MGD Flashcards
Functions of cytoplasm
Metabolism of carbohydrates, amino acids and nucleotides
Fatty acid synthesis
Functions of Golgi body
Export of proteins
Detoxification reactions
Functions of lysosomes
Cellular digestion
Functions of mitochondria
ATP synthesis
Functions of endoplasmic reticulum
Export of proteins
Membrane synthesis
Lipid and steroid synthesis
Detoxification reactions
Functions of nucleus
DNA synthesis and repair
Functions of nucleolus
RNA processing and ribosome assembly
Functions of plasma membrane
Cell morphology and movement
Transport of ions and small molecules
Functions of ribosomes
Protein synthesis
free in cytoplasm or RER
Key differences between
prokaryotic and eukaryotic cells
Organism (E-PAF) (P-B)
Chromosomes (many vs one circular strand of DNA/RNA)
Ribosomes larger in E
Cytoskeleton in E vs flagella only in P
P- cell wall
E - nucleus, ER, lysosomes, Golgi complex, mitochondria, vacuoles (small/ none in animals)
OCCCRNELGMV
Name organisms with mammalian eukaryotic cells
Plants
Animals
Fungi
Name organisms with prokaryotic cells
Bacteria
Do eukaryotic cells have a cell wall?
No
Do eukaryotic cells have a nucleus?
Yes
Do eukaryotic cells have chromosomes?
Many (23 pairs in humans)
Do eukaryotic cells have an endoplasmic reticulum?
Yes
Do eukaryotic cells have ribosomes?
Yes (large)
Do eukaryotic cells have lysosome?
Yes
Do eukaryotic cells have a Golgi complex?
Yes
Do eukaryotic cells have vacuoles?
Ys (small/ none in animals)
Do eukaryotic cells have mitochondria?
Yes
Do eukaryotic cells have a cytoskeleton ?
Yes
Do prokaryotic cells have a cell wall?
Yes
Do prokaryotic cells have a nucleus?
No
Do prokaryotic cells have chromosomes?
One circular strand of DNA/RNA
Do prokaryotic cells have an endoplasmic reticulum?
No
Do prokaryotic cells have ribosomes?
No
Do prokaryotic cells have lysosomes?
No
Do prokaryotic cells have a Golgi complex?
No
Do prokaryotic cells have vacuoles?
No
Do prokaryotic cells have mitochondria?
No
Do prokaryotic cells have a cytoskeleton?
Flagella only
Describe macromolecular structure
Monomeric units
Joined by covalent bonds
Describe macromolecular interaction
Non-covalent interactions
Types of non-covalent (weak) interactions in macromolecules
Hydrogen bonds
Ionic interactions
Hydrophobic interactions
Van der waals interactions
What are ionic interactions ?
Can be attraction or repulsion of charged ions
What are hydrophobic interactions?
What is a hydrophobic molecule?
Non polar molecules
Unable to interact with water
E.g. Lipids
Storage without water and can pass through bilayer
What are van der waals interactions ?
Any two atoms in close proximity
What are hydrophilic molecules?
Polar molecule
Interact with water
Reduces storage ability (e.g. Glycogen)
Cannot pass through bilayer unassisted
What is pH?
Measurement of conc of H+ ions in a sol
Strong acids and bases completely dissociate in sol
Weak acids and bases barely dissociate
What is pK?
Stronger the tendency of an acid to dissociate
the lower the pKa value
What are buffers?
Weak acid + conjugate base
Resist changes in pH
If pH> pK
Deprotonated form dominates
If pH<pK
Protonated form dominates
If pH =pK
Amount of acid and conjugate base is equal
If deprotonated form dominates
pH>pK
Protonated form dominates
pH<pK
Amounts of acid and conjugate base are equal
pH=pK
Amino acid with benzene rings
Aromatic
Amino acid without benzene rings
Aliphatic
What is a zwitterion?
Neutral molecule
Has both positive and negative charge
What is pI?
Isoelectric point
pH at which the protein has no overall net charge
What is the pI of acidic protein?
Low pH
Many neg charged aa
Low pI
Many pos H+ ions needed to become neutral
What is the pI of basic protein?
High pH
Many pos charged aa
High pI
Few pos H+ ions needed to become neutral
Key features of peptide bond
All atoms of the bond are in the same plane
No rotation about peptide bind due to double bond characteristics
Carbonyl oxygen and amide hydrogen in trans orientation
Define alpha helix
Type of regular protein secondary structure
Right handed helix
3.6 aa residues per turn
Pitch of 0.54 nm
Define amino acid
Building blocks of proteins
Composed of central carbon attached to 4 other chemical groups:
Amino group (-NH2), carboxyl group (-COOH), hydrogen atom and variable group (R)
Define amphipathic
Molecule that has both a polar (hydrophilic) and non-polar (hydrophobic) end
Define beta sheet
Type of regular secondary structure
Polypeptide chains are on extended conformation
Parallel or anti parallel
Define denaturation
Disrupting the normal folded conformation of a protein to produce an unfolded polypeptide chain
Define domain
Region of a protein that folds into a distinct globular unit
Will often have a specific functional role within the protein
E.g. Ligand binding, interaction with other proteins
Define disulphide bond
Covalent bond
formed between two sulphur atoms
of cysteine residues in a protein
Define fibrous protein
Insoluble class of proteins Elongated structure Repeating elements
Define globular protein
Water soluble class of proteins Compact highly folded structure
Define hydrogen bond
Weak electrostatic interaction
between a hydrogen atom bound to an electronegative atom (N,O) and another electronegative atom
What is a protein?
Polymer composed of aa monomers joined by peptide bonds
How many different types of aa are there?
20
In which aa is the carbon not a chiral centre?
Glycine
Variable group of hydrogen
Two types of spatial arrangements of aa
Chiral centre - shows stereoisomerism
D- and L- form
Naturally found spatial arrangement in proteins
L-form
Aromatic R groups
Phenylalanine
Tyrosine
Tryptophan
PTT
Polar, uncharged R groups
Cysteine Asparagine Threonine Serine CATS
Non polar, aliphatic R groups
Glycine, alanine, methionine, proline
Lucine, isoleucine, valine
GAMPLIV
Negatively charged R groups
Glutamate
Aspartate
GA
Positively charged R groups
Histidine
Arginine
Losing
HAL
Define peptide bond
Type of covalent bond
Joins amino acids in proteins
Forms between carboxyl group of one and amino group of 2nd
Peptide bond formation is a condensation reaction (release H2O)
Define primary structure
Linear amino acid sequence of polypeptide chain
Determines the overall structure and function of protein
Define secondary structure
Local spatial arrangement of polypeptide backbone
Define tertiary structure
Overall 3-dimensional arrangement of all atoms in a polypeptide
Involves folding secondary structure
So far apart aa on primary sequence can interact
Larger proteins have distinct domains- serve particular roles
Define quarternary structure
3-dimensional arrangement of multi-subunit proteins
Bonds in primary structure
Covalent (peptide) bonds
Common secondary structures of polypeptides
Alpha helix
Beta sheet
Bonds involved in secondary structure
Hydrogen bonds
Bonds involved in tertiary structure
Hydrogen bonds Van der waals Hydrophobic interactions Covalent disulphide bonds Ionic interactions
Bonds involved in quaternary structure
Hydrogen bonds Van der waals Hydrophobic interactions Covalent disulphide bonds Ionic interactions
What is a homomeric protein?
Protein where quaternary structure consists of identical polypeptide chain subunits
What is a heteromeric protein?
Protein where polypeptide subunits in quaternary structure are different
Role of globular proteins
Enzymes
Regulatory proteins
Role of fibrous proteins
Structure
Support
Protection
What can assist some proteins with folding?
Molecular chaperones
What are molecular chaperones?
Assist some proteins with folding
Define allosterism
The binding of a ligand/ substrate
At one site in a multi subunit protein that influences the subsequent binding of other ligand/substrates to other subunits
Define competitive inhibition
Enzyme inhibition Inhibitor competes with substrate For binding at the active site Increase in the Km for the substrate Vmax remains unchanged
Define Bohr effect
Decrease in oxygen binding affinity of haemoglobin
caused by decrease in pH
Define enzyme
Biological catalyst
Increases rate of reaction
Lowering the activation energy
Define haem
Poryphyrin derivative
Found in haemoglobin and myoglobin
Central iron atom
Site of reversible oxygen binding
Define Km
Substrate concentration that will give
Half the maximal rate (Vmax)
Michaelis constant
Define non-competitive inhibition
Enzyme inhibition
Inhibitor binds at a site other than the active site
Decreased vmax
Km for substrate remains the same
Define sickle cell anaemia
Haemoglobinopathy
Glu-to-Val mutation in B-haemoglobin molecules
resulting in deformed red blood cells
Define thalassaemia
Group of haemoglobinopathies
caused by mutations in globin genes
resulting in an imbalance between the globin proteins
Define Vmax
Maximal velocity of an enzyme catalysed reaction
Role of myoglobin and haemoglobin
Oxygen transporting proteins
Name two oxygen transporting proteins
Myoglobin (Mb)
Haemoglobin (Hb)
What part of haemoglobin do oxygen atoms bind to?
Haem group
How many oxygen molecules can myoglobin carry?
One
Single subunit protein
How many oxygen molecules can Hb bind to?
Four
Tetrameric molecule
Describe oxygen binding curve for Mb
Hyperbolic oxygen binding curve
No cooperativity
Describe the oxygen binding curve for Hb
Sigmoidal oxygen binding kinetics
Cooperativitiy
What does 2,3-BPG stand for?
2,3-bisphosphoglycerate
How does 2,3-BPG affect oxygen binding curve?
Increasing amount of BPG
Decreases affinity of Hb for oxygen
Curve shifts to the right
How does CO2 and H+ affect oxygen binding curve?
Decreases affinity of Hb for oxygen
Curve shifts to the right
How does CO affect oxygen binding curve?
Carbon monoxide binds tightly to Hb
Prevents oxygen binding
What catalyzes nearly all chemical reactions in the body?
Enzymes
Protein catalysts
What does lactase do?
Hydrolysed lactose into glucose and galactose
What does DNA polymerase do?
Polymerises nucleotides to form DNA
Name 6 classes of enzymes
Transferases Hydrolases Oxidoreductases Lyases Isomerases Ligases THOLIL
Role of oxidoreductase enzymes
Oxidation-reduction reactions
Transfer of electrons
Role of transferase enzymes
Transfer C-,N- or P- containing groups
Role of hydrolases
Catalyse cleavage of bonds by the addition of water
Role of lyase enzymes
Addition or removal of groups to form double bonds
Role of isomerases
Transfer of groups within molecules to form isomers
Role of ligase enzymes
Formation of bonds between C and O, S and N, at the expense of ATP
What is a cofactor?
Prosthetic group
Inorganic ion
E.g. Fe2+, Mn2+
What are coenzymes?
Prosthetic group
Organic compounds
Act as temporary carriers of groups in reaction
E.g. NAD, CoA
What is NAD?
Nicotinamide Adenine Dinucletide
Coenzyme
What is CoA?
Coenzyme A
Examples of prosthetic groups
Coenzymes
Cofactors
What are prosthetic groups?
Tightly or covalently linked to enzyme protein
Additional chemical components to catalyse reactions
Properties of enzymes
Highly specific
Increase rate of reaction
Left unchanged after reaction has occurred
Explain specifity of enzymes
Interact with one or only a few substrates
Catalyse one type of reaction
How do enzymes affect equilibrium of a reaction?
They don’t.
How do enzymes work?
Lower activation energy for reaction to occur
Substrate bind to active site on enzyme
Increase local concentration of reactants
Stabilised formation of high energy transition state
What is an active site?
Site of reaction on enzyme
Coded by few aa of the long polypeptide chain
Usually clefts or crevices in protein
Why is active site usually crevice/cleft?
Allow substrate to bind
Exclude water from reaction
What is the lock and key hypothesis?
Only molecules that have a complementary shape to the active site will be able to bind
Specificity of enzyme
What is the induced fit hypothesis?
Binding of substrate results in changes in shape of enzyme
To enhance binding
How is substrate held in active site?
Multiple weak bonds with aa in this part of enzyme
What does the Michaelis Menten equation show?
How the reaction velocity v0 varies with the substrate concentration
What is v0?
Initial reaction velocity
What is the intercept at y-axis of a Lineweaver-Burk plot?
1/vmax
What is the slope of a Lineweaver-Burk plot?
Km/Vmax
What is the intercept at x-axis of a Lineweaver-Burk plot?
-1/Km
What are the axis of a Lineweaver-Burk plot?
X-axis 1/[S]
Y-axis 1/V
How do irreversible enzyme inhibitors work?
Bind covalently to the enzyme molecule to destroy enzyme function
Types of reversible enzyme inhibitors?
Competitive and non-competitive inhibitors
How do competitive enzyme inhibitors work?
Binds at active site
Affects Km not Vmax
Can be overcome by increasing substrate concentration
How do non-competitive inhibitors work?
Bind at site other than the active site
Affects Vmax not Km
Cannot be overcome by increasing the substrate concentration
Define base
Nitrogenous base
Component of nucleic acid involved in base pairing
I.e. guanine (G), adenine (A), thymine (T), cytosine (C), uracil (U)
Define base pair (BP)
A pair of nitrogenous bases held together by hydrogen bonds
I.e. G-Cbase pair with 3 hydrogen bonds
And A-T base pair (or A-U base pair ) with 2 hydrogen bonds
Define chromatin
DNA, proteins and RNA needed to package DNA into the cell nucleus
‘Loose’ chromatin is ‘bead on a string DNA’
Highly condensed chromatin is a chromosome
Define chromosome
Structure in the cell nucleus containing DNA wrapped around histones in a highly folded structured fashion
(1 double stranded DNA molecule or 2 identical double stranded molecules after DNA replication)
Define covalent modification
Addition of a chemical group to a protein
Reversible covalent modification is often used as means of regulating the activity of enzymes and other proteins
Define DNA
Deoxyribonucleic acid
Polymer of deoxyribonucleotides
Define genome
Total genetic content (DNA sequence) of an organism
In case of diploid organisms the DNA sequence of one set of chromosomes
Define isoenzymes
Enzymes that catalyse the same reaction but have a different amino acid sequence
Define nucleoside
Nitrogenous base
And a pentose sugar
Define nucleotide
Nitrogenous base
Pentose sugar
And a phosphate
Define nucleosome
Basic repeat unit of eukaryotic chromatin
Bead-like structure formed by DNA wrapped around histones
Define phosphorylation
Addition of a phosphate group
Define protein kinase
Enzyme that can catalyse addition of a phosphate group from ATP to Ser/Thr/Tyr residues in a protein
Define protein phosphatase
Enzyme that catalyses removal of a phosphate group from Ser/Thr/Tyr residues in a protein
Define purine
Two-ring nitrogenous base
E.g. Guanine (G) and Adenine (A)
Define pyrimidine
One-ring nitrogenous base
E.g. Cytosine (C), thymine (T) and uracil (U)
Define RNA
Ribonucleic acid
Polymer of ribonucleotides
Define zymogen
Inactive precursor of a proteolytic enzyme
What are the main forms of enzyme regulation?
Substrate and product concentration
Changes in enzyme conformation
Changes in the amount of enzyme
Ways to change enzyme conformation
Allosteric control
Covalent modification
Proteolytic activation
What is an allosteric enzyme?
Multi subunit enzymes that contain more than 1 active site for the substrate
What is positive cooperativity?
Binding of substrate to one active site enhances subsets binding to other active sites
What does an allosteric activator do to the Michaelis Menten curve?
Increases activity of enzyme
Curve shifted to the left
Shifts the R to T conformational equilibrium toward R
What does an allosteric inhibitor do to the Michaelis Menten curve?
Decreases activity of enzyme
Curve shifted to the right
Shifts the R to T conformational equilibrium toward T
Which enzyme catalyses attachment of a phosphate group?
Kinase
Which enzyme catalyses removal of phosphate groups?
Phosphatase
What is the an enzyme’s inactive protein precursor known as?
Zymogen
How are zymogen a activated?
Removal of part of the polypeptide chain
Many proteases produced in this form
What is a protease?
Enzyme that can break peptide bonds
How is premature proteolytic cleavage prevented?
Many proteases are produced in inactive zymogen form
Transported safely to sites of action
Then activated
E.g. Blood clotting factors
Ways to change amount of enzyme for regulation purposes?
Regulation of enzyme synthesis
Regulated protein degradation
How is enzyme synthesis regulated?
Increasing or decreasing rate of transcription of mRNA
How is protein degradation regulated?
Proteins can be tagged for destruction by the addition of a small protein molecule known as ubiquitin
Ways to regulate metabolic pathways
Feedback inhibition
Feed forward activation
Counter regulation
What is feedback inhibition in metabolic pathway regulation?
End product of a pathway inhibits its own rate of synthesis by inhibiting enzymes earlier in the pathway
E.g. High [ATP] inhibit catabolic pathways
What is feed forward activation?
Increased amounts of initial substrate increases first step in pathway
E.g. High concentration of ethanol induce microsomal ethanol oxidising enzymes
How does counter regulation of metabolic pathways work?
If a catabolic pathway breaking down compound A is activated
Then the opposing anabolic pathway making compound A will be inactivated
E.g. Glycogenolysis and glycogenesis
Main mechanisms which regulate the blood clotting cascade
Inactive zymogens present at low concentration
Amplification of an initial signal
Localisation of clotting factors to the site of damage
Feedback activation by thrombin
Termination of clotting by multiple processes
How are clotting factors localised to site if damage?
Factor with Gla domains bind to damaged endothelial cell lining
Allow rapid activation of downstream effector molecules
Explain feedback activation by thrombin
Activated thrombin enhances conversion of
Factor V, VII and XI to activated forms
Ways to terminate clotting
Removal of activated proteins
Proteolytic digestion
Binding of inhibitor molecules
Factors of extrinsic pathway
Factor VII
Factor X
Factors of intrinsic pathway
Factor XI Factor IX VIII V Factor X
What is prothrombin converted to?
Thrombin
By factor Xa
What is fibrinogen converted to?
Fibrin
By thrombin
Which factor is necessary to form cross linked fibrin?
Factor XIIIa (activated by thrombin)
How many chromosomes do humans have?
23 pairs
Examples of polynucleotides
DNA
RNA
What links nucleotides?
Covalent phosphodiester bonds
Molecule at 5’ end?
Free phosphate
Molecule at 3’ end
Free OH- group
Conventionally, which way is DNA strand written?
5’ to 3’
Mutation in CF
3bp deletion (
Mutation in SCA
A>T
Glu>Val
In human beta haemoglobin protein
What is achondroplasia ?
Form of short limbed dwarfism
Mutation in achondroplasia
G>A
GLY>Arg
In cDNA of fibroblast growth factor gene
Mutation in PKU
G>A
Premature stop codon
Unstable mRNA transcript
Of human phenylalanine hydroxylase gene
Define allele
Alternative form of a gene
Each individual has two alleles for every gene
which can either be the same or different
Define autosome
Chromosome other than the sex chromosomes
I.e. human chromosome 1-22
Define centromere
Region of a duplicated chromosome where two sister chromatids touch
Region of a chromosome to which the micro tubule spindle attach during cell division
Define chiasmata (pl chiasmata)
Point where two chromatids exchange genetic information during crossing-over in meiosis
Only when 2 non-sister chromatids (chromatids from different homologous chromosomes within a chromosome pair) exchange information will there be a genetic consequence
Define chromatid
One of two identical components of a duplicated chromosome
Each containing a single (identical) double stranded DNA molecule
Define dominant
Phenotypic trait is dominant when it occurs in both homozygotes and heterzygotes
Define gene
Unit of heredity
Transcription unit
Length of DNA on a chromosome that contains the code for a protein (or RNA)
And sequences necessary for its expression e.g. Promoter and terminator sequences and introns
Define genotype
Genetic make-up of an individual
Either as a whole or for one specific genetic locus
Define heterozygous
Having two different alleles for a specific genetic locus
Define homozygous
Having two identical alleles for a specific genetic locus
Define locus (pl loci) Or genetic locus or gene locus
Specific position on a chromosome
Define meiosis
Process of cell division by which gametes are produced
Define mitosis
Process of cell division for somatic cells
Define phenotype
All observable characteristics of an individual
The observable trait as a result of genetic make-up of one (or more) specific gene locus (loci)
Define recessive
Phenotypic trait is recessive when it occurs in homozygotes only
Define somatic cell
Cell other than the gametes
Stages in the cell cycle
G1
S
G2
M
What happens in G1?
Cell prepares for DNA replication
Cellular contents, excluding the chromosomes are duplicated
What happens in the S phase?
Semi-conservative DNA replication
Each of the 46 chromosomes is duplicated by the cell
What happens in G2 phase?
Cell prepares for cell division
The cell ‘double checks’ the duplicated chromosomes for error making any needed repairs
What happens in M phase?
Cell division (mitosis)
What substrates are used by the enzyme DNA polymerase?
Deoxyribonucleoside triphosphate (dNTP)
What reaction is catalysed by DNA polymerase?
(dNMP)n + dNTP -> (dNMP)n+1 + PPi
5’ to 3’ chain growth
What is semi-conservative DNA replication?
Each strand of the parent molecule is maintained in the two daughter molecules
What does DNA helicase do?
Unravels the DNA double helix
What are Okazaki fragments?
DNA fragments of the lagging strand which is made discontinuously
What does DNA ligase do?
Joins the Okazaki fragments
Which strand is synthesised continuously in DNA replication?
Leading strand
Composition of human somatic cells
Diploid
23 pairs of chromosomes
(22 pairs of autosomes and 1 pair of sex chromosomes XX/XY)
Composition of human gametes and examples
Sperm and egg
Haploid
One set of 23 chromosomes
Chromosome properties after replication
Classical X-shape
Two identical sister chromatids joined by centromere
What is the centromere?
Region of a chromosome that joins two identical sister chromatids
How many daughter cells produced after mitosis?
2 diploid cells
Each have same chromosomal content as parent cell
How many daughter cells produced after meiosis
4 haploid cells
Each has half the number of chromosomes as the parental cell
What happens during meiosis?
Diploid cell undergoes
One round of replication
Two rounds of division (meiosis I and meiosis II)
Why does mitosis take place?
Growth
Maintenance
What is the M phase?
Mitosis
Steps of mitosis
Prophase Prometaphase Metaphase Anaphase Telophase Cytokinesis
What happens in prophase?
Break down of nuclear membrane
Spindle fibres appear
Chromosomes condense
What happens in prometaphase?
Spindle fibres attach to chromosomes
Chromosomes condense
What happens in metaphase?
Chromosomes align
What happens in anaphase?
Centromeres divide
Sister chromatids move to opposite poles
What happens in telophase?
Nuclear membrane reforms
Chromosomes decondense
What happens in cytokinesis?
Cytoplasm divides
Parent cell become 2 daughter cells with identical genetic information
What do all people in a horizontal line in a pedigree have in common?
Same generation
What are the main patterns of inheritance?
✔Autosomal recessive ✔Autosomal dominant Sex-linked inheritance ✔X-linked recessive/dominant Y-linked
What is autosomal inheritance?
When the gene in question is located on an autosome
What is sex-linked inheritance?
When the gene in question is loaded on a sex chromosome
What is X-linked inheritance?
When gene in question is located on the X-chromosome
Sex-linked inheritance
What is Y-linked inheritance?
When gene in question is located on the Y-chromosome
Inherited directly from father to son
Sex-linked inheritance
Unaffected Male
In pedigree
White Square
Unaffected female
In pedigree
White circle
Affected individual
In pedigree
Black
Carrier
In pedigree
Half black shading
Or black dot in centre
Deceased individual
In pedigree
/
Through symbol
Unknown sex
In pedigree
Diamond
What are linked genes?
2 genes close together on the same chromosome
Don’t show independent assortment during meiosis
They co-segregate
How can linked alleles separate during meiosis?
Crossing-over
Recombination
Define anticodon
Three nucleotides on tRNA molecule that are complimentary to a codon on the mRNA template
Define codon
Three nucleotides on mRNA molecules that code for a specific amino acid in the protein sequence
(Except stop codon)
What is a stop codon?
Does not code for an amino acid
Signals termination of polypeptide chain growth
Define genetic code
Universal ‘conversion table’ from DNA information into amino acid information
I.e. how sets of three nucleotides (codons) encode the information for the different amino acids
Define mature mRNA
End product of transcription
I.e. after capping, polyadenylation and splicing has occurred
Serves as template in translation
Define ORF
Open reading frame
A area of gene that holds code for all amino acid residues of the gene product (the protein)
Define promoter
Area of gene upstream of the ORF which regulates transcription
And therefore gene expression
Include sequences for binding of transcription factors and FNA polymerase and many regulatory factors (activators and repressors)
Define ribosome
Two-unit particle composed of many protein and ribosomal RNAs
Site of protein synthesis
mRNA
Messenger RNA
rRNA
Ribosomal RNA
tRNA
Transfer RNA
Define transcription
Process by which the DNA ‘transcribed’ (‘copied’) into RNA message
Define transcription initiation
Start of mRNA production
Dependent on RNA polymerase binding
Define transcription termination
End of mRNA production
Which is sequence dependent
Define translation
Process by which RNA message is ‘translated’ into an amino acid code (protein)
Define translation initiation
Start of protein production
I.e. at the AUG codon
Define translation termination
End of protein production
I.e. at a stop codon (UAA, UAG, UGA)
Where is mRNA made, what is this process called?
Nucleus
Transcription
DNA to mRNA
Where is protein made?
Cytoplasm
Translation
mRNA to protein
What are the three phases in transcription process?
Initiation
Elongation
Termination
What happens in initiation of transcription process?
Promoter recognition
And binding
What happens in elongation of transcription process?
Actual process of ‘transcribing’ by RNA polymerase
What happens in termination of transcription process?
Sequence-dependent termination of RNA chain growth
Post-transcriptional processes
Transform pre-mRNA into mature mRNA
Capping
Tailing
Splicing
What is capping?
Addition of 5’ cap
What is tailing?
Polyadenylation
Addition of 3’ polyA tail
What is splicing?
Removal of introns
Exons ‘spliced together’
What are the types of RNA?
rRNA ribosomal 80+%
mRNA messenger
tRNA transfer
What codon is AUG?
Methionine
What is the initiation codon?
AUG
Methionine
What are the stop codons?
UAA
UAG
UGA
What are the three phases in the translation process?
Initiation
Elongation
Termination
What happens in the initiation process of translation?
AUG codon recognition and binding
And formation of functional ribosome
What happens in the elongation process of translation?
Actual process of ‘translating’ the RNA message into protein
mRNA read codon by codon from 5’ to 3’
While polypeptide chain growth is from amino to carboxyl terminus
What happens in the termination process of translation?
Stop codon recognition and dissociation of ribosome
Define glycosylation
Attachment of carbohydrate groups (oligosaccharide) to protein (side chain of certain aa residues) via glycosidic linkages
Can be N-linked (via amino side chains of Asn residues)
Can be O-linked (via hydroxyl side cabins of Ser/Thr residues)
Define preproprotein
Protein containing both a signal peptide and a propeptide
Define proprotein
Inactive precursor of a protein
To become fully active the protein must undergo limited proteolytic to remove the propeptide
Define protein targeting
Mechanisms involved in directing proteins to their correct site of action inside (or outside) the cell
Define scurvy
Disease caused by vitamin C (ascorbic acid) deficiency that results in inadequate formation of hydroxyproline residues in collagen, reducing the stability of the collagen fibres
Define signal peptidase
Enzyme that removes the signal peptide from secretory proteins after entry into the ER
Define signal peptide
Short N-terminal peptide sequence that directs a newly formed secretory or membrane protein into the ER
Define signal recognition particle
Complex containing protein and RNA that binds the signal peptide of newly synthesised secretory proteins and escorts it to the ER for translocation
Define tropocollagen
Collagen triple helices formed extracellularly where the N- and C-terminal regions have been removed
Which proteins are more likely to be made on ribosomes on the ER?
Proteins destined for insertion into the plasma membrane, lysosomes, Golgi/ER or secretion
What addition do secreted proteins have?
Signal sequence at the N-terminus that targets them to the ER
What do signal sequences contain?
10-15 hydrophobic residues
1+ positively charged residues near amino terminus before hydrophobic sequence
Few polar amino acids in the C-terminal region
Protein secretion pathway part 1 - signal sequence
1 Protein synthesis initiated on free ribosomes
2 N-terminal signal sequence produced
3 Signal sequence of newly formed protein is recognised by the signal recognition particle (SRP)
Protein secretion pathway part 2 - SRP
4 GTP-bound SRP directs the ribosome synthesising the secretly protein to SRP receptors on the cystolic face of the ER
5 SRP dissociates
Protein secretion pathway part 3
6 Protein synthesis continues and the newly formed polypeptide is fed into the ER via a pore in the membrane (peptide translocation complex)
7 Signal sequence removed by a signal peptidase
8 Ribosome dissociates and is recycled
Modification to proteins in lumen of ER
Disulphide bond formation
Glycosylation
N-linked glycosylation
Carbohydrate added via N-glycosyl link to amide nitrogen of Asn
Occurs in ER
O-linked glycosylation
Carbohydrate added via glycosidic link to hydroxyl of Ser or Thr
Occurs mainly in Golgi
Types of secretion
Constitutive secretion
Regulated secretion
What is constitutive secretion?
Continuous process
Not regulated
Proteins packaged into vesicles and release continuously by exocytosis
E.g. Serum, albumin, collagen
What is regulated secretion?
Proteins released in response to a signal/ stimulus e.g.hormone
Proteins packaged into vesicles but not released until stimulus received e.g. Insulin
What cuts pro insulin into 3 peptides?
Proteases
Biosynthesis of collagen part 1
1 Synthesis and entry of chain into lumen of rough ER
2 Cleavage of signal peptide
3 Hydroxylation of selected proline and lysine residues
4 Addition of N-linked oligosaccharides
5 Addition of galactose to hydroxylysine residues
Biosynthesis of collagen part 2
6 Chain alignment, formation of disulphide bonds
7 Formation of triple helical pro collagen from C- to N- terminus
8 Completion of O-linked oligosaccharide chains by addition of glucose
Biosynthesis of collagen part 3
9 Transport vesicle
10 Exocytosis
11 Removal of N- and C- terminal peptides
Biosynthesis of collagen part 4
12 Lateral association of collagen molecules followed by covalent cross-linking
13 Aggregation of fibrils
Define antibody
Protein which is produced by the body in response to a foreign compound (antigen) usually a protein.
Antibodies can be used diagnostically to detect specific proteins.
Define cloning
Production of exact copies of a piece of DNA
Define cloning vector
DNA molecule such as a plasmid, virus or artificial chromosome into which a piece of foreign DNA can be added for cloning
Define ELISA
Enzyme-linked immunoabsorbent assay
Technique using an antibody linked to an enzyme used to quantify the amount of a molecule
Used in diagnostic tests to measure the concentration of biological molecules in solution
Define gel electrophoresis
Technique which separates macromolecules (proteins/DNA/RNA) on the basis of their size or charge
Molecules are separated in gel and migrate due to the presence of an electric charge played across the gel
Define PCR
Polymerase chain reaction
Very powerful and sensitive technique whereby small fragments of DNA are amplified using a DNA (or RNA) template
Amount of template needed is minute
Amount of amplified DNA end-product is enormous
Enough for further experimental procedures
E.g. Gene cloning, restriction analysis and DNA sequencing
Define primer
Short oligonucleotide (10-25nt)
Can be 3’-extended by DNA polymerase
Primers of specific sequence used in molecular techniques like PCR and DNA sequencing
Define restriction endonuclease (restriction enzyme)
Enzyme that recognises and cuts double stranded DNA at a specific sequence (the restriction site)
Define restriction site
Sequence of DNA that can be recognised by a restriction endonuclease
Enzyme can then specifically cut the double stranded DNA molecule within (or adjacent to ) the recognition sequence
Define SDS-PAGE
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Gel electrophoresis of proteins in the presence of the detergent SDS
Proteins are separated on the basis of their size
Define western blotting
Technique where a protein separated by electrophoresis is transferred to a membrane filter and is detected by the binding of an antibody directed against it
Techniques for DNA analysis at gene level
Restriction analysis/ gene cloning
Gel electrophoresis
Polymerase chain reaction
How many disulphide bonds does insulin have?
3 disulphide bonds
Formed in pro insulin
Eer sent in mature insulin too
Proteolytic processing of insulin
Preproinsulin
= pro insulin + signal sequence
= mature insulin + C peptide
