RECAPS Flashcards
give 4 ways in with cells differ from each other
size, shape, chemical requirements, and function
two examples of how cells differ in shape
- nerve cells are extended and branched to transmit electrical signals
- paramecium is shaped like a submarine and covered with cilia, whose coordinated beating sweeps the cell forward
cell specialisation
- in multicellular organisms, division of labour allows for efficiency
- this does not occur in single celled organisms
- some cells become so specialised that they cease to proliferate
what do all living cells share?
- a similar basic chemistry; composed of the same sorts of molecules which participate in the same types of chemical reactions
- genetic information carried in genes
define a living cell
a self-replicating collection of catalysts
viruses and reproduction
- do not have ability to reproduce by their own efforts
- parasitise reproductive machinery of the cells they invade to make copies of themselves
where have all living cells evolved from?
the same ancestral cell, which existed between 3.5 and 3.8 billion years ago
- mutation
- sexual reproduction
- natural selection
3 major domains of the tree of life
eukaryotes (smallest domain), bacteria, and archaea
how is the tree of life organised?
analysis of the genome
which cells are larger; eukaryotes or prokaryotes? what about their genomes?
eukaryotic cells, and also have much larger genomes
most of the earth’s biomass is stored in
plants
which domain of life is most diverse and why?
bacteria
- small
- have been around for longest
- reproduce very quickly (so evolve fast)
draw and label a bacterial cell
- cytoplasm
- plasma membrane
- outer membrane
- cell wall
which domain is most poorly understood?
archaea
describe archaea
- differ from bacteria by chemistry of their cell walls, types of lipids that make up the membrane, and range of chemical reactions they can carry out
- archaea live everywhere, including extreme environments
- predominant form of life in soil and seawater
- play a major role in recycling nitrogen and carbon
- genomes closely related to eukaryotes
nucleus
- information store of cell
- enclosed in 2 concentric membranes (nuclear envelope)
- contains molecules of DNA
mitochondria
- enclosed in 2 membranes, with inner membrane invaginated
- generate chemical energy for the cell via cell respiration
- harness energy from oxidation of food molecules to produce ATP
- contain own DNA and reproduce by dividing
chloroplasts
- two surrounding membranes and stacks of membranes containing chlorophyll (green pigment)
- carry out photosynthesis
- contain own DNA and reproduce by dividing
endoplasmic reticulum
- irregular maze of interconnected spaces enclosed by a membrane
- site where most cell-membrane components, as well as materials destined for export from the cell are made
Golgi apparatus
- stacks of flattened, membrane-enclosed sacs
- modifies and packages molecules made in the ER that are destined to be secreted from the cell or transported to another cell compartment
lysosomes
small organelles in which intracellular digestion occurs, releasing nutrients from ingested food particles into the cytosol and breaking down unwanted molecules for recycling within cell or excretion
peroxisomes
small, membrane-enclosed vesicles that provide an environment for a variety of reactions in which hydrogen peroxide is used to inactivate toxic molecules
transport vesicles
ferry materials between one membrane-enclosed organelle and another
draw diagram for continual exchange of materials in a cell
pg 24
endocytosis
portions of plasma membrane tuck inward and pinch off to form vesicles that carry material captured from the external medium into the cell
exocytosis
vesicles from inside the cell fuse with the plasma membrane and release their contents into the external medium
cytosol
concentrated aqueous gel of large and small molecules; site of many chemical reactions
cytoskeleton
- responsible for directed cell movements
- composed of three major filament types; actin filaments, intermediate filaments, and microtubules
- role in cell division
actin filaments
thinnest filaments; particularly abundant in muscle cells, where they serve as a centre part pf the machinery responsible for muscle contraction
microtubules
thickest filaments; form of minute hollow tubes; help pull chromosomes apart during mitosis
intermediate filaments
thickness between actin filaments and microtubules; serve to strengthen most animal cells.
motor proteins
use energy stored in molecules of ATP to move along cytoskeleton filaments
protozoans
free-living, motile, unicellular eukaryotes
Didinium
- large, carnivorous protozoan with a diameter of 150 micrometers
- uses beating cilia to swim at high speed
- when it encounters prey (usually another protozoan) it releases numerous small, paralysing darts from its snout
- attaches to and devours the other cell, inverting like a hollow ball to engulf its victim
model organisms
representatives species studied by biologists
E. coli
- small, rod shaped
- lives harmlessly in the gut of humans and other vertebrates, but will also grow and reproduce in simple lab nutrient broths
what knowledge has come from studying E.Coli?
- how cells regulate gene expression
- how cells replicate their DNA and how they make proteins from DNA
- ‘recombinant DNA’ revolution, enabling us to manipulate genes and DNA in the laboratory
- harnessed as a biological factory for producing large quantities of therapeutic proteins, including insulin
S. Cervisiae
- small, single-celled fungus that is more closely related to animals than plants
- rigid cell wall, relatively immobile, many organelles (nucleus, GA, ER, mito) but no chloroplasts
- can grow and divide almost as rapidly as bacteria
- carries out basic tasks that every eukaryotic cell must perform and can even mate w opposite sex
what knowledge has come from studying Baker’s yeast?
- genetics of sexual reproduction
- cell division cycle
arabidopsis thaliana
- model plant
- can be grown indoors in large numbers
- thousands of offspring within 8-10 weeks
what knowledge has come from studying arabidopsis thaliana?
- understanding of the mechanisms that enable plants to grow toward sunlight, to flower in spring, and to coordinate development with season cycle
- insights into the development and physiology of crop plants
C. Elegans
- nematode worm
- develops with clockwork precision from a fertilised egg cell into an adult that has exactly 959 body cells, an unusual degree of regularity for an animal
- understanding of sequence of events of development
- understanding of apoptosis (programmed cell death for disposal of surplus cells)
Drosophila melanogaster
- fruit fly
- study of animal genetics
- genes for development similar to those of humans; human development and basis of genetic disease
- genetic analysis provided definitive proof that genes are carried on chromosomes
- shown how DNA directs development of zygote into adult
- mutants w body parts used to characterise genes needed to make normal adult
zebrafish
- developmental processes, particularly in vertebrates
- easily bred and maintained in lab
- transparent for first 2 weeks of life, allowing observation of how cells behave during development
- insights into development of heart and blood vessels
mouse
- mammalian genetics, development, immunology, cell biology
- possible to breed mice with deliberately engineered mutations in any specific gene, or with artificially constructed genes introduced into them
- test the function of any gene and determine how it works
why is it that many human cells can be studied in vitro?
when grown in culture, they continue to display the differentiated properties appropriate to their origin
organoids
- used to study developmental processes
- certain human embryo cells can be coaxed into differentiating into multiple cell types, which can self-assemble into organ like structures that closely resemble a normal organ
four types of weak interactions that help bring molecules together in cells
- electrostatic attraction: between oppositely charged molecules
- van der Waals: when two atoms approach each other
- hydrophobic force: generated by a pushing of non polar surfaces out of the hydrogen-bonded water network, where they would otherwise physically interfere with the highly favourable interactions between water molecules
application of hydrophobic interaction
promote molecular interactions in building cell membranes, constructed from lipid molecules with long hydrocarbon tails
define nucleotides
nitrogen-containing ring linked to a five-carbon sugar that has a phosphate group attached to it
bases
under acidic conditions, can bind an H+ and thereby increase the concentration of OH- ions in aqueous solution
pyrimidines
cytosine, thymine, uracil
derived from a six-membered pyrimidine ring
purines
guanine and adenine
bear a second, five-membered ring fused to the six-membered ring
base plus sugar (no phosphate group)
nucleoside
ATP
adenine, sugar, three phosphates linked in series by two phosphoanhydride bonds
rupture of these bonds releases free energy
structure of nucleic acids
long polymers in which nucleotide subunits are linked by covalent phosphodiester bonds between the phosphate group of one nucleotide and a hydroxyl group of the next
how are nucleic acid chains synthesised?
from energy-rich nucleotide triphosphate by a condensation reaction that releases pyrophosphate
distinguish between the roles of DNA and RNA
- DNA is more stable due to double helix so acts as a long-term repository for hereditary information
- RNA serves as more transient carriers of molecular instructions
draw the 5 bases
base/nucleoside names for the 5 bases
adenine - adenosine
guanine - guanosine
cytosine - cytidine
uracil - uridine
thymine - thymidine
AMP
adenosine monophosphate
dAMP
deoxyadenosine monophosphate
UDP
uridine diphosphate
how are phosphates usually bound sugars?
joined to the C-5 hydroxyl of the sugar
3 functions of nucleotides and their derivatives
- carry chemical energy in their easily hydrolysed phosphoanhydride bonds
- combine with other groups to form coenzymes
- used as small intracellular signalling molecules in the cell
between which carbons do phosphodiester bonds take place?
5’ and 3’ carbon atoms of adjacent sugar rings
3’ end
ends with OH (hydroxyl)
5’ end
ends with phosphate
A pairs with
T or U
G pairs with
C
describe the structure of the double helix
- strands antiparallel to each other (oriented with opposite polarities)
- anti-parallel sugar-phosphate sytands twist around each other to forma. double helix containing 10 base pairs per helical turn
why does twisting of the double helix take place?
renders conformation of DNA’s helical structure energetically favourable
do prokaryotes have cilia?
no
origins of mitochondria (endosymbiosis) - entangle - engulf - endogenies (E^3) model
- archaean cell was anaerobic
- bacterial ectosymbiont was aerobic
- surface protrusions on archaea expanded over time
- this led to enclosure of ectosymbiont by archaeal membrane fusion
- escape of endosymbiont into cytosol and formation of new intracellular compartments
- over time, this evolved into modern-day mitochondria
did prokaryotes or eukaryotes form first?
prokaryotes formed much earlier, and then eukaryotes
what is an alternative model for endosymbiosis?
some models are more predatory, where the aerobic bacterium is engulfed via a process similar to phagocytosis
common features of both types of endosymbiosis models
- ancient anaerobic archaeal cell
- ancient aerobic bacterium
- over evolutionary time, a symbiotic relationship
Asgard cell
- type of archaea belonging to the group Asgard
- has a cell body and protrusions with ectosymbionts on their surface
describe and draw the sequence of the tree of life
ancestral prokaryote (3.5-3.8b years ago)
bacteria and archaea separated
1b years later, the first single-celled eukaryote was formed
lines of evidence to support endosymbiont hypothesis
- mitochondria and chloroplasts still have remnants of their own genomes, which are circular. their genetic systems resemble that of modern-day prokaryotes
- mitochondria and chloroplasts have kept some of their own protein and DNA synthesis components and these resemble prokaryotes too. they have their own ribosomes and multiply by pinching in half — the same process used by bacteria. are also sensitive to similar antibiotics.
- membranes in mitochondria and chloroplasts often similar to those in prokaryotes and appear to have been detached from engulfed bacterial ancestor.
general attributes of model organisms
- rapid development with short life cycles
- small adult (reproductive) size
- readily available (collections or wide-spread)
- tractability - ease of manipulation or modification
- understandable genetics
central dogma of molecular biology
DNA -(transcription)-> RNA -(translation)-> protein
tRNA
- transports amino acids
- protein synthesis
rRNA - ribosomal RNA
- part of the ribosome
- does catalytic work of making protein by creating peptide bonds
- has a structural role as part of RNA
refined central dogma
not all RNA is translated into protein - it has many other uses
draw a diagram for the elaborated central dogma information flow
genome
cell’s complete set of DNA, including mitochondria and chloroplasts
transcriptome
all the RNA in a particular cell at a particular point in time
genome vs transcriptome/proteome
transcriptome/proteome are much more dynamic
proteome
entire set of proteins in a cell at a particular point in time
how are the proteome and transcriptome related?
the proteome feeds information into the transcriptome
interactome
set of all protein-protein interactions taking place in a cell at a single point in time
metabolome
full set of small molecules that can be found in a cell at a certain point in time (anything that is generally smaller than a protein, like ATP, sugars, vitamins, some hormones)
example of metabolome affecting transcription
lac operon
phenome
comprised of everything (all the -omes), and together with the observable characteristics of what you’re looking at (cell, organ, etc)
describe the directionality of transcription and translation
- DNA, RNA, and proteins are synthesised as linear chains of information with a definite polarity
- info in RNA sequence is translated into an amino acid sequence via a genetic code which is essentially universal among all species
what are nucleic acids?
an organism’s blueprints - the genetic material in a cell
- DNA: deoxyribonucleic acid
- RNA: ribonucleic acid
three parts of a nucleotide
- pentose sugar - scaffold for base
- nitrogenous base
- phosphate group - backbone. There may be 1 (mono), 2 (di), or 3 (tri) phosphate groups
pyrimidines
UC The pyramids?
- single ring
- uracil
- cytosine
- thymine
purines
pure Animals Gobble
- double ring
- adenine
- guanine
where is the base attached?
1’
distinguish between ribose and deoxyribose in terms of functional groups
2’ carbon in ribose has a hydroxyl group - in deoxyribose it just has a hydrogen
nucleoside monophosphate
nucleoside vs nucleotide
nucleoside = base + sugar
nucleotide = base + sugar + phosphate
adenosine
sugar + adenine
guanosine
sugar + guanine
cytidine
sugar + cytosine
uridine
sugar + uracil
thymidine
sugar + thymine
nucleoside monophosphate
sugar + base + 1P
nucleoside diphosphate
sugar + base + 2P
nucleoside triphosphate
sugar + base + 3P
dNTPs
deoxyribonucleoside triphosphates
- DNA is synthesised from them
- N stands for A, C, T, G
NTPs
- N stands for A, C, U, G
- ribonucleoside triphosphates
- RNA is synthesised from them
nucleotides are linked by
phosphodiester bonds
overall charge on nucleic acids
negative all the way along
interactions between individual molecules are usually mediated by
noncovalent attractions:
1. electrostatic attractions (happen within and between large molecules) - weakened by water
2. hydrogen bonds - strongest in a straight line
3. van der Waals attractions - two atoms very close together, causing temporary dipole due to uneven distribution of electrons. not weakened by water
4. hydrophobic force - water pushing non-polar things away from itself. individually weak, but add up.
individually, very weak forces - BUT can sum to generate strong binding between molecules
Cell theory
- the cell is the basic organisational unit of life
- all organisms are comprised of 1 or more cells
- cells arise from pre-existing cells: the ability to reproduce is characteristic of living matter, which must be able to duplicate DNA, create proteins, etc
prokaryotic
- no nuclei
- single celled (but communities may exist)
- bacteria and archaea (domains)
eukaryotic
- nuclei with membrane
- single-celled (eg algae) or multicellular
- plants, fungi, animals, humans, protozoans
describe and draw a prokaryotic cell
- no membrane-bound organelles; localised DNA may be in nucleoid or not localised at all
- smaller size than eukaryotes (~1micrometer)
- less DNA than eukaryotes
draw and describe a eukaryotic cell (plant)
- nucleus
- several membrane-bound organelles
- larger size and more complex (~5micrometers)
draw and label a eukaryotic cell (animals)
what is the need for cytoskeletons in eukaryotic cells
simple diffusion is not enough to guide transport9
explain the existence of amino acids as optical isomers
- all amino acids (except glycine) exist as optical isomers termed D and L forms.
- only L forms are ever found in proteins
- D-amino acids occur as part of bacterial cell walls and in some antibiotics, and D-serine is used as a signal molecule in the brain
how many amino acids carry a charge?
five of the 20 amino acids - including lysine and glutamic acid - have side chains that form ions in solution. the rest are uncharged.
two types of abbreviations given to amino acids
three-letter and one-letter abbreviations
what structural feature of amino acids allows for the presence of L and D isomers?
the alpha carbon is asymmetrical
why are chains of amino acids very flexible?
the two single bonds around the C in the amide bond allow rotation
list the amino acids with basic side chains
- lysine (Lys, K)
- arginine (Arg, R)
- histidine (His, H)
positive charge
list the amino acids with acidic side chains
- aspartic acid (Asp, D)
- glutamic acid (Glu, E)
negative charge
list the amino acids with uncharged polar side chains
- asparagine (Asn, N)
- glutamine (Gln, Q)
amide N is not charged but is polar
- serine (Ser, S)
- threonine (Thr, T)
- tyrosine (Tyr, Y)
the OH group is polar
list the amino acids with non polar side chains
- alanine (ala, A)
- valine (val, V)
- leucine (leu, L)
- isoleucine (Ile, I)
- proline (pro, P)
- phenylalanine (Phe, F)
- methionine (met, M)
- tryptophan (trp, W)
- glycine (Gly, G)
- cysteine (Cys, C)
how is the polypeptide backbone formed?
from a repeating sequence of the core atoms (-N-C-C-) found in every amino acid
what constrains the shape of folded, long polypeptide chains/
many sets of weak non covalent bonds that form within proteins (hydrogen bonds, electrostatic attractions, and van der Waals)
how does the distribution of polar and non polar amino acids in a protein an important factor governing the folding of a protein?
- non polar (hydrophobic) side chains tend to cluster in the interior of the folded protein to avoid constant with the aqueous surroundings
- polar side chains arrange themselves near the outside of the folded protein, where they can form H bonds with water and other polar molecules
when polar amino acids are buried within the protein, they are usually hydrogen bonded to
other polar amino acids or to the polypeptide backbone
define the conformation of a polypeptide chain
the final folded structure
conformation is determined by
the shape in which its free energy is minimised.
the folding process is energetically —–. why?
favourable; it releases heat and increases the disorder of the universe
how can a protein be denatured and what does this mean?
protein is unfolded into a flexible polypeptide chain by treatment with solvents that disrupt the noncovalent bonds holding the folded chain together.
renaturation
when the denaturing solvent is removed, and the proper conditions are provided, the protein often refolds spontaneously into its original conformation
2 ways in which chaperone proteins work
- bind to partly folded chains and help them to fold along the most energetically favourable pathway
- form isolation chambers in which single polypeptide chains can fold without the risk of forming aggregates
function of chaperone proteins
assist protein folding in cells
function of bacterial transport protein HPr
facilitates transport of sugar into bacterial cells
4 types of 3D structure models
- backbone model
- ribbon model
- wire model
- space-filling model
in what protein was the alpha helix found?
in the protein alpha-keratin
in what protein was the beta sheet found?
in the protein fibroin
describe handedness of a helix
- depending on the way it twists, a helix is said to be either right-handed or left-handed
- handedness is not affected by turning the helix upside down, but it is reversed if the helix is reflected in a mirror
where are short regions of alpha helix especially abundant?
- in proteins that are embedded in cell membranes, such as transport proteins and receptors
- the portions of a transmembrane protein that cross the lipid bilayer usually form an alpha helix composed largely of amino acids with non polar Sid chains
- polypeptide backbone is hydrogen bonded to itself in the alpha helix, where it is shielded from the hydrophobic lipid environment of the membrane by the protruding non polar side chains
coiled coil
two or three alpha helices wrap around one another to form this stable structure. forms when the alpha helices have most of their nonpolar/hydrophobic side chains along one side, so can twist around each other with their hydrophobic side chains facing inward - minimising contact with the aqueous cytosol
distinguish between parallel and antiparallel beta sheets
parallel: when the neighbouring segments run in the same orientation (eg from N-terminus to C-terminus)
antiparallel: when they run in opposite directions
properties of beta sheets
- give silk fibres their tensile strength
- form basis of amyloid structures (beta sheets stacked together in long rows with their amino acid side chains interdigitated)
use of amyloid structures
- cells specialised for secretion store peptide or protein hormones in transport vesicles
- hormones adopt an amyloid structure to allow efficient packaging and unfold again once they reach cell exterior
how can amyloid structures cause disease?
- when proteins fold incorrectly, they sometimes form amyloid structures that can damage cells
- brain is particularly vulnerable to the damage caused by an accumulation of amyloid aggregates
- most neurone cannot regenerate -> neurodegenerative diseases like Alzheimer’s, Parkinson’s, Huntington’s
prions
- misfolded proteins
- infectious: amyloid form can convert properly folded molecules of the protein into the abnormal, disease-causing conformation
- move up the food chain and find their way to the brain where they form aggregates that spread from cell to cell
- scrapie in sheep, BSE/mad cow, CJD in humans
draw a diagram for how prions lead to amyloid fibrils
- normal protein can adopt an abnormal misfolded prion form
- prion form can bind to normal form, inducing conversion to abnormal conformation
- abnormal prion proteins propagate to form amyloid fibrils
give an example of the different functions of different protein domains
bacterial catabolite activator protein (CAP) has two domains:
- small domain that binds to DNA
- large domain that binds to cyclic AMP, an intracellular signalling molecule
- when the large domain binds cyclic AMP, it causes a conformational change in the protein that enables the small domain to bind to a specific DNA sequence and thereby promote the expression of an adjacent gene.
unstructured sequences
- larger proteins can contain many domains which are often connected by relatively short, unstructured lengths of polypeptide chain
- continually bend and flex due to thermal buffeting
why is only a minuscule fraction of the unimaginably large collection of potential polypeptide sequences actually made by cells?
- most biological functions depend on proteins with stable, well-defined 3D conformations
- functional proteins must not engage in unwanted associations with other proteins
- vast majority of potential protein sequences has been eliminated by natural selection through trial-and-error process
an example of a protein family
serine proteases, a family of protein-cleaving enzymes including digestive enzymes
- portions of amino acid sequences nearly the same
- most of the detailed twists and turns in their polypeptide chains are virtually identical
- distinct enzymatic activities
define a binding site
any region on a protein\s surface that interacts with another molecule through sets of noncovalent bonds
define a subunit
- if a binding site on one protein binds to a second protein, this will form a larger protein with a quaternary structure
- each polypeptide chain in such a protein is called a subunit
dimer
two identical, folded polypeptide chains form a symmetrical complex of two protein subunits held together by interactions between two identical binding sites
proteins can assemble into three main assemblies:
filaments (eg helical actin filaments), sheets, spheres, hollow tubes, rings
globular proteins
polypeptide chain folds up into a compact shape like a ball with an irregular surface
fibrous proteins
- have roles in the cell that require them to span a large distance
- have a simple, elongated 3D structure
intermediate filaments
- coiled-coil regions are capped at either end by globular domains containing binding sites that allow them to assemble into ropelike intermediate filaments
- component of the cytoskeleton that gives cells mechanical strengths
what is the role of fibrous proteins outside of the cell?
form the gel-like extracellular matrix that helps bind cells together to form tissues
describe collagen
- most abundant fibrous extracellular protein in animal tissues
- consists of 3 long polypeptide chain, each containing glycine (non polar) at every third positioin
- regular structure allows chains to wind around one another to generate a long, regular triple helix with glycine at its core
- collagen molecules bind to one another forming strong fibrils
describe elastin
- formed from loose and unstructured polypeptide chains that are covalently cross-linked
- resulting elastic fibers enable skin, arteries, lungs to stretch and recoil without tearing
how do protein molecules attached to the surface of a cell’s plasma membrane/secreted as part of the extracellular matrix maintain their structures among the potentially harsh extracellular conditions?
- stabilised by covalent cross-linkages
- can either tie together 2 AAs in the same polypeptide chain or join together many polypeptide chains in a large protein complex
- most common are disulphide bonds
how are disulphide bonds formed?
before a protein is secreted, by an enzyme in the endoplasmic reticulum that links two -SH groups from cysteine side chains
example of disulphide bonds
lysozyme retains its antibacterial activity for a long time due to disulfide cross-links
why do disulfide bonds generally not form in the cell cytosol?
proteins do not require this type of structural reinforcement in the relatively mild conditions inside the cell
how do proteins proceed in a singular direction (eg when walking along a cytoskeletal fibre)?
- conformational change must be unidirectional
- one of the steps must be made irreversible
- this is achieved by coupling one of the conformational changes to the hydrolysis of an ATP molecule tightly bound to the protein
- great deal of free energy is released when ATP is hydrolysed, making it very unlikely that the protein will undergo the reverse shape change needed to move backward
motor proteins are also known as
ATPases
the most complex tasks within cells are carried out by
large protein assemblies formed from many protein molecules
how do protein machines work?
the hydrolysis of bound nucleoside triphosphate (ATP or GTP) drives an ordered series of conformational changes in some of the individual protein subunits, enabling the ensemble of proteins to move coordinately
how do proteins locate their partners - and assemble into complexes that are activated only when and where they are needed - within the crowded conditions inside the cell?
many protein complexes are brought together by scaffold proteins
define a scaffold protein
large molecules that contain binding site recognised by multiple proteins
how do scaffold proteins work
they bind a specific set of interacting proteins, greatly enhancing the rate of a particular chemical reaction while also confining the chemistry to a particular area of the cell
give an example of cells that use scaffold proteins
nerve cells use scaffold proteins to organise the specialised proteins that localise at the synapse between one cell and the net
describe the structure of scaffold proteins
- though some scaffolds are rigid, the most abundant ones are very elastic
- contain long unstructured regions that bend, enhancing the collisions between the specific molecules bound to the scaffold
define a bimolecular condensate
- assemblies which often contain RNA and protein, forming fluid, membrane less subcompartments that perform a particular biochemical function
- contains at least one type of scaffold protein or scaffold RNA molecule that can interact w ‘clients’ which become concentrated
give an example of a biomolecular condensate
nucleolus
phase separation
property where the dynamic network of weak interactions that allows individual molecules to come and go, while the condensate as a whole remains intact and separated from its surroundings
the existence of condensates is
transient but stable
describe the process of purifying proteins from cells or tissues
- breaking open the cells to release their contents
- initial fractionation procedure to separate out the class of molecules of interest
- isolating the desired protein
- can be used in biochemical assays to study the details of its activity
cell homogenate or extract
resulting slurry after breaking open cells to release their contents
how does chromatography purify the protein
different materials are used to separate the individual components of a complex mixture into fractions, based on the properties of the protein such as size, shape, or electrical charge
affinity chromatography
- most efficient form of protein chromatography
- separates polypeptides on the basis of their ability to bind to a particular molecule
electrophoresis
- used to separate proteins based on size and net charge
- yields a number of bands or spots that can be visualised by staining; each band or spot contains a different protein
how was protein sequencing done in earlier years?
- protein broken down into smaller pieces using a selective protease
- identities of the aas in each fragment determined chemically
describe how mass spectrometry is performed
- peptides derived by digestion with trypsin are blasted with a laser - this heats the peptides, causing them to become ionised and then ejected as a gas
- peptide ions are accelerated by a powerful electric field and fly toward a detector
- time it takes them to arrive is related to their mass and charge
tandem mass spectrometry
- complex mixture of proteins
- after peptides pass through first mass spec, they are broken into even smaller fragments and analysed by a second mass spec
what methods do scientists use to determine experimentally the structure of purified proteins?
X-ray crystallography, NMR spec, cry-electron microscopy
how do we know the vast majority of proteins may fold up to a limited number of structural domains?
although the number of multi domain families in protein databases is growing rapidly, the discovery of novel single domains is levelling off
how are proteins useful in the industry?
bacteria, yeast, and cultured mammalian cells are now used to mass-produce a variety of therapeutic proteins, such as insulin, human growth hormone, and even the fertility-enhancing drugs used to boost egg production in women undergoing IVF
give an example of how investigators have designed proteins with completely novel functions
they have built a synthetic protein that contains a special cage that can be made to swing open like a latch when exposed to a compound that serves as its molecular key. this can be used to dispense a drug or to deliver a molecule
parts of an amino acid
- alpha carbon to which all other atoms and groups are attached
- amino group (NH2)
- carboxyl group (COOH)
- R group - side chain
how is a peptide bond formed?
- there is a reaction between the carboxyl group on one amino acid and the amino group on the other.
- the OH group on the carboxyl end of one amino acid reacts with the hydrogen atom on the amino group of the other to eliminate a molecule of water
what two features are always present in polypeptides, even in short chains?
an amino end (N-terminus) and a carboxyl end (C-terminus)
residues
what amino acids are referred to as once they have joined together into a polypeptide chain
in an alpha helix, where does hydrogen bonding occur between residues?
- there is hydrogen bonding between an oxygen atom of the carbonyl group of residue ‘n’ and the hydrogen atom of the amide group of the residue ‘n+4’ on the same polypeptide chain
- this is repeated in a regular fashion (1 and 5, 2 and 6, 3 and 7)
- the peptide chain thus twists around on itself and forms a cylindrical structure (a stable alpha helix)
are R-groups involved in the formation of alpha helices?
no
give the hierarchy of protein structure and examples for each
- primary; AA sequence
- secondary; local folding, like alpha helix and beta sheet
- tertiary; long-range folding, essentially 3D structure
- quaternary; multimeric organisation (the organization of multiple polypeptide chains with respect to each other)
- multiprotein complexes
major categories of amino acids
- acidic: negatively charged
- basic: positively charged
- uncharged polar: tends to form H=bonds, interact with h2o on the outside of proteins
- non polar: on the inside of proteins, ‘hydrophobic core’ due to hydrophobic interactions. found in lipid bilayer
what type of amino acids usually have enzymatic functions?
polar amino acids
what is unique about the structure of cysteine?
- contains interchain disulphide bonds
- whether these occur can be controlled by the cell based on redox conditions
- helps the protein hold its shape with physical or chemical stress
how is the primary structure of a protein numbered?
from the amino group (N-terminus)
give an example of how differences in primary amino acid sequence matter
- vasopressin and oxytocin
- both are 9AA long neuropeptide hormones
- AA sequence is identical except at 2 positions
- vasopressin controls urine production rates and oxytocin is involved in birth, lactation, and pair bonding
give an example of how the order of AA’s is important too
- Leu-Enkephalin (pentapeptide N-Tyr-Gly-Gly-Phe-Leu-C) is a natural opioid peptide which down modulates the perception of pain
- the pentapeptide N-Leu-Phe-Gly-Gly-Tyr-C, basically a reversal, has no pharmacological effects
- the NH2-COOH orientation of the peptide is essential for function
describe the structure of the beta sheet
- H-bonding between carbonyl oxygen (C=O) of 1aa and amide hydrogen (N-H) of aa in neighbouring strand
- R groups not involved but alternately project up and down
- beta sheets typically contains 4-5 beta strands but can have more than 10
types of beta sheets
- anti-parallel
- parallel
where are beta sheets found
strong, rigid structure found in silk
compare and contrast hydrogen bonding in alpha helices and beta sheets
- H bonds formed between carbonyl oxygen, amide hydrogen in peptide backbone
Alpha: - 4 AA’s apart and within the same segment of
pp chain
Beta: - Between AA’s in different segments or
strands of pp chain
coiled coil
- when alpha helices are twisting together
- amphipathic alpha helix
- these are found in alpha-keratin of skin, hair, and also myosin motor proteins
- helices wrap around each other to minimise exposure of hydrophobic amino acid side chains to aqueous environment
tertiary structure
- 3D overall structure of a protein
- held together by hydrophobic interactions, non-covalent bonds (hydrogen bonds, dipole-dipole and van der Waals), and covalent disulphide bonds
What determines the confirmations into which proteins fold?
proteins generally fold into the conformation that is the most energetically favourable
what helps fold proteins?
Proteins will fold into the shape dictated by their
amino acid sequence, but chaperone proteins help make the process more efficient and reliable
in living cells.
3 types of hydrogen bonding within tertiary structure of a protein
backbone to backbone: hydrogen bond between atoms of two peptide bonds
backbone to side chain: hydrogen bond between atoms of a peptide bond and an amino acid side chain
side chain to side chain: hydrogen bond between atoms of two amino acid side chains
What are protein domains?
- portion of a protein that has its own tertiary structure, often functioning in a semi-independent manner
- eukaryotic proteins often have 2 or more domains connected by intrinsically disordered sequences
- domains are important for the evolution of proteins
domains are often specialised for
different functions
Src protein kinase
- contains SH3 domain, SH2 domain, and kinase domain with 2 lobes
- phosphorylates amino acids to change the activity of proteins
- SH2 regulates kinase domain
- SH3 regulates kinase domain in a different way
- kinase domain phosphorylates the amino acids
protein families
- have similar amino acid sequences and tertiary structures
- however, members have often evolved to have different functions
- most proteins belong to families with similar structural domains
quaternary structure
proteins that have more than one polypeptide chain
describe haemoglobin
- 4 separate polypeptide chains
- 2 alpha subunits and 2 beta subunits
- each subunit is a separate polypeptide
- sickle cell anaemia is caused by a mutation in the beta subunit
give 3 types of multi protein complexes
- many identical subunits (eg actin filaments)
- mixtures of different proteins and DNA/RNA (eg viruses and ribosomes)
- very dynamic assemblies of proteins to form molecular machines (eg machines for DNA replication initiation or for transcription)
scaffold proteins
assemble other proteins needed for a particular process, getting them close together so work can be carried out
how are proteins studied?
- first purify protein/proteins of interest via various types of electrophoresis and affinity chromatography
- then determine amino acid sequence (eg mass spectrometry)
- discover precise 3D structure using techniques such as x ray crystallography, nuclear magnetic resonance spectroscopy or cry-electron microscopy
what properties can be exploited to separate proteins from one another so they can be studied individually?
- size, shape, charge, hydrophobicity, and their affinity for other molecules.
AI used to predict protein structure
alphafold to predict protein structure from linear amino acid sequences
proteomics
large scale study of proteins
- identity and structure of proteins
- protein-protein interactions, regulation of these interactions and their position within a pathway
- abundance and turnover of proteins
- location within a cell or tissue
- bioinformatics, statistics, and artificial intelligence often in combination with other ‘omics’ data
1KB
1000 base pairs
cross-talk
over time, some genes from mitochondrial and chloroplast sequences have migrated to the nucleus
why do organisms across the tree of life have such noticeable differences in size?
as complexity increases, there seems to be a trend toward larger genomes
what is included in the DNA that we have?
- 50% of the genome is made up of repetitive DNA
- 50% is unique sequences
- less than 1% of your genome encodes proteins
label a diagram for the percentage of the human genome and their functions
describe the necessity of packaging of DNA in the cell in prokaryotes
- in a non-packaged state, even the small prokaryotic genome would occupy a considerable portion of the cell volume
- DNA is condensed through folding and twisting and is complexed with proteins
- this forms the prokaryotic nucleioid
the chromosome solution
method of getting eukaryotic genome packed into cell
fluorescence in situ hybridisation (FISH)
- heat up chromosomes
- DNA helix will loosen a bit
- mix in a probe (short sequence of DNA) that is complementary to some of the sequence on the chromosome
- fluorescent dye is stuck onto the probe
- some of the strands will find their complementary sequence and bind there
describe the constitution of a chromosome
- each chromosome contains a single, long, linear DNA molecule and associated proteins called chromatin
- chromatin is tightly packaged, but dynamic as the DNA must remain accessible for transcription, replication and repair
draw interphase chromatin
draw M phase chromatin
centromere and sequencing
difficult area to sequence as it is very compact
telomeres and sequencing
compact so hard to sequence
levels of organisation of chromatin
- short region of DNA double helix
- ‘beads on a ring’ form of chromatin (double wrapped around nucleosome with some linker DNA)
- chromatin fibre of packed nucleosomes (30nm fibre)
- chromatin fiber folded into loops
- entire mitotic chromosome
describe the types and arrangement of histone proteins
- small proteins - rich in lysine and arginine
- positive charge neutralizes negative charge of DNA
- four core histone proteins (H2A, H2B, H3 & H4)
- pair of each in octamer core
- one linker histone (H1)
- H2A and H2B form a dimer, H3 & H4 form a dimer
- each core histone protein has a tail that can be covalently modified (reversibly) to be methylated, phosphorylated, acetylated; this is important for regulation of nucleosomes and how compact the DNA is
H1
acts as a paper clip and changes the trajectory of the linker DNA so it is bent and compacted better
how are chromatin loops made
sequence-specific clamp proteins and cohesions are involved in forming chromatin loops
how are chromatin loops altered during mitosis?
as cells enter mitosis, condensins replace
most cohesins to form double loops of
chromatin to generate compact chromosome
each DNA molecule has been packaged into a mitotic chromosome that is ——- times shorter than its extended length
10,000
function of chromatin re-modeling complexes an histone modifying enzymes
proteins that work together to make changes in chromatin structure and alter access to DNA for replication or transcription, so that the DNA is in a state where the cell can actually use it.
heterochromatin
highly condensed chromatin
- meiotic and mitotic chromosomes
- centromeres and telomeres
- time spent highly condensed varies (constitutive vs facultative)
heterochromatic regions of interphase chromosomes are areas where gene expression is
suppressed
euchromatin
relatively non-condensed chromatin
- degree of condensation varies
- level of activity varies (ie quiescent vs active)
active euchromatic regions of interphase chromosomes are areas where genes tend to be
expressed
constitutive heterochromatin
highly condensed pretty much all the time (eg centromeres/telomeres)
facultative heterochromatin
easier to decondense; genes that may need attention sometimes, like regulatory sequences
quiescent euchromatin
a level of condensation that is not as loose as active euchromatin but not as tight as heterochromatin
active euchromatin
genes are being expressed (transcribed)
heterochromatic vs euchromatic
a continuum that is dynamic
what is the degree of chromatin condensation controlled by?
localised covalent modification of histones, the presence of chromatin remodelling complexes, and RNA polymerase (transcription) complexes model the reversible switching from euchromatic to heterochromatic regions along chromosomes
how are interphase chromosomes arranged in the nucleus?
- discrete regions
- these regions can vary from cell to cell
gene off -> gene on
- homologous chromosomes detected by hybridisation techniques
- part of the chromosome is loosened so specially marked gene is moved to loosely compacted area of chromosome
is DNA replication conservative or semi-conservative? describe both
conservative: daughter cells have both parental DNA strands in one daughter cell and both of the newly synthesised strands in the other cell
semi-conservative: two daughter cells each have one parental DNA strand and one newly synthesised strand
why is conservative DNA replication problematic?
- if there is a mistake in the newly synthesised strands, there is no other strand to rectify this
what is the direction of DNA replication?
there are three main models that occur in nature, but we will focus on bidirectional growth from one starting point - DNA is growing in two directions from every point of origin. Bacteria and eukaryotes use this methods
describe the start of DNA replication
- double helix is opened with the aid of initiator proteins at the replication origin
- single stranded DNA templates are thus ready for DNA synthesis
where does DNA replication start?
always starts from the same location on DNA
What are some of the characteristics of the
sequences at replication origins?
Easy to open, A-T rich
Recognized by initiator proteins that bind to the DNA
how many origins of replications do origins have?
bacteria have a single one, eukaryotes have multiple (DNA is a lot bigger, so this is more effective)
Rolling Circle Replication
bidirectional; only applies to circular genomes
what happens at DNA replication forks?
5’ to 3’ direction (rule of DNA polymerase) growth in DNA. Okazaki fragments are formed in the lagging strand. The replication fork is asymmetrical - leading strand (5’ to 3’) replicated continuously, lagging (3’ to 5’) replicated discontinuously
homologous genes
when genes from different organisms have very similar nucleotide sequences, it is highly probable that they descended from a common ancestral gene
the vast majority of our DNA does not code for proteins or functional RNA molecules; instead, it includes
a mixture of sequences that help regulate gene activity, plus sequences that seem to be dispensable
how is genome size measured?
in nucleotide pairs of DNA per haploid genome
list the 6 basic types of genetic change that are crucial in evolution
- mutation within a gene
- mutation within regulatory DNA sequences
- gene duplication and divergence
- exon shuffling
- transposition of mobile genetic elements
- Horizontal gene transfer
mutation within a gene
- may change, delete, or duplicate one or more nucleotides
- thus alter the splicing of a gene’s RNA transcript or change the stability, activity, location or interactions of its encoded protein
mutation within regulatory DNA sequences
gene expression may be affected by a mutation in the DNA sequence that controls transcription of the gene
gene duplication and divergence
- a cell can make an extra copy of a gene/genome
- as the cell continues to divide, the original DNA sequence and duplicate sequence can acquire different mutations and assume new functions and patterns of expression
exon shuffling
two or more existing genes can be broken and rejoined to make a hybrid gene containing DNA segments that originally belonged to separate genes
transposition of mobile genetic elements
can move from one chromosomal location to another, thus altering activity or regulation of a gene
horizontal gene transfer
a piece of DNA can be passed from the genome of one cell to that of another, even to that of another species
how have biologists constructed a phylogenetic tree that goes all the way back to the origins of life?
- focused on the gene that codes for the ribosomal RNA (rRNA) of the small ribosomal subunit
- because the process of translation is fundamental to all living cells, this component of the ribosome has been highly conserved in all living species
human genome sequence
the complete list of nucleotides contained in our 23 chromosomes
other than its primary aim, how has the Human Genome project helped us?
- improvements in sequencing technologies
- new tools for handling large amounts of data
- cost of DNA sequencing has dropped
significance of transposons in our DNA
almost half of our DNA is made up of transposons that have colonised our genome over evolutionary time. most can no longer move
the number of protein-coding genes in the human genome may be unexpectedly small, but their relative size is unusually large. what does this mean?
- only about 1300 nucleotide pairs are needed to encode an average-sized human protein of about 430 amino acids; yet the average length of a human gene is 26,000 nucleotide pairs.
- most of this DNA is non-coding introns or regulatory DNA sequences
what does in-situ hybridisation allow us to do?
allows a specific nucleic acid sequence - either DNA or RNA - to be visualised in its normal location
how does in-situ hybridisation work?
makes use of single-strand DNA or RNA probes labeled with either fluorescent dyes or radioactive isotopes to detect complementary nucleic acid sequences within a cell, tissue, or organism
state 2 main applications of in-situ hybridisation
- explore how transcription regulators guide the development of multicellular organisms, providing important info ab when and where these genes carry out their function
- detect particular DNA sequences in an individual chromosomes (eg diagnose genetic abnormalities)
why do females and males have different numbers of types of chromosomes?
males, with their Y chromosomes, have an extra type of chromosome
homologous chromosomes
maternal and paternal versions of each chromosome
karyotype
an ordered display of the full set of an organism’s chromosomes
define a gene
a segment of DNA that contains the instructions for making a protein or RNA molecule
different possible functions of an RNA molecule
- used to produce a protein or may be final product
- eg may have structural or catalytic roles
- may play a part in regulating gene expression
how do we know junk dna is important?
comparisons of the genome sequences from many different species reveal that small portions of junk DNA are highly conserved among relative species
give an example of how there is no simple relationship between gene number, chromosome number, and total genome size
- human genome is 30 times smaller than some plants and 10 times smaller than some species of amoeba
- humans have a total of 46 chromosome, but some species of deer have 7 and some carp have more than 100
interphase chromosomes
exist as long, thin threads of DNA in the nucleus and cannot be easily distinguished in the light microscope
replication origin
site where DNA replication begins
telomeres
mark the ends of each chromosomes and serve as a protective cap that keeps the chromosome tips from being mistaken by the cell as broken DNA in need of repair
centromere
allows duplicated chromosomes to be separated during M phase
how are interphase chromosomes specially organised?
- each tends to occupy a particular region of the nucleus to prevent entanglement
- some chromosomal regions are physically attached to sites on the nuclear envelope or lamina which help chromosomes remain within their distinct territories
proteins that bind to DNA to form eukaryotic chromosomes are traditionally divided into
histones and non-histone chromosomal proteins
function of histones
responsible for the first and most fundamental level of chromatin packing - the nucleosome
how did investigators determine the structure of the nucleosome core particle?
treated chromatin in its unfolded form with enzymes called nucleases, which cut the DNA by breaking phosphodiester bonds. when nuclease digestion is carried out for a short time, only the exposed DNA between the core particles (linker DNA) will be cleaved, allowing the core particles to be isolated
SMC
structural maintenance of chromosomes proteins
functions of SMCs
associate with additional proteins to form an SMC ring complex that uses the energy of ATP hydrolysis to motor along the DNA, pushing out a loop of DNA in its wake
cohesin
SMC ring complex that organises the structure of interphase chromosomes
- cohesin rings will travel along the DNA, extruding loops until they run up against a sequence-specific clamp protein
- these proteins stall the cohesins and bind to one another, which draws together the DNA at the base of each loop
- it is the spacing and location of the clamp proteins that dictates the size and contents of each chromosomal loop
condensins
SMC ring proteins containing different SMCs and accessory proteins than cohesins
function of condensins
- as cells prepare to divide, condensins replace most of the cohesins that formed the loops in the interphase chromosome
- then use the energy of ATP hydrolysis to form loops of their own, which wind the chromatin into a tighter mass of coils
two ways to adjust local structure of chromatin
- ATP-dependent chromatin-remodelling complexes
- histone-modifying enzymes
ATP-dependent chromatin-remodelling complexes
large protein machines, present at about one copy for every 5 nucleosomes, which can use the energy of ATP hydrolysis to change the position of nucleosomes on the DNA. they can render DNA more or less accessible
histone-modifying enzymes
- generate reversible chemical modification of histones
- tails of all four of the core histones are particularly subject to these covalent modifications, which include the addition and removal of acetyl, phosphate, methyl groups
acetylation of lysines
can reduce the affinity of the tails for adjacent nucleosomes, thereby loosening chromatin structure and allowing access to particular nuclear proteins
how do modifications serve as docking sites on histone tails for a variety of regulatory proteins?
different patterns of modifications attract specific sets of non-histone chromosomal proteins to a particular stretch of chromatin. some of these proteins promote chromatin condensation whereas others promote chromatin expansion and facilitate DNA access
heterochromatin
- most highly condensed form of interphase chromatin
- makes up around 40% of typical interphase chromosome
- half remains permanently condensed (eg centromeres)
- the remaining contains genes whose activity has been silenced (ie embryogenesis genes)
euchromatin
- 60% of interphase chromatin
- a lightly packed form of chromatin (DNA, RNA, and protein) that is enriched in genes, and is often (but not always) under active transcription.
why is DNA replication called semiconservative?
each parent strand serves as the template for one new strand, so each of the daughter DNA double helices ends up with one of the original strands plus one completely new strand
what types of proteins begin DNA synthesis?
initiator proteins at replication origins
why is DNA rich in A-T base pairs typically found at replication origins
A-T base pair is held together by fewer hydrogen bonds than a G-C base pair. therefore, it is easier to pull apart
why is beginning DNA replication at many places at once helpful?
it greatly shortens the time a cell needs to copy its entire genome
why is it critical that DNA synthesis be initiated only once at every replication origin?
failure to regulate this process could cause genes to be copied too many times or even lost
