- Regulatory gene sequence includes: Promotor (where the RNA polymerase binds), operator (where the repressor binds)

What happens when there is too much tryptophan in the cell?

-When there are tryptophan levels within the cells, the tryptophan present will bind to the trp repressor protein
- When the tryptophan binds to the repressor, it activates the repressor
- The now activated repressor binds to the trp operator, upstream from the trp genes, but just downstream of the promotor region
- Because the repressor protein is attached on the operator, the RNA polymerase when it attaches to the promotor, will not be able to transcribe the trp genes because the repressor blocks the way of the RNA polymerase

What happens when there is too little tryptophan in the cell?

- Since the repressor needs tryptophan to be bound to it to be active and repress the genes, if there are low levels of tryptophan, then there isn't tryptophan to keep the repressor active, thus it becomes inactive
- So, without the tryptophan binding to the repressor, in becomes inactive
- Therefore, there is no repressor bound to the operator region of the gene, meaning that the RNA polymerase can move along since it has a clear path and transcribes the genes

Condensation Reaction Nucleotides

- DNA nucleotides have a missing oxygen at the 2nd carbon
1. The OH at the 3' carbon bonds where the OH on the phosphate group is on the next nucleotide
2. The H is lost from OH on the 3' carbon
3. The OH on the phosphate group is also lost
4. The lost H from the 3' carbon and OH from phosphate groups join to make H20, water
- The creation of water gives the name condensation reaction, also the lost of the H and OH give the name condensation
-Hydrolysis is the reaction to reverse this

Condensation Reaction Amino Acids

- Amino acids are made up of a central carbon, r side chain, amine group and carboxyl group
- There is a hydroxyl in the carboxyl group
- In this example there are two amino acids, amino acid A and amino acid B following directly after
- In amino acids A, the hydroxyl found in the carboxyl group is broken off.
- The hydrogen found in the anime group of amino acid B breaks off
- This breaking off forms a covalent bond (peptide bond) between amino acid A and B
- The Hydroxyl (OH) joins with the H and forms water

Peptide Bonds

- Peptide bonds are a covalent bonds that are formed during condensation reactions between amino acids
- Bonds between amino acids in a polypeptide chain
- Holds amino acids together

Phosphodiester Bonds

- A bond between a sugar group and a phosphate group
- Bond between 2 nucleotides that holds them together
- Links two nucleotides in DNA and RNA
- Created in condensation reactions of nucleotides

Trp Attenuation

- Happens when there isn't heaps of tryptophan, but there are still some attached to tRNA molecules
- Because in prokaryotes, transcription and translation happen in the cytoplasm, once the gene is being transcribed, the ribosome begins translating. So happens almost simultaneously.
- Remember, this process happens in the region BEFORE the GENES for TRYPTOPHAN. The RNA polymerase has not yet READ THE GENES FOR TRYPTOPHAN, HAPPENS IN THE LEADER
- Within the leader region, a region between the promotor and the operon (series of genes) there are 4 domains (1-4)
- Parts of these domains are complimentary to other parts of domains (first few bases of domain 1 might be complimentary to last few bases to domain 2). So, if given the opportunity theses domains will stick together
- In domain there are two codons for tryptophan
- Within domain two there is a stop codon
- When the ribosome reads the mRNA for the tryptophan in domain 1, the tRNA molecules will bring the amino acid. But as stated before, the ribosome will stop at the stop codon in domain 2. The ribosome siting there between domain one and two, prevents them from their complementary parts sticking together
- This means that the RNA polymerase gets "ahead" of the ribosome and transcribes domains 3 and 4 and the attenuator (section of AAA bases which has complimentary mRNA UUU before the trp genes)
- But because three and four are complimentary they stick to each other creating a loop like a hairpin. That tension of 4 pulling to stick to three, also separates the attenuator from the DNA
- Because attenuator pulled from DNA, RNA polymerase detaches and the genes for trp are never transcribed

What happens if there is no tryptophan?
- There no trp in the cell needs some. No tRNA brings amino acids for the two trp codons in domain one
- So when the ribosome reaches these codons in domain one, the ribosome stop waiting for the tRNA to bring the amino acids, but because there isn't enough, it will not bring it
-This means that the RNA Polymerase will continue to transcribe domains 2-4
- But because domain 2 and 3 are complimentary, they will stick together
-But because 2 and 3 are stuck together 3 and 4 and not stick together as shown in the other scenario
- Meaning that the attenuator is not pulled off since 4 is also no pulled off, thus the RNA polymerase does not detach from the DNA and continues transcribing and allows trp to be mad e
- So because the tRNA does not bring the amino acids at the trp codon in domain 1, it stops before the stop codon in domain 2, meaning it prevents domain 3 and 4 from sticking keeping the attenuator intact since not pulled by 4 anymore and the RNA polymerase can continue transcribing

Primary Structure

- Refers to the sequence of amino acids in a polypeptide chain
- Can provide information on how protein will fold to form specific structures.

Secondary Structure

- Formed when a polypeptide chain folds and coils by forming hydrogen bonds between amino acids of its different sections creating alpha helices, beta pleated sheets and random coils
- Results form hydrogen bond formation between amino acids with in its different sections
- Due mainly to hydrogen bonding
- Can be multiple within a protein
-The secondary level of structure of an enzyme is determined by hydrogen bonds, and is the folding and coiling of a section of the polypeptide chain.

- Alpha helix
- Generated when a single polypeptide chain turns around itself to make a rigid cylinder
- Bends in a helical manner
- Made of INTRA- molecular hydrogen bonds. This mean that hydrogen bonds are made within itself not other secondary structures
- In high temperatures these bonds break
- Is stretchy
-When pressure is applied bonds can break but can also go back when pressure is removed. Hair solons manipulate the hydrogen bonds within hair


-Beta pleated sheets
- Form from neighbouring polypeptide chains that run in the same or opposite direction
- Multiple beta pleated sheets
- Made of INTER- molecular bonds meaning that these hydrogen bonds are between other beta pleated sheets
- Does not stretch like alpha
- Needs more pressure to break

- Random coiling also is a secondary structure
- Where function begins

Tertiary Structure

- All proteins have tertiary structure as least
- Main 3D structure begins here
- Overall 3D shape of protein
- Due to attraction between R-groups of amino acids
- Attractions between alpha helices and beta pleated sheets
- Secondary structure further fold by forming interactions and bonds between amino acids and r-groups with different SECTIONS of the polypeptide chain.
-The tertiary structure of proteins is determined by hydrophobic interactions, ionic bonding, hydrogen bonding, and disulfide linkages
- For example, hydrophilic R groups on amino acids attract other hydrophilic R groups
- If in hydrophobic environment, then hydrophobic r-groups would be on the outside of the protein while the hydrophilic r-groups would be on the inside and visa versa.
- Chaperonins are used to help create this structure

Quaternary Structure

- When two or more polypeptide chains with tertiary structure join together
- Not all proteins have this
- Each polypeptide chain is referred to as a subunit of the protein

Chaperonins Enzymes

- Chaperonins are proteins that help fold other proteins

Gene Expression

-The process of reading the information stored within a gene to create a functional product, typically a protein
- Complex series of events which results in the formation of functional gene products such as proteins or non-coding strands of RNA
- Information in a gene is synthesis
- Transpiration and translation play major roles in gene expression

- The expression or synthesis of a genes product can be affected through:
- Repressors
- Activators
- Alternate splicing

Gene regulation

- The control of gene expression, typically achieved by switching transcription on or off
- Regulatory genes code for proteins that influence the expression of structural genes, preventing the over or under expression of a particular protein and allowing the cell to adapt to its needs
- Helps conserve energy, material and time
- Also helps specialise a cell, ensuring that only genes related to cell's function are expressed and not they ones that are not needed. For example, genes in eyes needed to perform its function, is not expressed in heart cells.

Characteristics of DNA

- There are 4 properties
- Universal: In almost all organisms the same DNA triplet is translated to the same amino acid and is read the same way

- Unambiguous: Each codon is only capable of one amino acid. For example UUA will only code for leucine

- Degenerate: Each amino acid may be coded for by multiple different codons. For example, UUA and UUG both code for leucine. Changes to the original DNA sequence through mutations may not necessarily lead to the insertion of a different amino acid. UUA, a changed to G so UUG still will code for wanted amino acid Leucine

- Non-overlapping: Each triplet or codon is read independently, without overlapping from adjacent triplets o codons.

What is an anti-codon

- 3 bases found on an tRNA which are complimentary to its corresponding codon in the mRNA sequence

What is the Proteome?

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

What are the types of proteins and their functions?

-STRUCTURAL
•Provide support and structure
•Examples: Collagen & Keratin

-ENZYMES
•Speed up chemical reactions by providing a site for the reactants & lowering the activation energy required.
•Examples: Amylase & RNA Polymerase

-IMMUNOGLOBULIN
•Defend the body against disease
•Example: Antibodies

-HORMONES
•Regulate activities within the body
•Examples: Insulin & Melatonin

-RECEPTORS
•Respond to external and internal stimuli
•Example: Acetylcholine Receptors

-TRANSPORT
•Carry molecules around the body
•Examples: Carrier & Channel Proteins in the cell membrane

-STORAGE
•Storage of metal ions and amino acids
•Examples: Casein & Ferritin

What are enzymes?

- An organic molecule, typically a protein, that catalyses (speed up) specific reactions
- Generally tertiary structure
- Soluble and globular in shape which have tertiary structure or at times quaternary structure
- Act biological catalysts

How do catalysts and enzymes work?

- Enzymes are considered as a catalyst
- A catalyst is something that speeds up or triggers a chemical reaction by lowering the "activation energy" of a chemical reaction
\-
- The activation energy is the minimum amount of energy needed for a reaction to occur
- These reactions will eventually happen in the body if given time, but catalyst just make them happen sooner or quicker
- Catalysts are not consumed by the reaction

How do enzymes work?

- Each enzyme has a specific molecule that they can react with
- That molecule is called the substrate
- The substrate fits into the active site of the enzyme
- The shape of the active site is crucial, because if the enzyme's active site is not complementary to the substrate, it will not bind and will not be able to catalyse the reaction
- When the substrate and enzyme bind, it is called a enzyme-substrate complex

What are the ways substrates can fit into an active site?

- There are two types of ways the substrate can fit into the active site
- Lock and key
- Principal of a lock and key, the substrate (the key) fits in perfectly since it has the right shape to the active site of the enzyme (the lock)

Induced Fit
- When the enzyme and substrate interact, the interaction changes the shape of the active site to better fit the substrate (like how a baseball glove changes it shape to better hold the ball when being caught)

What is a promoter?

A promoter is a region of DNA where RNA polymerase begins to transcribe a gene

What is an operator

A segment of DNA where the repressor binds to, thereby preventing the transcription of certain genes.
- prokaryotes only

What is an operon?

a group of closely linked genes that produces a single messenger RNA molecule in transcription and that consists of structural genes and regulating elements
- prokaryotes only

What is the leader?

The 5' untranslated region between the promotor region and operon genes

Why and how are enzymes regulated?

- Enzymes can be regulated by other molecules which increase or reduce there activity such as:
- pH
- Temperature
- Concentration of substrates, enzymes and product

What affected enzyme function?

The factors that affect the rate of enzyme action are:
-Temperature
-pH
-Concentration of substrate and product
-Inhibitors & Feedback Inhibition
-Phosphorylation
-Cofactors and Coenzymes

How does temperature affected the rate of reaction in enzymes?

- As a general, when the temperature increases molecules move faster
- So, substrates will move more and will have a higher chance of colliding with an enzyme and increasing the chances of binding to its complimentary active site
- As the temperature increases so does the rate of reaction
- BUT, ENZYMES and PROTEINS
- Beyond the optimal temperature there is a narrow range called the tolerance range in which enzymes cans still function
- Each enzyme has its own optimal temperature where its activity is the greatest
- Proteins denature at a point called the critical temperature which past their optimal temperature
- This point makes the enzymes denature, meaning that bonds that create tertiary and quaternary structures are broken down
- This denaturation results in conformation change (change in the shape of proteins) in the active site of the proteins
- As a result the substrate no longer fits
- Denaturation is irreversible

What happens when it is too cold?
- Enzymes activity decrease below the optimal temperature as the substrate and enzyme move slower decreases the chances of colliding and binding
- They can experience little to no activity or freeze
- But, unlike denaturation, it can be reversed

- NOTE: Make sure you can identify what is affecting the rate of reaction in a graph

How does ph affected the rate of reaction in enzymes?

- Enzymes also have an optimal pH
- If an enzyme if exposed to an environment where the pH is above (too acidic) or below (alkaline) its optimum, then it can denature
- Location of where the enzyme is found greatly affects its pH tolerance, for example, pepsin, an enzyme found in the stomach is based in an acidic environment and therefore would have a higher pH optimum
- Denaturation affects the secondary and tertiary structure of proteins
- Denaturation can occur in both ends of the scale, to alkaline or too acidic, this results in a bell curve graph

How does concentration affected the rate of reaction in enzymes?

- Substrate concentration
- If the enzyme concentration remains constant while the substrate concentration increases, then the reaction rate will increase
- This is because more reactants are available to undergo the reaction
- HOWEVER, if there are so many substrate molecules that continuously occupy all active sites, meaning that the enzymes are saturated with substrate
- This is considered as the saturation point, where even if the amount of substrate increases, the reaction can no longer increase, because all the active sites are occupied all the time
- So if you brought in more substrate, there wouldn't be enough enzymes to catalyse the reactions

Enzyme concentration
-If enzyme concentration is high, then reaction rate will be high because there is a large number of active sites available for the substrate to bind to
- If the enzyme concentration rises (substrate concentration stays the same), then the reaction rate will increase
- This is true until enzymes are in excess, the reaction rate will plateau regardless of any continued increase in enzyme concentration (adding more enzymes won't help if there isn't any substrate)

What are limiting factors and what do they affect?

- It is whatever limits just how fast the reaction can occur
- This can be the enzyme or substrate
- This is a way cells can regulate their enzyme production - the product of the reaction itself inhibits enzyme function

What are the types of enzyme inhibition?

- Enzyme inhibitors are molecules that bind to an enzyme and prevent if from performing its function
-There are two types
- Competitive inhibition
- Non-competitive inhibition

What are non-competitive inhibitors?

- Inhibitor binds to an enzyme at a site other that the active site (allosteric site)
- This binding forms conformational change in the active site of the enzyme, and the change in structure prevents substrate from binding to it, preventing the reaction

What are competitive inhibitors?

- Molecule binds the enzyme's active site
- This directly occupies and blocks the active site, meaning that the substrate is now unable to bind with the enzyme and no reaction will occur
- To block the an active site, a competitive inhibitor has to have a shape that is complementary to the active site, and must also be similar to the shape of the substrate
- UNLIKE the substrate the inhibitor binds to the active site, but does not trigger a reaction
- Both substrate and inhibitor are attempting to bind to the active site

What does reversibility of enzymes inhibition mean?

- Inhibition can be reversible or irreversible
Reversible
•The inhibitor weakly interacts with the active site of the enzyme.
•Decreases the frequency at which the substrate can bind to the active site

Irreversible
•The inhibitor binds strongly either at the active site or at another region on the enzyme.
•Causes the active site to be blocked or a conformational change (shape change of the enzyme) so that the active site is no longer complementary to the substrate.

What is feedback inhibition/ enzyme regulation biochemical pathway mean?

- Biochemical processes within the body can be controlled by something called end-product inhibition
- This is a form of negative feedback
- In end-product inhibition, the final product of the series of reactions inhibits enzyme from an earlier step in the sequence
- Lets say, there are 3 enzymes
- Enzyme 1 produces a product, product A
- Product A is the substrate (reactant) for enzyme 2
- Enzyme 2 uses A to produced, product B.
- B is the substrate for enzyme 3, and enzyme 3 creates the final product, product c
-Using end-product inhibitions, product c, the final product, can be used to inhibit enzyme 1, thus, stopping the production of all the other products needed to create the final product

What is phosphorylation?

-Adding a phosphate will activate
-This is the binding of a phosphate group to the enzyme.
-It is a common way of activating or deactivating enzymes.
-Dephosphorylation of the enzyme can be used to achieve the reverse.
- For example, ATP, usually found as ADP, but when a third phosphate is added, it becomes ATP and can help release energy

What are coenzymes and how do they work?

- A non-protein organic cofactor that assist enzyme function. They release energy and are recycled during a reaction
- Usually derived from vitamins
- Usually donate a proton or electron
- ATP is an example, provides energy for chemical reactions. Before the reaction, it is ADP, adds a phosphate, ATP, then goes to the reaction provides energy by breaking off that phosphate and is left as ADP again, and is reused
-Works alongside enzymes

What are cofactors and how do they work?

- Cofactors are metallic cations that are need by the enzymes to catalyse a reaction
- To catalyse means to bring about the enzyme which will speed up or trigger (catalyse) the chemical reaction
- Assist in enzyme activity
- Cofactors have the word Factor which is similar to factory, and metals are used in factories, so cofactors are metallic

What is a coenzyme and how do they work?

- A non-protein organic cofactor that assist enzyme function. They release energy and are recycled during a reaction
- ATP is an example, provides energy for chemical reactions
-Works alongside enzymes

What are some loaded and unloaded enzymes?

Unloaded Loaded
ADP ATP
NAD+ NADH
FAD+ FADH2
NADP+ NADPH

What does anabolic mean?

-Building up of larger molecules from smaller molecules
- Requires energy

What does catabolic mean?

- Breaking down big molecules into smaller ones
- Releases energy

What are the regions of the gene?

In the Upstream Region is an important section called the Promoter.
Promoter Region
Where RNA polymerase binds for transcription.
Identifies the DNA strand to be transcribed
Identifies where transcription of the gene starts
Identifies the direction of transcription
In Eukaryotes is usually the sequence TATAAA (called a TATA box)
Also can contain regulatory regions for gene expression.
The Downstream region also has sections involved in gene regulation.- Genes have different sections within them
- Firstly there are two major regions
- Coding regions: Areas that will be transcribed and produce a protein (exons)

- Flanking regions: Contain non-coding regions: Areas that will be included in the pre-mRNA but will be spliced out and not make a protein. Does not code for proteins (introns)
-These flanking regions are placed before and after the coding regions of a gene.

Upstream
- Flanking region before the gene is the upstream
-This is the Segment of DNA that Hormones attach to.
-It contains the DNA Code with the instructions where to begin the transcription process
- Has the promotor region
-Where RNA polymerase binds for transcription.
-Identifies the DNA strand to be transcribed
-Identifies where transcription of the gene starts
-Identifies the direction of transcription
- In Eukaryotes is usually the sequence TATAAA (called a TATA box)
- Also can contain regulatory regions for gene expression.

Downstream
Region and the region after the gene is the downstream
- It contains the DNA Code with the instructions where to Stop the transcription process
- The Downstream region also has sections involved in gene regulation.

What is protein denaturation?

-The unfolding and disorganization of a proteins structure
- The disruption of a molecule's structure by an external factor such as heat or pH
- Is irreversible as it affects the bonds with in the protein structure
-Relating this to enzymes, enzymes are a type of protein and can also denature
- Denaturation causes unfolding of proteins this leads to a change in shape. For enzymes, shape has a direct correlation to its function. Since a substrate must be complimentary to the active site, a change it the shape will meant that the active site and substrate are no longer complimentary. This results in the enzyme not being able to catalyse the reaction and not carrying out its function

What are the 3D protein structural classification?

- Can have 2 3D structures
- Fibrous Proteins
•Elongated and insoluble
•Function is usually structural eg. Collagen

- Globular Proteins
•Compactly folded and coiled into a spherical overall shape
•Function as enzymes and hormones eg. amylase

How does protein structure affect its function?

- For proteins, the shape is defines is function. The shape determines what it can do and thus, determines it function
- Enzymes are a good example of how shape affects function
- The enzyme's active site and substrate are complementary
- A change in the active site's shape means that it is no longer complimentary to the shape of the substrate, thus the substrate can no longer fit into the active site and catalyse the reaction, which is what its function is

What does essential amino acids mean?

- Means that is only obtained via diet
- Not made in humans

What is the structure of an amino acid

- The are 4 main components
- Central Carbon
- Amine group (gives the amino part of amino acid)
- R-group which varies from amino acid to amino acid and affects interactions
- Carboxyl (gives the acid in amino acid)

What contributes to gene regulation?

-Gene Regulation is gene expression (usually protein synthesis) and is tightly regulated or controlled so that only the right amount of product (protein) is synthesised by a given cell.

- Alternative splicing contributes to gene regulation
- It changes the order and combination of exons. For example the gene's exons 1, 2, 3, 4, 5, might be that order that one time in transcription, but in another time, the same exons, could undergo alternative splicing resulting in the combination 2,4,5,1,3. This different combination leads to a different sequence of amino acids as the previous one and thus creates a different protein

- Transcription factors also play a role
- Activators bind to the enhancer part of the gene which turns on the gene. This means encourages transcription.
- So, if a gene needs to be expressed less or more it can be regulated by this.
- Repressors bind to the silencer part of the genes which turns the gene off. This means that transcription is not encouraged.
- So if a gene does not need to be expressed, it is regulated by the presences of a repressor

What is the function of the rough endoplasmic reticulum?

- Involved in synthesis and transportation of proteins to other parts of the cell through channels
- Has ribosomes that produces proteins that are generally taken out of the cell. These proteins are put into the cisternal space of the ribosome and carbs (glycoproteins), lipids (lipoproteins) and phosphates (phosphoproteins) can be added
- Involved in INTRACELLULAR transport
- Proteins from the RER are put into transport vesicles and taken to the Golgi complex

What is the function of the Golgi complex?

- Involved in the packing and modifications of proteins and lipids, especially proteins being exported out of the cell
- Has two ends, cis (closer to the RER) and the trans end (further from the RER)
- Has cisternal spaces where proteins are modified and finally packed into vesicles in the trans end of the Golgi body
- Once proteins have left the Golgi can't be modified further
- Involved in EXTRACELLULAR transport, outside the cell
- The number of Golgi is dependant on extracellular transport, so, glands, which secrete a lot, will have a lot of Golgi complexes
- Grows as more transport vesicles attach to give proteins and decreases in size at trans end as it buds off to make secretory vesicles
- Uses secretory cells to take proteins out of the cell

What is the function of the mitochondria?

- Is the site of ATP synthesis.
- Generates most of the energy to power process within the cell
- Energy is needed to moves vesicles around the cell and for exocytosis and endocytosis

What is a transport vesicle?

- Transport vesicle is a vesicle that contains proteins and buds off the RER and travels to the Golgi body.
- Used for transport within the cell

What is a secretory vesicle?

-Transport modified proteins from the Golgi complex to the plasma membrane to be exported out through endocytosis
- Used to transport molecules to the outside of the cell

What does DNA manipulation allow scientists to do?

- Understand the function of genes (think knock out genes, genes removed and see what the gene produces with out it. For example, a gene is knocked out, expressed and there is no pigment, the gene has something to do with pigment)
- Compare genetic information between species
- Solve crimes and paternity cases
- Produce transgenic plants and animals (to improve crop and animal breeds)

What are polymerases?

- Polymerases are enzymes that join monomers to form polymer chains
- For example RNA polymerase forms RNA by joining it monomer, RNA nucleotides into mRNA

What is reverse transcriptase?

-Type of DNA polymerase enzyme that transcribes single-stranded RNA into DNA.
-Creates a complementary DNA strand to a template RNA strand.
-Works by joining together nucleotides (deoxy-nucleotides) in a 5' to 3' direction according to the base-pairing rules.
-It is useful for producing cDNA (complementary DNA) to mRNA so that there are no introns.
-It is the reverse to transcription

What are endonucleases?

- Also known as restriction enzymes
- Enzymes that cuts at a certain point within a nucleic aicid
- That certain point is called the restriction site and each endonucleases has its own specific restriction site with a specific sequence for example, AAGCTT might be the restriction site of one endonuclease, so it will cut at that sequence
- Can be used for different species because DNA is universal
- This cutting can also be called digesting
- The cut will produce fragments which will have different types of ends
- It could have sticky ends or blunt ends
- Site must be palindromic, so same backwards and forwards
- For example: A A G C T T
T T C G A A

What are the types of ends?

- Two types
- Blunt or sticky

Blunt
- result of a straight cut
- No over handing nucleotides
- The nucleotides at the ends will have a base pair

Sticky ends
- Result of a staggered cut
- Has overhanging nucleotides that might not have a base pair
- Advantageous because ensures inserted genes are orientated correctly when manipulating DNA

What do ligase do?

-Linages act like molecular glue
- Enzymes that joins molecules, including DNA and RNA by forming phosphodiester bonds within the sugar-phosphate backbone
- Re-joins sugar-phosphate backbone

What are some DNA manipulation techniques and its applications?

- Gel electrophoresis
Applications
-Paternity and maternity testing (Seeing relatedness)
-Used in forensic science
- DNA profiling
- DNA fingerprinting

PCR
Application
-Used to amplify DNA for Gel electrophoreses

CRISPR Cas-9
- Gene editing

How does CAS-9 work in bacterial cells?

- Bacteria uses the CRISPR system as an immune system
- The CRISPR system capture snippets of DNA from invading viruses to create DNA segments known as CRISPR arrays
- The CRISPR array essentially "remembers" the virus, so when the virus comes a second time, the viral DNA that the bacteria preserved, will identify it and cut that DNA

- CAS9 is the enzyme that goes to the specific DNA (target DNA) and cuts it

CRISPR in bacteria
1. Cas1 and 2 are responsible for cutting the viral DNA in the initial attack
2. By locating the PAM, Cas1 and 2 cut out a short sequence of the viral DNA just before the PAM so it is not included into the protospacer and spacer. THis short sequence that has been cut is known as the protospacer
3.Bacteria then inserts the section of viral DNA into its own DNA to act like quick reference for future viral attacks.
4.When the virus(or a similar one) next attacks the previously copied section of viral DNA is transcribed into guide RNA (gRNA).
5.This gRNA is attached to a Cas9 Protein.
6.The Cas9 Protein is now able to seek out viruses with DNA that matches the gRNA they have.
7.Once a matching virus is found the Cas9 protein is able to cut up the viral DNA and inactivate the virus.

How does CRISPR work in gene editing?

- In gene editing, the gRNA is synthesised by people.
- What makes CAS9 so good is that if the gmRNA is made, it can go an cut at that sequence
- Since endonucleases can only cut at there restriction site, if there isn't a known endonuclease for a particular sequence, then it can't cut. But, if the sequence of the wanted gene is known, gRNA can be made,so as long as Cas9 has a gRNA is can make a cut
-Cas9 has a lot more variety in what it can cut and is not restricted. As long as you know the sequence of the gene that is to be cut, you can make the gRNA.
- When used in humans cells, the same concept applies, the gmRNA is created, goes looking for the DNA strand, if the pam and the gmRNA is complimentary to the DNA, then it will cut
- Once just, the cell will not just leave cut DNA, it could repair the cut in different ways

Non-homologous end joining
- Has no template or guide on how to repair the DNA, makes it prone to mutations, thus if wanting to slice the gene is great, since it is mutated, it will be silenced

Disrupt:
-If a single cut is made, a process called "non-homologous end joining" can result in the addition or deletion of base pairs, disrupting the original DNA sequence and causing gene inactivation.

-Delete:
-A larger fragment of DNA can be deleted by using two guide RNA that target separate sites. After cleavage at each site, non-homologous end joining unites the separate ends, deleting the intervening sequence.


Homologous end joining
- Template is used to help guide the repair of the DNA, reduces mutations
Correct or Insert
-Adding a DNA template alongside the CRISPR-Cas9 machinery allows the cell to correct a gene, or even insert a new, using a process called homology directed repair.
- Template helps guide the repair of the recently cut gene meaning it will correct the gene in a better way

What is a PAM

-Stands for protospacer adjacent (next to) motif
- It is found next to, not in the viral DNA in the protospacer
- The cas 1 and 2 come across a PAM, they are signalled to extract the protospacer from the invading DNA
- Cas 1 and cas 2 cuts the viral DNA just before the PAM, so the PAM is not included into the spacer, what is put into the CRISPR region
- Increases the efficiency of Cas9, cas9 looks for the PAM of a gene sequence and if it matches, it will unwind the DNA and the if the DNA sequence is complimentary with its gRNA

What is a protospacer?

- A protospacer is a short sequence of DNA extracted from a bacteriophage by a Cas1 and Cas2 which has yet to be incorporated into the CRISPR gene
- So, the sequence of DNA before it is placed into the CRISPR system

How is the CRISPR Cas-9 system applied?

- There are the 3 other applications bedside gene editing
- Research:
- Attaching a fluorescent protein to Cas9 to locate a specific gene in the genome
- Disrupting the expression of a gene to see the effect of that protein being knocked out. In turn, this helps identify the function of specific genes. For example, a gene is removed from mice, and these mice end up being albino, we know that in the absences of this gene, there is also absence of pigment, which suggest that the removed gene's function is related to pigment

- Dealing with diseases
- Replacing a deleterious allele with a healthy allele
- Adding genes that code for proteins that decrease the susceptibility to infectious diseases such as HIV/AIDS
- Modifying caner-promoting genes to make them less influential

Agriculture
- Introducing pest and herbicide-resistance genes to increase the yield of crops
-Altering genes to promote increased growth rates to improve the yield of crops

What are the limitations of Cas-9 gene editing?

- Difficult to deliver the CRISPR-Cas9 material to mature cells in large numbers, which remains a problem for many clinical applications. Viral vectors are the most common delivery method.

-Not 100% efficient, so even the cells that take in CRISPR-Cas9 may not have genome editing activity.

- Not 100% accurate, and "off-target" edits, while rare, may have severe consequences, particularly in clinical applications.

What are the bioethical issues with using Cas-9

Safety & Unknown Consequences
•Off-target effects (edits in the wrong place).
•Accidental Mosaicism.
•Unknown health consequences.

Informed Consent
•Affected Offspring can't consent.
•With potential unknown consequences how informed can consent be?

Justice & Equity
•Will the technology only be available to those who can afford it.
•Potential for new class system.

Embryos in Research
•Destruction of embryos raises moral and religious objections as destruction of potential life.

Vanity Editing
•Modifications could be used to enhance desirable traits instead of curing disease.

What is PCR

Polymerase chain reaction
- Used to amplify DNA if there is a small sample
- Denaturation: The DNA is heated up to 90 to 95 Celsius. This breaks the hydrogen bonds between the two strands and separates them leaving two strands of single stranded DNA.
- Annealing: The temperature is brought down to around 50 degrees Celsius. Primers which are short single strands of DNA complimentary to the DNA sequence is added at the 5' prime ends of the strands. Primers help provide a place for the DNA polymerase to bind and create the complimentary strand .
- Elongation: Then during elongation the strands are heated up to 72 degrees Celsius. This is the optimal temperature of the Taq polymerase. Taq polymerase will create a complimentary side of the DNA by reading the DNA sequence and grabbing and bonding complimentary free floating nucleotides. This will create two strands of double sided DNA and this cycle will continue growing exponentially until enough DNA is amplified.

What is the function of primers in PCR?

- DNA primers are added to the mixture to bind to complementary nucleotide sequences at the 5' ends of each single-stranded piece of DNA. This provides Taq polymerase with a binding site to begin building a complementary strand.
- Remember, primers attach to complementary DNA nucleotides. The addition of the primer and the joining to complementary nucleotides then allows the Taq polymerase to begin copying.
- Two primers are needed as the nucleotide sequence at the 5' end of the coding and template strands are different

What is gel electrophoresis?

- Gel electrophoresis is a DNA analysis technique used separate DNA bases of the size of its fragments
1.A Gel sheet is made and loaded into a tray.
2.A buffer solution is poured over the Gel sheet.
3.The DNA samples are loaded into sample wells at the top of the Gel sheet
4.Electrodes are placed at each end of the tray in the buffer solution.
5.The electrodes are connected to a power supply and an electric current is run through the gel/buffer.
6.While the electric current is running the DNA fragments will move towards the positive end of the Gel.
7.The Gel contains tiny pores which the DNA has to weave through to migrate.
8.Over time the fragments separate out based on their size.

What are GMOS?

- GMOs stand for genetically modified organisms
- These are any organisms whose genomes have been altered using genetic engineering technology.
- Two subcategories of GMOs are transgenic and cisgenic

Transgenic
•The added genes are from other members of the different species.
•Can now produce proteins that were not part of the host species Proteome.

Cisgenic
•The added genes are from other members of the same species.
•Will only produce proteins that are currently part of the species Proteome.

Why are GMOs used?

- There are two main reasons why GMOs are used
- Firstly to increase crop productivity
- Means to max out the benefits that the plant can provide such as its nutritional value, ability to grow in different conditions, larger /increased fruit production
- The golden rice case study is a great example of increasing crop productivity
- New genes from non-rice species
- These new genes cause the rice to store beta-carotene (a precursor of vitamin A) in the rice grains rather than in the leaves, thus increasing the nutritional value of vitamin A in rice

- Secondly, to increase resistance to damage
- An example of this is BT
- Transgenic - new genes come from bacteria.
- BT is the abbreviation of the bacteria's name that the gene comes from
- The gene form proteins crystals that kill bugs that attack the plant, but are harmless for humans to consume
- Done to help give plants in places in which food security is low more resistances to pests and increase food security

- Also used to make plants more resistant to pathogens and harsh environmental conditions
- This is done is to prevent crop loss and help increase food security. Food security is how reliable to food supply and source is.

What are the ways of gene delivery?

-To deliver a transgene into a host organism there are 2 key methods:
Bacteria Transfer
- Remember plasmids are meant to be disturbed to other bacterium, so great or sharing and transferring genetic information
-A plasmid with the gene of interest is transferred into the bacterium.
-Agrobacterium tumefaciens is a commonly used bacterium.
-The bacterium are then grown with the host cells.
-The bacterium then transfer the new DNA to the genome of the plant cells.

Particle/ Gene Gun
-Small metal particles are coated with the relevant DNA fragment
-These are then bombarded into the plant cells.
-This transfers the new DNA into the host cells.

What is transformed bacteria? How do we apply this concept?

-Transformed bacteria means when a bacteria has taken up a plasmid
- Bacteria transformation is used for DNA cloning mainly in the biopharmaceutical industry. Insulin is an example. Here is how it works
- A gene (insulin for example) is inserted into a plasmid.
- Insulin is made up of two polypeptide chains, so each chain will be made in its own plasmid. So two plasmid vectors are used
- The plasmids are opened up by using endonucleases to cut them
- The genes are inserted through heat shock electroporation and a DNA ligase
re-establishes the sugar-phosphate backbone of the plasmid creating recombinant plasmids
- The plasmid will have a reporter gene and an antibiotic resistant gene
- The bacteria are placed in a culture medium that has nutrients and antibiotics
- There will be three categories of bacteria produced, transformed with recombinant plasmid, transformed, and non-transformed
- Bacteria that are transformed (accept plasmid), will have a the antibiotic resistant gene and will no die (doesn't matter if the plasmid is has the insulin gene or doesn't, both plasmids have a anti-biotic resistant gene), those that die do no have a plasmid at all.
- Then the bacteria with the transformed recombinant plasmid will turn white because the lacZ gene (reporter gene) is not intact (keep in mind the insulin gene is next to the reporter gene) and the ones that turn blue mean that the reporter gene is intact thus is not the recombinant plasmid.

What are the implications of gene editing?

Social
•Consequences that affect economics, politics, or society.
•Examples:
•Gene Therapy may increase life span and quality of life.
•Not everyone may be able to access gene therapy.

Biological
•Consequences that affect ecosystems, environments, or public health.
•Examples:
•Those receiving gene therapy may have an immune response to the viral vector.
•Could other genes be affected by gene therapy?

Ethical
•Considerations based on moral or religious beliefs.
•Examples:
•Who should decide what disorders are treated with gene therapy?
•Gene therapy may allow people with genetic disorders to live long enough to have kids, that they may be able to pass the disorder on to.

What are the implications with GMOS?

Social
Pros
-Increased crop productivity leads to better food security.
-Tolerance of adverse conditions protects against famine, improving food security.
-Herbicide-tolerant crops reduce labour, farmers don't need to pull weeds by hand, instead spray chemicals that selectively kill weeds only.
-Increased crop yields result in larger profits.
-Improved flavour and texture, give consumers a more appealing product.
-Improved nutritional content leads to a reduction in nutritional deficiencies, creating healthier populations.

Con
-Having to buy new seeds each season may be costly for farmers.
-Complex legal issues around the use of GM products may cause farmers undue stress and anxiety related to regulation.
-There are strict packaging and marketing regulations for GMO producers that may not be complied with if either the producer or consumer are undereducated on these regulations.

Biological
Pro
-GM crops usually have better crop productivity than non-GM crops. This means that more food can be grown using less land, reducing habitat loss due to land clearing.
-Insect-resistant GM plants require fewer pesticides, which is better for the environment.
-GM foods can be made to have improved nutritional content, improving the health of individuals that consume them.

Con
-GM crops may lose their effectiveness if weeds or pests evolve resistance.
-Widespread use of GM crops could result in the loss of genetic diversity within crop populations.
-Cross-pollination between GM crops and wild species or weeds may cause genes to spread and lead to unforeseen consequences.

Ethical
Pro
- Some people believe that using genetic modification is an ethical imperative given the potential for widespread benefits, including nutrition, wealth, and the overall health of humanity, especially in developing nations.

Con
- Some people consider GMOs to be unnatural, or like we are 'playing God'.
-Some people believe that GM foods are unsafe to eat particularly if there is lack of long-term evidence of healthy use.
-Some people believe that genetically modifying animals for human benefit is inhumane.
-Companies can own the rights to GM and may make unfair demands of farmers.
-This ownership issue could result in:
-Cross-pollination of non-GM crops by nearby GM crops could result in the non-GM farmer being sued by the patent-owner.
Farmers can't reuse seeds from some GM crops and must buy new expensive seed supplies each year from biotechnology companies

What are factors that affect the movement of DNA in gel electrophoresis?

1.The size of the DNA molecule.
2.The charge of the DNA.
3.How long voltage is applied to the gel (the more time the furth the DNA will have the chance to move)
4.How concentrated the Agarose gel is (the denser the gel the slow the DNA will move)