Med 1002 Flashcards
1
Q
What is the fundamental unit of life?
A
A cell
2
Q
What are the characteristics of cells and life?
A
Being able to:
- Divide and Grow
- Convert Energy
- Respond to stimuli
- Evolve
3
Q
What are the 4 main macromolecules?
A
Proteins, lipids, carbs and nucleic acids
4
Q
What are the main constituents of the chemical part of a cell?
A
The 4 macromolecules along with ions and other small molcules
5
Q
What are the subunits of the 4 macromolecules?
A
Carbs - Monosaccharides/residues
Proteins - Amino Acids
Lipids - Fatty Acids
Nucleic Acids - Nucleotides
6
Q
What is ATP and why is it so important?
A
Adenosine Triphosphate contains 3 phosphate groups and has a high energy bond between two phosphate groups which when broken releases energy to do cellular work with
7
Q
What is the reaction when ATP is broken down?
A
Hydrolysis
8
Q
What is the reaction when ADP is turned into ATP
A
Phosphorylation
9
Q
Describe what viruses are
A
Viruses are not living cells however they contain genetic material
10
Q
What is the head of the virus comprised of?
A
An outer protein coating called a capsid which surrounds the nucleic acids
11
Q
What are the monomers of carbs or polysaccharides?
A
Monosaccharides
12
Q
What is the general formula of the monomers of carbs?
A
C(n)H(2n)O(n)
13
Q
What are the ways we can classify/name carbohydrates?
A
- Different functional groups can be attached to monosaccharides
- We need to know that there will be one carbonyl group and the rest of the carbon atoms will be attached to hydroxyl groups
- MS’s that contain aldehydes are called aldoses
- MS’s that contain ketones are called ketoses
- We can also classify based on number of carbons (trioses, tetroses)
14
Q
What do carbs look like in 3-D?
A
Monosaccharides form a ring which is almost like a puck, the ring is kind of bent
15
Q
What is chiralty?
A
When a carbon atom within a molecule has 4 distinct, different chemical groups attached to it.
This results in two molecules that can be formed, which are referred to as the D (right) or L (left) version.
16
Q
How do you assign D or L in chiral molecules?
A
Find the chiral carbon atom which is the furthest away from the carbonyl atom and see which side the hydroxyl group is. If right, then D and vice versa
17
Q
How do we assign numbers to atoms in an aldose or ketose?
A
The carbon atom attached to the carbonyl atom is numbered 1
18
Q
What do pyran and furan refer to?
A
In carbs, if an oligosaccharide forms a 6 member (not 6 carbon) ring, it will contain pyran in it’s name. If it has 5 members, it has 5.
E.g: Glucopyranose
19
Q
What are the 4 important carbohydrate derivatives we need to know?
A
Sugar alcohols
- No carbonyl groups, just hydroxyl groups
Deoxy sugars
- One or more carbon atoms may not have hydroxyl groups, just hydrogen
Amino sugars
- Sugar with amino group attached
Sugar phosphates
- Sugar with phosphate group attached
20
Q
What are oligosaccharides?
A
Carbs made of 2-10 monosaccharides (also called residues)
21
Q
How are oligosaccharides and polysaccharides made?
A
Two or more monosaccharides come together in a condensation reaction, forming an oligosaccharide. Water is expelled during this part. The bond formed is called a glycosidic bond. Energy is needed to form this bond.
Note: We can have different carbon in the monosaccharide bonding together.
22
Q
What kind of carbohydrate are lactose and sucrose?
A
Disaccharides
23
Q
Describe some properties of glycogen
A
- Glycogen is a polysaccharide and a storage molecule
- It contains thousands of glucose monomers
- It has branched chains
- It is a homopolysaccharide
24
Q
State the differences between homo and heteropolysaccharides
A
Homopolysaccharides are made of one type of monomer while heteropolysaccharides are made of multiple kinds of monomers.
25
What is formed when proteins interact with carbs?
If carbs bind to proteins, they can form glycoproteins.
- If the carbs attach to the oxygen of a protein, it's O-linked and similarly it's N-linked if it's nitrogen
- They can also form a subclass of glycoproteins called proteoglycans
26
What is formed when lipids bind to carbs?
Glycolipids
27
What kind of carbs interact with other macromolecules?
Oligosaccharides and polysaccharides
28
For what functions do carbs bind to other macromolecules? (CPRS)
- Cell Adhesion
- Protection
- Recognition
- Structure
29
What are the general characteristics of a lipid?
Lipids are not very soluble in water hence they are hydrophobic
30
What is the subunit of a lipid?
Fatty acids (except for steroids)
31
What are the two general types of lipids?
Complex lipids (contain fatty acids) and steroids (don't contain fatty acids)
32
What are the types of complex lipids and what are their general properties?
Storage lipids (neutral), also called triglycerides
Membrane lipids (polar)
- Phospholipids
33
What are the functions of lipids (aight)?
Store of energy
Insulation from environment (low thermal conductivity)
Hydrophobic
Contribute to membrane structure
Co-factors of enzymes
Signalling molecules
Pigments
Antioxidants
34
Why are lipids good stores of energy?
Specifically triglycerides, they have a lot of C-C and C-H as they are reduced compounds so lot's of available energy. They can pack together closely together.
35
Explain the components of a triglyceride molecule
Comprised of polar glycerol molecule and 3 fatty acids, not necessarily the same ones
36
What is the structure of a fatty acid?
A carboxyl group attached to a long hydrocarbon chain. The carboxyl group is polar while the hydrocarbon chain is non polar. Hence we say there's a polar head and non-polar tail.
37
What do most fatty acids in humans share in common?
They all contain an even number of carbon atoms
38
What are the two types of fatty acids based on bonds?
Saturated (no double covalent bonds)
Unsaturated (double covalent bonds)
39
Describe the different structural properties of saturated vs unsaturated fatty acids
Saturated FA's are 'straighter' while unsaturated FA's are 'bent', especially cis FA's
40
Describe the nomenclature of fatty acids (e.g: 16:1 [delta] 9)
The first number refers to the number of carbons in the chain. The number after the colon is the number of double bonds. The number after the delta symbol is the carbon which the double bond is on (the number one carbon is on the carboxyl group)
41
What is the difference between essential vs non-essential fatty acids?
Essential fatty acids such as omega 3 fatty acids cannot be produced by our body. Meanwhile, non-essential fatty acids can be produced from the food we eat
42
How does the nomenclature vary for omega 3 fatty acids?
The number one carbon becomes opposite, it's the furthest one from the carboxyl group and this is called the omega carbon
43
Describe the structure of a phospholipid
A glycerol molecule with two fatty acids, with a phosphate group attached to alcohol group on third arm. We have polar head, which is part of the phosphate group, and a non polar group which is the fatty acid group
44
What is a property of a phospholipid due to it’s structure and how does this explain a phospholipid’s interaction with other phospholipids?
- This means the molecule as a whole is amphiphilic or amphipathic (Meaning it has both polar and non polar or hydrophilic and hydrophobic components)
- Phospholipids naturally form bilayers
- In a membrane or micelle, the hydrophilic head containing the phosphate group is on the external part while the two hydrophobic tails are on the interior of the membrane
45
What are lipids which do not contain fatty acids called and what is their structure.
- Lipids which don’t contain fatty acids are called steroids
- They are regulatory molecules
- They are made of interconnected rings (called the steroid nucleus)
- They are nonpolar thus hydrophobic
46
What are some examples of steroids?
Some examples are vitamins, hormones (testosterone, estrogen) and cholesterol
47
What is cholesterol and what are the functions of cholestrol?
- A steroid
- It causes lipids to pack closer together, enhancing the order and rigidity of the membrane or structure
- Thus it also decreases membrane permability
- It is used in production or synthesis of bile acids
- Steroid hormone synthesis
48
What is the subunit of a protein?
Proteins are made of polymers of amino acids
49
What is the structure of an amino acid?
- An amino acid has an alpha carbon which has 4 groups attached to it
- A carboxyl group which can lose a hydrogen (acid part) (COOH connected to alpha carbon)
- An amino group which can gain a hydrogen (base part)
- A hydrogen atom
- And a side chain (R chain) which is what differentiates all the AA’s from each other
50
What are the type of amino acids that can exist (r groups) (and recite the names of the acids we’re supposed to know)?
- Polar (AG)
- Acidic
- Aspartic Acid
- Glutamic Acid
- Basic (HAL)
- Histidine
- Arginine
- Lysine
- Nonpolar
51
What is a property of amino acids that they share with some monosaccharides?
- A lot of amino acids are chiral
- NOTE NOT ALL OF THEM ARE (GLYCINE DOESN’T HAVE 4 DISTINCT GROUPS)
52
Where are you most likely to find L and D amino acids?
- L amino acids are the ones which usually form our proteins
- D amino acids can be found in some bacterial cell walls and can be the targets of antibiotics
53
How are D and L assigned to amino acids?
Based on relationship to D - and L- glyceraldehyde
54
Why is chiralty important when it comes to amino acids?
- Proteins like enzymes in the body speed up the reactions which occur in the body
- However, enzymes only work for specific reactions because they work for specific reactants
- The reactants have a specific shape that the enzyme can bind to and so the enzyme cannot bind to any other shape after
- Hence, only one enantiomer can bind with the enzyme
55
What are the 3 amino acids which are unique when it comes to their side groups and how does this affect their structure?
- Glycine
- Glycine isn’t chiral because there’s two groups attached to the alpha carbon which are the same
- It’s very flexible
- Proline
- It has a cyclic side chain which binds to the amino group
- Not very flexible
- Cysteine
- It’s side chain contains sulfur and it can form S-S covalent bonds with other amino acids, might be known as disulphide bridging
56
What is the difference between ampipathic and amphoteric?
- Ampipathic means a molcule or substance has a polar and non polar component
- Amphoteric means a substance can act as an acid or base
57
What is special about amino acids when they’re in varying pH levels?
- Amino acids have a different structure at different pH levels
- In acidic conditions, the amino acid becomes a cation, with a positive NH3+ group and the hydrogen being attached at the carboxyl group
- In basic conditions, the amino acid become an anion, with a NH2 and the hydrogen is lost from the carboxyl group
58
What is an isoelectric point?
- The pH at which more than 99.9% of an amino acid is a zwitterion
- This means there’s no overall charge
- It’s different for each amino acid
- NOTE: The isoelectric point is when an amino acid is LEAST SOLUBLE
59
Describe the concentrations of amino acids in cation, zwitterion and anion forms as pH increases
- As pH increases, cation concentration decreases
- Zwitterion concentration increases, peaking at the isoelectric point before decreases
- Anion form doesn’t increase for a while, then is slowly increases
60
What happens to the solubility of an amino acid at pI?
- Zwitterions have no net charge, cations have +1, anions have -1 charge
- At the isoelectric point, the solubility of an amino acid is the least
- This is because the positive end of a zwitterion attracts the negative end of another zwitterion
- Water therefore cannot overcome these interactions so the AA doesn’t dissolve as easy
61
In amino acids, what kind of side chains form H-bonds and where do these bonds occur
- Polar side chains will form hydrogen bonds
- Hydrogen will h-bond with NOF and vice versa
62
What are hydrophobic interactions?
- When a hydrophobic molecule such as a nonpolar amino acid comes into contact with water, the molecules of these hydrophobic molecules tend to stick together and cluter together
- This reduces the surface area or contact area with the water
- So in the whole protein, the nonpolar amino acids tend to be in the centre
63
What are peptide bonds?
- Amino acids bind to form chains called peptides
- Peptide bonds are covalent bonds between carboxyl group and amino group
- The C-N (peptide bond) is rigid and there's no rotation about this bond
- It's shorter than expected and most peptide bonds have a trans configuration
- There are 2 rotatable backbone bonds per residue
64
What reaction forms peptide bonds?
Condensation reactions
65
Draw the sturcture of Gly, Ala, Phe, Tyr (on paper)
Check by search
66
List all the sturctural levels of proteins
Primary
Secondary
Tertiary
Quarternary
67
Explain what the primary structure of a protein is responsible for
The primary structure of a protein is comprised of the specific order of AA's in that protein. They are connected by peptide bonds
68
Describe secondary structure of proteins
When AA close to each other fold to form repeating patterns such as alpha helices and beta pleated sheets
69
Detail tertiary structure of proteins
- Folding and bonding of AA that are ‘further’ apart to form the 3-D structure
- Cell’s water environment drives protein shape with folding partly driven by hydrophobic forces
- Hydrophilic R-groups on the outside and the hydrophobic groups on the inside
- This increases the proteins stability
- Sometimes disulfide bonds
70
What is the quaternary structure of proteins
- Proteins that contain more than one peptide chain (subunit)
- Arrangement of these subunits determines the quaternary structure
71
What are transmembrane proteins and what are some properties of them which suit their function?
They're proteins which interact with the lipid bilayer to maybe let stuff in and out. These membranes are formed from alpha helices which allows them to stick into the lipid bilayer. These proteins also have the hydrophobic R-groups on the outside which allows them to interact with membrane
72
What are the important properties of a protein determined by?
- Primary structure or sequence of amino acids
73
What is the difference between a simple and conjugated protein?
A simple protein only contains amino acids and conjugated proteins contain other chemical proteins
74
What is a prosthetic group regarding proteins?
The non-amino acid part of conjugated proteins
75
What are the types of conjugate proteins you can have?
Lipoproteins: Lipid + Protein
Metalloproteins: Specific metal + Protein
Glycoprotein: Carb + Protein
76
What do we mean when we say proteins belong to families?
Certain proteins have similar sequences so they cluster together on a tree where clusters represent proteins with similar sequence and function
77
What is the natural state of a protein called?
When a protein isn't denatured, it's called a native protein
78
What happens when a protein's conformation is altered?
It becomes denatured and the protein is no longer a native protein. Protein can no longer perform it's function. Note: A protein's primary structure isn't altered but the other structures are.
79
What are the ways a protein can be denatured, make sure to describe?
Heat: Breaks H-bonds so denatures protein
Chemicals: Can break bonds via reducing agents
Acids, bases and salts: Affect pH and affect salt bridges + h-bonds
Heavy metals: Interact with sulfhydryl groups which denatures them
80
What are all the types of proteins and what are their functions + example (PREMSST)
- Transport proteins
- Carry things around
- Hemoglobin
- Storage proteins
- Store something that cells needed
- Ferritin (stores iron), ovalbumin
- Also includes nutrient proteins
- Glutenin
- Protective proteins
- Defend against injury and infection
- Antibodies and fibrogen
- Movement or motile proteins
- Actin, myosin, tubulin
- Help move things around
- Regulatory proteins
- Control cellular and physiological activity, signaling and receptors
- Hormones, cytokines
- Structural proteins
- Support cell and organ shape
- Collagen
- Elastin
- Keratin
- Enzymes
- Specialized proteins which speed up metabolic processes by increasing rate of reaction
- Other proteins
- Some have exotic functions like elastic proteins or anti freeze proteins
81
Can denaturation of a protein be reversed?
Not always
82
What are the kinds of shapes proteins can take?
- The shape of proteins influences their function (as discussed earlier) and suits their roles in cells
- E.g: Some proteins are fibrous (straight) and structural like keratin
- Meanwhile some proteins are globular (spherical)
- Enzymes are spherical or globular
83
Explain how ligands (what are these) interact with proteins and provide an example
- Proteins will bind to molecules in specific ways
- Usually a ligand (a signal) (or a smaller molecule than the protein) will come in and bind with the protein in a specific way
- Remember protein has specific shape so ligand must also have specific shape
- The ligand can be an ion, small molcules (ATP) or macromolecules
- Ligands bind with proteins via NON COVALENT BONDS
- For example:
- Anti-bodies or immunoglobulins
- There’s many different types of immunoglobulins
- They bind many different types of antigens
- The antigen is a ligand
- Immunoglobulin is protein
84
What are enzymes and how do they work?
- Enzymes are globular or spherical proteins
- Enzymes bind to specific ligands *also called substrates , altering it’s configuration/shape so that it is more easily changed into the product, while the enzyme itself is unchanged
- Enzyme doesn’t take part in a reaction, so it’s not altered or used up
- Substrate binding involves noncovalent interactions happening in the enzyme’s active site
- Even very simple reactions need enzymes to speed up the process
- Enzymes do not change the equilibrium of a reaction, they just speed up the reaction rate
85
What are metabolic pathways?
conversion of one compound (substrate), into another compound (product) which becomes the substrate of another reaction which keeps on going
86
How is enzyme activity regulated?
- Enzymes need to be controlled (regulated) to ensure the correct reactions are happening in a cell
- This involves involved binding an enzyme, thus controlling it
- Some enzymes are controlled by phosphorylation:
- Adding or removing ATP groups, changes the size and shape of an enzyme, thus affecting it’s binding site, which affects the behavior's of the enzyme as it cannot bind to it’s specific ligand anymore
87
How do we name enzymes?
- The name of an enzyme usually follows this specific structure:
- Typically the first part of the name reflects the name of the substrate and then clarifies the reaction catalysed. ends in -ase
- E.g: Lactate dehydrogenase
- Lactate substrate
- Dehyrogen means remove hydrogen from lactate as the reaction
- Exceptions: Trypsin, pepsin, thrombin
- These exceptions are trivial names
88
What are the enzymes we need to know as of 23/03/2023?
- Ligase
- Catalyze formation of bonds
- Polymerase
- Catalyze polymerization reactions
- Kinase
- Addition of phosphate groups to molecules
- Phosphatase
- Hydrolytic removal of phosphate group
- Protease
- Hydrolyze peptide bonds in proteins
89
What are the enzymes we need to know as of 23/03/2023?
- Ligase
- Catalyze formation of bonds
- Polymerase
- Catalyze polymerization reactions
- Kinase
- Addition of phosphate groups to molecules
- Phosphatase
- Hydrolytic removal of phosphate group
- Protease
- Hydrolyze peptide bonds in proteins
- Oxidoreductase
- Redox reactions
90
What is heredity?
- The transfer of characteristics from one generation to the next
- The info to make a new organism is passed on in in the reproductive cells (sperm and eggs)
- The genetic instructions are contained in genes
- Genes are part of our genetic material
- DNA found in nucleus as part of the chromosomes
91
What's in the nucleus?
- Basic (positively charged) compounds
- Polymers of amino acids (proteins)
- Acid (negatively charged) compounds
- Polymers of nucleotides (nucleic acids)
92
Describe the two types of nucleic acids
- Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
- Responsible for information storage and retrieval
- They determine the amino acid sequence for proteins
- Can be structural (rRNAs)
- Functional (tRNA, snRNA…)
93
What are some important nucleotides/derivatives?
- Nucleotides aren’t only for information storage - making RNAs & proteins
- Metabolic - ATP is the cellular energy ‘currency’
- Cofactors in biochemical reactions - NADH/NAD+
- Cell signaling - cAMP
94
What is the general structure of a nucleotide?
Comprised of nitrogenous base + pentose monosaccharide + phosphate
95
Why are nucleic acids considered negative compounds?
The phosphate groups have hydrogen atoms disassociated so molecule becomes negative and this is attached to the rest of the molecule
96
What are the two kinds of pentose monosaccharides used in nucleic acids?
Deoxyribose and ribose (for DNA & RNA respectively)
97
When ribose is free in solution, what is special about it?
- When free in solution, ribose exists in equilibrium between two forms:
- Straight chain aldehyde (aldose)
- Furan ring form
98
In what form does a pentose monosaccharide exist in a nucleotide?
Exists as a ring in a nucleotide, (1’ and 2’ etc are carbons in monosaccharide)
99
What are the two kinds of nitrogenous bases found in DNA and RNA + give examples:
Pyramidine: Hexagon benzene ring with nitrogen at one point (T, C and U)
Purine: Hexagon benzene ring as above but with pentagon as well (A and G)
100
In what pairs do nitrogenous bases come in?
A and T/U
C and G
101
What are some of the properties of the nitrogenous bases involved in nucleotides?
- Bases are hydrophobic are relatively insoluble in water at cellular pH.
- They tend to exist as tautomers
- In tautomerism, the placement of the hydrogen and the double bonds varies
- It occurs when two isomers can physically interchange with each other in rapid dynamic equilibrium
- We usually only consider the form at pH of 7.0
- One tautomer is usually favoured, so in the equilibrium equation, we indicate the favoured form by pointing a long arrow towards it
- In bases, chemical properties of the base affect structure and hence the function of nucleic acids
102
What is the significance of the double bonds in pyrimidine and purines?
- Results in bases being highly conjugated
- The electron aren’t localized between two atoms (shared between two atoms) but spread over several
- In other words, the electrons delocalised
- Here, because the double bonds aren’t actually ‘double’ because the electrons are delocalised, most bonds have a partial bond character
- Resonance causes the molecule to be more stabilised
- Double bonds in bases make pyrimidine planar and purines nearly planar
- Due to resonance, all bases absorb UV light
- Strong absorption at 260nm wavelength
103
What is a nucleoside?
When a deoxy/ribose (pentose monosaccharide) attaches to a base, it’s called a nucleoside, note phosphate ain't attached here
104
What reaction occurs when a nucleotide is being formed?
Condensation reaction
105
How can we modify bases?
- We can modify bases
- Methylation and demethylation (adding and removing methyl groups)
- Cells can also have minor bases
- Cells also have nucleotides where the phosphate group is not on the 5’C and this phosphate can be involved in forming a ring like structure
106
What are the bonds involved in nucleotides?
Phosphodiester bonds
107
How do we differentiate between the carbon atoms in nucleotides?
The carbons in the pentose monosaccharide are labelled from 1' to 5' and the bases are labelled from 1 etc
108
On which carbons on the pentose are certain structures attached?
On the 5' carbon it's phosphate group, 3' is attached to next nucleotide (another phosphate group) and 1' has base
109
Describe the backbone of DNA
- Phosphate groups are ionised, so they’re negative at cellular pH
- Covalent backbone of DNA (sugars + phosphate groups) is hydrophilic
- Bases are hydrophobic (face inwards)
110
How do we write DNA or RNA sequences?
- Start from 5’ end to 3’ end
- Each base is symbolised by a letter
- If a sequence has less than 50 nucleotides, it’s called an oligonucleotide
111
Outline the hierarchal structure of DNA
- Primary structure
- Sequence of nucleotides from 5’ to 3’ (AGTTTACTCCT)
- Secondary structure
- Two strands of DNA run antiparallel
112
What is the chemical evidence that pointed to DNA being a double strand helix shape?
- NA is made up of a hydrophilic backbone and hydrophobic base (bases face inwards)
- All DNA contains same amount of phosphate groups and pentose sugar but different amounts of bases
- Same amount of sugar molecules and phosphate groups
- Sum of purines (hex and pent) is the same as sum of pyrimidines
- Sum of Adenine (A) is the same as sum of thymine (T)
- Sum of Guanine (G) is the same as sum of cytosine (C)
- Exact base composition varies between species which is why all of us are different
- Base composition of DNA from different cells in a specimen is identical
- Base composition does not change with age, nutritional status and environment
113
What was some of the physical evidence for DNA?
- Using x-rays, we can see a distinct shape
- There’s a regular structure:
- Helix with complete turn every 3.4 nm with a diameter of about 2nm
- We know it’s a helix because of earlier evidence and also because of density measurements which hinted that helix must have two chain
114
How do the strands within DNA interact?
- DNA has two strands which form a helix
- These two strands interact via hydrogen bonding
- Hydrogen bonds are strongest when 3 atoms are in a straight line
- The other strand is the complement of the other
- The bases in DNA form complementary base pairs
- A and T
- C and G
- When A and T hydrogen bond, there are two hydrogen bonds
- When C and G bond, there are 3 hydrogen bonds
115
Are other base pairings within DNA possible such as A and G?
- ther pairing are technically possible
- but there are less hydrogen bonds available
- you cannot bind two purines or two pyramidines together
- because to have that 2nm diameter the two molecules would have to overlap and create a kind of stepladder
- this is energetically unfavourable
116
What is stacking within DNA?
- Because purines are nearly planar and pyramaides are planar, in water these hydrophobic bases cluster together essentially stacking due to IMFs which stabilises the 3-d structure
- stacking minimises contact with water and it increases DNA stability
117
What are some of the physical characteristics of DNA helix
- 10 bases make a complete turn in the Watson- Crick Model (double helix)
- The helix has a major and minor groove
- It also can be left or right handed
118
What are three forms of DNA and where are they found?
- A, mostly found in solutions which don’t have much water so not our cells
- B, which is most stable under physiological conditions
- Z, short tracts of DNA can take on this shape in cells
119
Is DNA flexible?
- DNA is actually flexible and has several configurations
- There is rotation around the sugar and base bond
120
Describe how DNA can be denatured
- DNA can denature or anneal REVERSIBLY
- When the H bonds and hydrophobic interactions are disrupted
- When this happens, two strands are formed (note no covalent bonds are broken)
- Is reversible especially if more than 10 bases are still held together, if this is the case then spontaneously the strands will reanneal
121
What is hybridization?
- Different DNA samples (human and mice) can anneal and then join together
- You can do this with RNA too
- This process is called hybridization
122
What are some properties of DNA (not including Tm)?
- DNA at room temp and pH 7.0 is actually very viscous
- At extreme pH or elevated temp, the DNA will denature which leads to decreased viscosity
- We can figure out the percentage of how much stuff has denatured by studying the change in viscosity
- Single stranded DNA absorbs light better than double stranded DNA
- Looking at a light absorption graph, a single strand will absorb more light so as DNA denatures, absorbance increases
- This is known as hyperchromic shift
123
Explain what is Tm and what are some factors which affect it? There are 5 factors
- DNA as something known as T(m), which is the temperature when DNA has reached half total maximum denaturation
- We measure this by looking at viscosity change and hyperchromic effect (by measuring UV light absorbed)
- Differences in melting temperature can be accounted for a few different reasons
- DNA length
- Base composition
- As GC pair has 3 hydrogen bonds, more energy needed to break them apart so melting temp increases
- So as GC content increases, melting temp increases
- Salt concentration
- The higher the concentration of salt, the more stable the DNA
- Ions interact with DNA backbone (phosphate and sugars), particularly with phosphate group
- This interaction reduces the repulsion force between two phosphate groups, increasing stability
- pH
- Ionisation of molecules changes with pH
- As pH decreases, protonisation happens and the reverse is true
- Changes in protonisation can lead to loss of H bonds between complementary strands
- Note: If you decrease the pH to too much acidity, glycosidic bonds can be broken, breaking the actual covalent bonds
- So to denature, milk alkaline solutions preferred
- Other solutes in solution
- Some solutes can form H-bonds, essentially ripping the molecule apart if in high concentration
- Urea and formamide are examples
- This decreases melting temperature and these solutes also prevent re-annealing
124
What is a hairpin in DNA?
When a DNA strand forms intrastrand bonds rather than H-bonds with another strand, a bend or hairpin is formed
125
What is a cruciform?
When two strands who H-bond to each other also have intrastrand bonding, they get cruciform
126
Compare and contrast nuclear vs mitochondrial DNA
- Nuclear
- Long double helices of DNA wound up around proteins (containing lots of basic AA) to allow packaging into nucleus
- Mitochondria
- Also double stranded but it’s circular rather than helical
127
Why is DNA replication known as semiconservative?
- We need to break the DNA down into two strands so we can use one as a template
- Then we need to make a new DNA strand which is complementary to the original one
- Each new cell contains one strand from parent cell and one new strand which is made
- That means that DNA replication is semiconservative
128
How can we break apart the two DNA strands for replication?
- The strands are held together by H bonds and hydrophobic interactions
- So to overcome these, we need energy (ATP)
- High temp is needed to denature or we use an enzyme
- The enzyme used is DNA helicase to unwind the double helix
129
List the things needed to form a DNA polymer and why do we need this?
e need a few things to form a new DNA polymer
- We know that phosphodiester bonds on 3’ and 5’ carbon are needed but how do they form in the case of DNA replication
- Elongation of the DNA chain occurs by adding stuff to the 3’ OH (hydroxyl) group
- An enzyme, DNA polymerase, is used to speed up this condensation reaction
- The substrate the enzyme acts on is deoxyribonucleoside triphosphate (dNTP)
- We need a template or one parent strand
- We need a short, double stranded nucleic acid which is supplied by a RNA primer which anneals or joins to the DNA polymer
- We need Mg (2+) as it’s a cofactor for this reaction
- Well first the dNTP has 3 phosphate groups but for DNA we need one phosphate group to form a nucleotide
- So the primer strand, which contains RNA and has an OH group
- The dNTP has a base on it which is complementary to the base on the parent strand
- So it comes close to the primer strand, the electrons on the OH of the primer ‘attack’ the triphosphate of the incoming dNTP, chopping off the 2 phosphate groups and leaving a singular phosphate group and forming a phosphodiester bond
- This process repeats
- The polymerase is the builder
- We need 2 Mg(2+) ions because the phosphate group is negative, we can imagine that the magnesium brings together the reactants which need to be joined together (3’ OH and dNTP)
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Compare and contrast the three types of DNA polymerase
- Alpha
- Has 5’ to 3’ polymerase activity
- Starts DNA synthesis and DNA repair, has primase activity
- Delta
- Has 5’ to 3’ polymerase activity
- Involved in leading and lagging strand synthesis
- Has 3’ to 5’ exonuclease activity (basically can proofread)
- Epsilon
- Has 5’ to 3’ polymerase activity
- Involved in leading strand synthesis
- Has 3’ to 5’ exonuclease activity, basically can proofread
- Epsilon and delta can proofread and replace mismatched bases
- They have high fidelity
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Explain the consequences of DNA replication happening from 5' to 3' direction and how these consequences are overcome
- Then primer will be placed by epsilon and and delta
- Now the thing is because synthesis happens from 5’ to 3’, one of the strands will be formed opposite to the direction the strand unravels
- This means one strand will have one continuous strand being built (leading strand) which is where epsilon polymerase is used
- The other one keeps having to make new fragments because the strand is ending
- These fragments are called Okazaki fragments
- Because unwinding keeps happening and fragments can’t continue, we continuously need to provide RNA primer
- Once 2 new strands are formed, we need to remove the RNA, connect the Okazaki fragments
- To remove, an enzyme known as FEN1 (flap structure-specific endonuclease 1) or RNAseH removes the single stranded RNA primer
- Then DNA ligase joins the adjacent Okazaki fragments together by forming phosphodiester bonds
- We need ATP for this reaction
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What proteins are involved in stabilising the DNA?
- Sliding clamp protein
- Also called PCNA
- Allows DNA polymerases to bind well so allows for more efficient strand production
- Clamp loading proteins
- Also called RFC
- Aids in attaching sliding clamp proteins
- Single stranded DNA-binding
- Also called Replication Protein A (RPA)
- Stabilises single stranded DNA so it doesn’t join with it’s original strand while replication is happening
- It also protects SSDNA (single stranded DNA) from degradation and winding up on itself
- DNA gyrase
- Type of topoisomerase
- Relieves torsional stress and helps to prevent DNA from over-twisting
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What is DNA organised into in the nucleus?
Chromatins and chromosomes
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What are the roles of chromosomes?
- Faithful transmission (passing on traits accurately)
- Make sure there is appropriate expression of genes
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What are sister chromatids?
- Each chromosome in a cell is replicated and two halves appear, joined at a point called the centromere
- The two halves of idential chromosomes are known as sister chromatids BEFORE SEPARATION
136
Describe the structure of a chromosome
- On the ends of it we have telomeres
- These are the ends of the chromatin which contain G (guanine) rich repeated DNA sequences
- A chromosome has two arms, one longer and one shorter
- Shorter arm is known as p arm
- Longer arm is known as q arm
137
Describe the nomenclature and classifications of chromosomes
- Numbered from 1-22
- Types of chromosomes depend on the location of centromere
- Metacentric
- Centromere is in the centre with equal length p and q arms
- Chr 1, 3, 16, 19, 20
- Submetacentric
- Centromere sligtly offset from centre, leading to slight assymetry between arms
- 2, 4 to 12, 17, 18
- Acrocentric
- Centromere very offset from the centre so very distinct p and q arms
- 13, 14, 15, 21, 22
- Telocentric
- Centromere at the very end of chromosome
- No human chromosome exhibits this
- The smaller the number, the larger the chromosome
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What are some important measurements we take when it comes to chromosomes?
- In chromosomes we can measure the number of chromosomes (n) and associated DNA content (C)
- n is the amount of chromosomes in a full set
- In human cells, we have n=23 and C= 3.5 pg
- pg is picograms
- Most cells in humans contain two full sets of chromosomes, so 2n or 46 (1 set from dad and one from mum) and 7pg of DNA
- However some cells like sperm cells carry 1n or 23
- Sometimes we can have 4n, in G2 and M phase of cell cycle
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What does ploidy mean?
- This means most cells in humans are diploid and some are haploid
- Ploidy is a measure for how many copies of a chromosome set we have
- Haploid is 1 copy
- Diploid is 2 copy
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What are karyograms and karyotypes?
- When you take all the chromosomes from a person’s cell you can line them up and see all of them
- This is called their karyogram
- The size, shape and number of chromosomes is called a karyotype and varies from species to species
141
What are homologous pairs of chromosomes? (what does it mean?)
homologous pairs are similar in structure and size and carry genetic info for the same set of hereditary characteristics. One chromosome from mum and another from dad.
142
Explain what chromosome banding achieves
- It’s when we stain a chromosome with a dye that binds to DNA, areas of light and dark appear
- Some dyes want to dye more with AT and others to GC hence the pattern
- Thus it allows us to see the relative composition of AT and GC bonding within DNA
143
What is chromatin and what are the two types?
- It’s the combo of proteins and DNA
- There’s two types
- Euchochromatin: less densely packed and the DNA is more accessible
- Heterochromatin: Highly condensed state and DNA isn’t as accessible
144
What are the kinds of proteins that are involved in chromatin structure?
- The most aubdant proteins in chromatin are histones
- 5 major types of histones: H1, H2A, H2B, H3, H4
- Comprised mainly of arginine and lysine residues (positively charged AA)
- Histones carry net positive charge which helps to attract negative phosphate groups
145
Explain how DNA is packed into chromatin and subsequently chromsomes (hierarchal approach)
- The double stranded DNA has two turns of DNA around a histone octamer (8 histone proteins joined together)
- The DNA keeps going and keeps wrapping around more histone octamers
- Makes this almost bead necklace
- When DNA turns itself around on the octamers, it forms nucleosomes
- Then a nucleosome with a H1 protein (which helps with packaging) becomes a chromatosome
- Chromatin condenses in metaphase
- To break a nucleosome, the bead like structure, you can use a nuclease to break the phosphodiester bonds present
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What is a nucleosome?
When DNA turns itself around on histone octamers, it forms nucleosomes
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How can we break apart nucleosomes in chromatin?
Use nuclease to break phosphodiester bonds
