Biochemistry Flashcards
1
Q
What is mRNA? tRNA? rRNA?
A
Carries genetic information from DNA - read in codons
tRNA - molecules that take amino acids and anticodons to the ribosomes, this anticodon complements to the codon.
rRNA - form ribosomes.
2
Q
Crick’s Adapter Hypothesis
A
Adapter is tRNA, two sides. One side is for amino acid specific. A separate part recognises the nucleotide sequence.
3
Q
Degenerate
A
More than one codon codes for an amino acid
4
Q
1 start codon
3 stop codon
A
AUG - methionine (START)
UAA, UAG, UGA (STOP)
5
Q
Translation
3 RNA
A
Process by which mRNA forms a polypeptide chain in the correct order
- mRNA
- tRNA
- rRNA
6
Q
Open reading frames
A
No termination codon for 50 codons
7
Q
Mutations- deletions or insertions, point mutations
A
Deletions or insertions - dangerous, change reading frame
Point mutations - not dangerous, single base substitution
8
Q
Aminoacyl tRNA synthesases
A
Recognise a amino acid, and help bind the anticodon to the codon.
9
Q
What is tRNAfmet
A
Recognises the AUG codon (bacteria)
10
Q
Shine - Dalgarno sequence
A
sequence that guides mRNA to the correct position to start initiation
Only in prokaryotes
8 base pairs upstream of AUG
11
Q
P site
A site
E site
A
A-site : where initial binding of AUG and MRNA occurs
P-site : mRNA binds to AUG
E-site : exit path for tRNA molecule
12
Q
Translocation
A
Ribosome moves single codon downstream
13
Q
Post-translational modification
A
- Removal of signal sequence
- Folding
- Modification of amino acids
- Addition of groups
14
Q
Recombinant DNA
A
Artificial recombinant molecule created from 2 or more sources
15
Q
Reporter proteins
A
Organism that expresses a gene, it can be identified due to the fluorescence.
GFP -
16
Q
GFP
A
barrel structure, B sheet, a helix
Shows green light, when exposed blue light
Hydrogen bonding
17
Q
Viruses
A
Replicate in host cells,
18
Q
cDNA
A
Made from mRNA template, plasmid put into host cell, amplified.
19
Q
Vectors
A
Are DNA molecules used as a vehicle to transfer foreign genetic material
20
Q
cloning vectors - ORI
Selectable markers
A
Plasmid is replicated into the cell
Selectable markers - allows u to tell if the plasmid is present in the host cell (reporter gene gfp)
21
Q
Cloning vectors - multiple cloning site
A
Is used to insert gene of ur interest (has cut sites)
22
Q
Procedure for cloning
A
- Find vector
- Cut DNA (restriction endonuclease)
- Joining DNA (ligase)
- Amplify PCR
- Identify host cells with recombinant DNA
23
Q
What is PCR?
A
PCR is the process of amplifying DNA of interest multiple times .
24
Q
What is required for PCR?
A
Taq polymerase Primers Nucleotides DNA template Buffer at ph = 7
25
What is taq polymerase?
Can survive 95C and is used to separate the DNA double strands and catalyse more DNA at 72C
26
How is PCR used in medicine?
It identifies infectious diseases and diagnoses genetic disorders and cancers
27
What are the biological role of proteins?
```
Catalytic roles - speed up chemical reactions
Structural support - strength
Ligand binding roles -
Storage facility - ferritin for iron
Transport role - Hb movement
Defensive functions - act as antibodies
```
28
The central dogma
DNA -----------------> mRNA ----------------> protein
| Transcription. Translation
29
Configuration? Chirality? Enantiomers?
Spatial arrangement of groups around a carbon atom
x/y/z/a all 4 groups differ therefore they are chiral or asymmetric.
Enantiomers - 2 diff arrangement of groups around the C atom, mirror images, cannot be superimposed
Glycine is the only molecule that is not chiral, due to H atoms.
30
What does this mean; "biological systems are stereo-specific"
They can tell the difference between left and right hands. So shape (topology) is also important
31
If pH
PRONATED FORM
COOH
NH3+
32
If ph > pKa
DEPRONATED FORM
COO-
NH2
33
Zwitterion
At ph =7; acts as a base and acid (amphiprotic)
COO- and NH3+
34
Titrations show the pI, what is the pI?
Is the pH at which the molecule has a zero net charge,
35
Difference between pKa and pH?
pKa values are fixed, a constant that cannot change
Ph can be changed by adding an acid or base
36
Describe polar molecules
Hydrophilic molecules
Interact with water in an energetically favourable way
Reduce delta G and increase entropy
37
Describe non polar molecules
Hydrophobic
Interact with water in an energetically unfavourable way
Increase G and decrease S
Burry themselves in the core of enzymes
38
Give examples for non polar, aromatic, polar uncharged, basic and acidic.
non polar, glycine gly
aromatic, phenylalanine Phe
polar uncharged, Serine Ser
basic Lysine Lys
acidic. Aspartate. Asp
39
Importance of histidine (BASIC)
Only standard amino acid with pKa of 7; therefore it can act as an acid or base
40
Carboxylic acid pKa
| Amino acid pKa
A- COOH pKa - 2. Side chain pKa - 4
A- NH2 pKa - 9. Side chain pKa- 11
41
Describe protein turnover
Protein synthesis and protein degradation
42
How to calculate MW of proteins?
MW of proteins = no of residues x 110
110 (weighted average mass for smaller amino acids)
43
Where is rotation allowed on proteins?
Only on single COVALENT bonds
44
What are NATIVE proteins?
Native proteins are thermodynamically stable; low free energy (G)
45
Primary structure
The linear sequence of amino acids with covalent bonds (includes peptide and disulphide bonds)
46
Secondary structure general
Include short chain interactions in the LOCALISED region of primary structure.
47
Rotation
No rotation is possible in double bonds or bonds with partial double bond character, so peptide is rigid.
Ca - C = N - Ca
No rotation in C-N bond, but allowed in Ca - C (psi) and N - C (phi)
Both psi and phi are +180
48
What reduce the range of conformations (spatial arrangement for groups around a carbon atom) for protein chains?
Rigid - no rotation in double bonds
planar arrangement - co planar (arranged in 2D plane)
Steric hindrance
49
Secondary structure a-helix
🔴 simplest form of secondary structure
🔴 has a tight, compact structure with R groups pointing outwards
🔴 has rotations in all bonds expect the peptide bond (C=N)
🔴 held together by internal H - bonds formed every 4 residues
Stabilising - polar amino acids interact in favourable way
Destabilising - glycine - small molecule and forms different coils to a helices
Proline - 'imino' N atom has no H atom, so can't form H -bonds
50
Secondary structure - B conformation
Is the formation of strands and pleates in an antiparallel or parallel arrangement.
- B strands: the polypeptide forms zig zag shape (not inherently stable)
- B pleates: multiple B strands arranged side by side forming pleats (inherently stable)
- B turns: turns and coils allows protein to reverse direction
Held together by H-bonds
Proline and glycine - allow for tight turns and kinked peptide formation
51
Tertiary structure
Multiple secondary structures coming together to form a 3D structure
A helices, b sheets, turns, pleates.
Defines globular and fibrous proteins.
Stabilised by hydrophobic interactions (internal- less interaction with water)
52
Quaternary structure
```
Is the overall arrangement of multiple separate protein chains coming together to form a complex subunit structure.
Monomer - 1 subunit - ribonuclease
Dimer - ADH
Trimer - collagen
Tetramer - haemoglobin
```
Held together by hydrophobic interactions
53
⬆️ products ⬇️ reactants = G positive, goes backwards, energetically unfavourable Keq 1
Delta G = 0, when Keq = 1
54
When equilbrium is established equation
delta G = -R . T . Log e (Keq)
55
Transition state
Is the intermediate between ️products and substrate
S*
A high energy molecule, but unstable
56
Activation energy
An energy barrier that must be overcome in order for a reaction to occur
57
Denature or enhance!
ph extremes can denature or enhance rxn rate
Temperature (heat) to denature molecule or increase rxn rate
Chemical solvents to denature molecules or enhance reactions
58
What are catalysts
Speed up chemical rxns by lowering the activation energy, are not consumed in the process and can be reused again. Do not effect delta G or Keq
59
What is binding energy
Is the sum of free energy released from weak interactions
60
Lock and key model
| Induced fit model
Lock and key - substrate binds to the enzyme with a perfect fit at the active site
Induced fit - substrate induces a fit at the active site, involves formation of transition state
61
Define Km
Is the maximal rate of rxn (the speed of the rxn)
62
Define Vmax
Is the substrate concentration when V0 = 1/2vmax, describes the affinity of the enzyme to bind to its substrate.
63
PH graphs
Are symmetrically shaped
Bell shaped curve
High or low ph can denature enzymes but breaking weak interactions and hence losing its biological function
64
Temperature graphs
At low temps; enzyme activity is low
At extreme temperatures; weak interactions distabilise, thermal desaturation, loss of structure, function and enzyme activity
65
The wobble case
The third base is different in codon
66
Chemical denaturents
Acetate (organic solvents), detergents and urea denature proteins by interacting with internal hydrophobic interactions. This disrupts hydrophobic interactions that stabilise the protein and hence loss of function.
67
Define specificity
enzyme can tell the difference between its normal substrate and another molecule
68
What is an enzyme inhibitor?
Interfere with the catalytic action of an enzyme and finished the reaction rate
69
Competitive inhibition
Competes with the substrate and binds at the active site, this reduces reaction rate as products aren't formed
Vmax is unaffected
km increases
70
Uncompetitive
Inhibitor does not compete with the substrate and binds at a place other than the active site, which changes the shape of the active site and this dimishes the activity of the enzyme.
Vmax and km reduced
71
Define protein purification
Isolation of a single protein via the physical separation and complete removal of other proteins,
72
What properties need to be exploited to seperate proteins
Size
Charge (pI)
Binding affinity
73
Tech 1 - COLUMN CHROMATOGRAPHY (exploit all 3 properties - charge, binding affinity and size)
2 phases and separation
Stationary phase - solid porous material
Mobile phase which contains buffer solution is what passes through the stationary phase (eluate)
Proteins travel through the coloumn at different rates, how seperation occurs
Large proteins come out quick, small proteins come out slow
74
Tech 2 - ion exchange chromatography
Exploits differences in magnitude of the net charge of proteins at a ph.
Anion (-) moves towards the anode (+)
Cation (+) moves towards the cathode (-)
75
Tech 3 - affinity chromatography
Seperation depends on binding affinity of protein to a ligand.
Colomn has ligand, and protein that binds to that ligand will stay in the coloumn, proteins that don't bind will pass through (eluted). E.g antibody antigen seperation
76
How Do we know when proteins are PURE .
Use seperation techniques with high power
Visualise protein with stains
Ur protein is pure if only one species is appearing
77
SDS page
Separates based on size and shape
- makes all proteins carry -Ve charge and linearise them
- then seperates them (-Ve charges move to anode)
- one protein shown means it's pure
78
Define homogeneity
Is 100% protein purification
79
isoelectric focusing
2D Page
IEF --- Seperation of proteins based on isoelectric points.
2D page --- seperation by IEP first then SDS page
80
Specific activity
Measure of the purity of enzyme preperation
| Units of enzyme activity /mg of protein
81
Turnover number of enzymes
Number of substrate molecules that are turned into products by an enzyme per second "kcat"
82
Laws of thermodynamics
1st - energy is neither created nor destroyed
2nd - all natural process increase the entropy of the universe
83
Why is second law broken?
Living organisms are open systems, they require food/energy to maintain order and structure.
84
ATP act as an energy source for
Chemical work - production of macromolecules
Physical work - muscle contraction
Transport - moving solutes against concentration gradients
85
Define free energy
The amount of energy available to perform work
Delta G - change in free energy
86
# Define entropy
Explain information as energy and NEGATIVE entropy
Delta S - degree of disorder and randomness
Randomised letters have no meaning, but are rich in entropy
Negative entropy - eg. information
87
Equation for delta G
Delta G = delta H - T . Delta S
88
What is standard free energy change?
```
Free energy change under standard conditions
1M
Ph= o
1atm
101.3 kpa
```
89
What is delta G prime?
Standard free energy at a ph other then 0. Most reactions occur at ph=7
90
Coupling
Drive unfavourable reactions by coupling reactions with a common intermediate
