W1

Prokaryotes No nuclei, single-celled, simpler (smaller, genome smaller)

- Plasma membrane, cell wall, flagellum (some), nucleoid, ribosomes
Eukaryotes 1000x volume of prokaryote cells
- Microtubules (gives structure), cytoskeleton, ribosomes made in nucleolus, endomembranes (e.g. ER,
golgi body, mitochondria (generates ATP)), fungi and plants have cell wall, central vacuole (turgor pressure, since no
cytoskeleton), and choloroplasts
Eukaryotes appeared 1B years after prokaryotes. Ancestral anaerobic eukaryote engulfed bacterium/prokaryote
through phagocytosis and the bacterium provided host w/ ATP or photosynthesis ability
-Endosymbion theory - Evidence: Mitochondria/chloroplasts have their own genome, looks very
prokaryotic. Housekeeping machinery, they have their own transport systems and synthesis machinery. Chloroplasts
and mitochondria have a double membrane (another membrane was put around the organelle during engulfment).
Model organisms rapid development w/ short life cycles, small adult size, readily available, easily manipulated,
understandable genetics
DNA to RNA = transcription. RNA to Protein = translation
Central Dogma DNA to tRNA for transferring proteins. DNA to rRNA, part of the ribosome (structural), catalyzes
synthetic reactions
Elaborated Dogma Genome, transcriptome, and proteome. Genome cells complete DNA sequence,
transcriptome All the RNA in a cell/tissue at a single point in time, proteome all the proteins in a cell/tissue at a
single point in time. Interactome set of protein-protein interactions in a cell/tissue at a single point in time (1000s
happening at a point in time) Metabolome collection of small molecules in a cell/tissue at a single point in time.
Phenome Just the phenotype, or interaction of phenotype with genetic & environmental factors
DNA, RNA, and all proteins are linear chains of info, having polarity/directionality. Info in nucleic acid sequence is
translated to AA sequence via a genetic code which is essentially universal for all species.
Genetic code 64 possible codes, but only 20 AA because the code is degenerate/redundant. Consequences can
possibly synthesize a new AA, all 20 AA are encoded by DNA, base pairing between codon and anticodon occurs
more easily, random mutations are reduced
Nucleic acids genetic material in a cell (the blueprints), both RNA and DNA are chains of polymers of repeated
subunits (usually negatively charged, as a result called acids)
3 parts: Pentose sugar (scaffold for the base), Nitrogenous base (varies, gives it its identity), Phosphate group (can
be 1, 2, or 3)
Bases Purines (double ringed A & G, 5C), Pyrimidines (single ring C, G & T, 6C). T differs from U by a
methyl group. * Pure things are good, U C The pyramids
Differences between DNA/RNA DNA: Deoxyribose vs ribose sugar. (OH on 2 C for ribose, H on 2 C for
deoxyribose) RNA: uracil vs thymine (thymine has methyl group, uracil doesnt)
Nucleic acid nomenclature Nucleoside when base attached to sugar (e.g. adenosine, guanosine), then AMP, ADP,
or ATP. *if H on 2 C, then deoxyadenosine, etc. Nucleoside monophosphate = sugar + base + 1P, etc.
* Nucleoside = sugar + base, nucleotide = sugar+base+phosphate grps
W2
Molecular interactions: electrostatic attractions, hydrogen bonds (strongest in a straight line), van der Waals
attractions (important for large macromolecules random dispersion for flickering dipoles), hydrophobic force
Nucleotides are linked by phosphodiester (covalent, quite strong) bonds. Free phosphate end is the 5 end
Base pairing: A-T: 2 hydrogen bonds (bonds straight across) G-C: 3 H bonds. Aka complementary base pairs

3 Forces keeping DNA together: van der Waals forces, hydrogen bonding, hydrophobic interactions (sugar backbone
is negatively charged, hydrophilic), base stacking, base are hydrophobic and pushed into the centre. .34nm each
section
DNA double helix has a minor groove & major groove. (for proteins to read helix @ major groove, more accessible)
Review: strands in double helix are complementary and antiparallel, 5 = phosphate group 3 = hydroxyl group
Denaturation to single strands from high temp or pH, important for DNA synthesis
Protein structure: Primary (sequence) e.g. AA sequence -> Secondary (local folding) e.g. alpha helix & beta sheet ->
Tertiary (long-range folding) e.g. 3D structure -> Quaternary (multimeric organization) e.g. when more than 1
polypeptide -> Supramolecular (large scale assembly) e.g. ribosome
Protein are composed of AAs, the AA side-chain/R group is variable, determining the type of AA, 4 categories: basic
e.g. histidine (positive charge @ neutral pH), acidic e.g. aspartic acid (negative charge @ neutral pH), polar
(Threinine), or non-polar (phenoalonine),
Cysteine when oxidized, created disulphide bonds/bridges, cell can control when these are made/broken (regulate
for protein function). E.g. in plants, reduced state is favoured
AAs that are similar to each other are grouped together. (so mutations may not result in a deleterious affect)
- xUy nonpolar, Arginine lysine histidine are basic,
* Proline introduce kinks, unable to adopt normal helical conformation, found in tight turns, unable to occupy many
conformations. **Big bulky ring
Methionine Aug start codon
Review: Codons with similar properties tend to have less mutational steps between them, one random mutation in a
codon is less likely to result in a dramatic change in AAs than 2 random changes
Synthesis of proteins: condensation rxn (creation of water after joining of 2 AAs), creating a peptide bond, carboxy
end of one AA join to the amino end of the other. Therefore it has a N(amino) term and C(carboxy) term. * R groups
are not involved in the making of peptide bonds (order of AAs is important, reversed order is different)
Alpha helix: .54nm, 3.6 amino acids/turn. Hydrogen bond between AAs 4 peptides apart. Single stranded, R groups
stick out, not in, made of proteins.
Beta (pleaded) Sheet: 0.7 nm, beta strands, antiparallel (most common, can be parallel), hydrogen bonding between
strands, curving around back and forth
H-Bonding in secondary structures: Carbonyl and hydrogen in amino group are H-Bonded
- Alpha helics: hydrogen bonding 4 AAs apart in the SAME strand, beta sheet: hydrogen bonding between
different strands (can be same chain)
Coiled coil: regular repetition in the linear chain, hydrophobic stripe, amphipathic (has hydrophobic and hydrophilic
side), multiple ones can group together to become a coil, the hydrophobic ones coiling together inside the coil.
Tertiary structure: e.g. Rhodopsin, held together by hydrophobic interations, non-covalent bonds, covalent
disulphide bonds. (3D structure can change dependent on circumstances)
Domains separate level of organization, domains are specialized for different functions, can independently fold,
and will have an independent function. * Kinase domain activates the protein
Quarternary structure: e.g. Hemoglobin, Sickle cell anemia conformational change in Beta subunits, each subunit a
separate polypeptide chain, different subunits can be of different secondary structure

Hemoglobin function transfers oxygen from lungs to tissues, heterozygous for this mutation are partially protected
against malaria, frequency highest levels in Afria/India (malaria prevalent), related molecule, myoglobin, has only 1
subunit found in muscle tissue
Large Scale protein assemblies: Dimers, domains group together and combine to create a shell.
*Domains: distinct unit of organization, can independently fold into stable and compact structures, usually about 40300 AAs long, usually associated w/ a specific function, involved in domain shuffling
Genomes & Chromosomes: 3 billion bps/genome, 25k genes spread across 3 chromosomes, XX = female, XY =
male. Genome size is not correlated with organism size or complexity
W3
50% of genome is repetitive DNA, 25% codes for genes, including genes (part of genes, but do not code for
protein), protein-coding is roughly 1.5%, some are promoters. Repeated sequences contain transposons (45%):
segments of DNA that are good at replicating themselves but have no particular function (LINEs and SINEs,
retroviral like elements which are from viruses that became apart of human genomes). Amount of transposons vary
between organisms.
Packaging of DNA in cell: in Prokaryotes, condensed through folding about 1000 fold, bound together to form the
nucleoid: components include positively charged polyamines, numerous nucleoid-associated proteins (NAPs),
supercoiling by the enzyme topoisomerase
Eukaryotes: 6 billion base pairs per cell (2m per cell), located in the nucleus which is about 6 micrometer in
diameter, number of cells in human body: 10^13, total length of DNA is 2x10^9 km
Genome is packaged into chromosomes (shown as a karyotype), using FISH (Fluorescence In Situ Hybridization)
Humans have 23 pairs of chromosomes, each contains a single, long linear DNA molecule & associated proteins
called chromatin.
- Chromatin is tightly packaged, but accessible (dynamic)
Nucleosomes: basic structural unit (beads on a string structure), linked by linker DNA
- Histones: small proteins rich in lysine and arginine, positive charge neutralizes negative charge of DNA,
4 core histone proteins, pair of each in octamer core (4 different proteins, dimers), one linker histone. * each histone
has a tail
H1 histone helps binds the DNA coming in and out of the spool, binding it together at a better angle for coiling into
a small space. *30nm chromatin fibre of packed nucleosomes. Net result has mitotic chromosome packaged 10000
times shorther than its extended length

W4
All parts grouped together, efficient because both polymerase is near the replication fork.
Review: Leading strand is synthesized continuously from a single RNA primer, Lagging strand is synthesized
discontinuously from multiple primers, Okazaki fragments: RNA primer + newly replicated DNA, synthesis
proceeds in 5 to 3 direction, Primosome: helicase + primase, Helicase can be present on both strands, but dominant
on the lagging strand.
PCR (Polymerase Chain Reaction): 1. Heat to separate strands (92-94), 2. Designed primers complementary to
amplified region, added in great excess, slowly cool (50), 3. Add DNA polymerase (taq), DNTPs, warm to (75-77)
for DNA synthesis, then repeat.
Issues in DNA replication:
What happens @ ends of eukaryotic linear chromosomes during replication?
- For lagging strand the primer will eventually run out of the template, and will end w/ loss of info

- problem solved by having repetitive DNA sequences at the ends of the chromosomes, called telomeres
- repetitive sequence added at 3 end determined by RNA template in telomerase (T loops is tucked in)
Telomere Replication: Telomere uses RNA template to add more nucleotides to the 3 end, then DNA polymerase
alpha comes in and complementary pairs.
- Uses RNA template, resembles a reverse transcriptase, generates G rich ends, adds nucleotides to the 3;
end of the parental template
Telomere length control: Experiments show that cells can regulate the telomere length. Cell can use telomere length
to regulate the life of a cell.
Cell can decide to give itself x # of replications before it is done. E.g. bone marrow stem cells have extensive
telomere length (expected to be around for a long time and replicates for a long time) while some somatic cells
arent expected to be around forever (replicative senescence, over time lose capacity to divide, short telomere
length)
* Telomere will shorten over time, cell can recognize short telomere length as damaged cell, goes through apoptosis
(suicide)
Telomeres & Cancer: Cancer cells have extremely long telomeres, no programmed death, most cancer cells produce
a high level of telomerase (normally telomerase activity decreases over time), modifying RNA template of
telomerase interferes cancer growth, prognosis of some cancer can be done by looking at telomerase levels in
tumour, interfering w/ telomerase activity and slow tumour growth
How is DNA unwound? Unwinding/replication can create supercoiling at the other end (torsional stress). Stress
released by topoisomerase type I, Type of break: single stranded, allows DNA to unwind around the broken strand
w/o stress. Spontaneous reformation of phosphodiester bond regenerates the DNA helix and DNA topoisomerase
Topoisomerase type II (used mostly in prokaryotes w/ circular DNA), untangles and separates. Forms a protein gate
to allow one ring to pass. ATP is required Type of break: double stranded, allows DNA to pass throw
How are mistakes found and corrected? RNA polymerases error rate of 1 in 10^4, DNA polymerase is 1 in 10^9
Human genome changed only about 3 nucleotides every time a cell divides. How?
- Exonuclease & strand directed mismatch repair
- Proofreading exonuclease (3 to 5): while new DNA strand is adding nucleotides, it can go back and
repair a wrong one. If error, hydrogen bonding flips the strand, blocking polymerase triggering exonuclease acitivity,
chews back to create new proper nucleotide. (wrong nucleotide flips template strand into editing domain)
Why 5 to 3? Prolly because of exonuclease activity. Once cell realized mistake it made, it needs to clip off the
nucleotide. If 5 to 3, then energy efficient and from incoming phosphate, but not enough energy if from 3 to 5.
Only way to have correction and chain growth
Strand directed mismatch repair: in eukaryotes, nicks in okazaki fragments have determine which strand is
new to fix. Prokaryotes methylate DNA, look for GATCs, and methylate the A, knowing new strand by which is
methylated. Mut enzymes looks for deformation in helix and make a nick in prokaryotes. MutS looks for and binds
@ deformation, MutL looks for nicks, clip out all the way back to the misincorporated nucleotide, then gap is filled
in w/ DNA polymerase and ligase
Damaged DNA: defects in repair mechanisms have been linked to variety of human diseases, breast colon and skin
cancers (important because these cells are dividing quickly)
DNA can be damaged by: oxidation, radiation (UV, xray, gamma), heat, chemicals, and cell stressors, depurination
(base is removed), deanimation (change cytosine to uracil)
How are they produced: If not corrected, will be found in daughter cells, A will replace G because U & A pairs
nicely, if repurianted, it will be skipped over.
Base excision repair: targets one nucleotide, looks for broken ring structures, deanimated structures, any problems
and deformations. If found, the glycosylase removes the base, and endonuclease and phosphodieasterase remove the
sugar phosphate

Nucleotide excision repair: many nucleotides, excision nuclease comes in to cut out chunk, then DNa polymerase
and ligase to fill in and stitch DNA
*Transcription coupled repair
Transcription (eubacteria): Gene definition: The entire nucleic acid sequence (usually DNA) that is necessary for the
synthesis of a protein and its variants or RNA. In other words, genes are segments of DNA that are transcribed into
RNA. Two types of genes that when transcribed: the resulting RNA codes for protein or resulting RNA stays as
RNA
Remember transcription is highly regulated. RNA polymerase (RNAP) (5 to 3)has rNTP & rNTP triphosphate
uptake channel, NO PRIMER, only needs template, has DNA/DNA duplex.
Generation of RNA transcript: Template: single stranded DNA, RNA linked by phosphodiester bonds, DNA-RNA
hybrid held together by base pairing (happens inside RNAP), has exonuclease repairing ability
W5
Transcription cycle: sigma factor protein lets RNAP know where to start. Sigma factor binds to RNAP (whole thing
called holoenzyme). Different sigma factors recognize different promoters (where to start). Holoenzyme slightly
unwinds DNA to start transcription. First few steps will be failures/repeats that are rough. Once it gets going the
factor dissociates and transcription gets going.
C-G rich regions make hairpin ship, A-T rich regions are not as strong. The combination pulls the RNA out of the
RNAP.. Steps: Initial RNA synthesis (many abortive attempts), factor releases, very rapid RNA elongation, RNAP
hits termination sequences creating loop, then hits AT rich areas , row independent.
Key points: Initial steps of RNA synthesis are relatively inefficient, quite different from elongation mode, which is
very highly processive, Termination characteristics is hairpin from GC rich regions followed by AT rich regions
weakening the duplex sites
Direction of transcription: Determined by the way RNAP is facing and orientation of the promoter. (Polymerase
itself moving 3 to 5, making the RNA 5 to 3.
12 subunits in eukaryotes RNAP, 5 in prokaryotes.
snRNA help with splicing, processing other RNA, miRNAs: microRNAs, regulate gene expression by blocking
translation, siRNA: small interfering RNA, turn off gene expression by degradation of mRNA (hydrogen bond w/
target RNA. Cell will then degrade the double strand)
Eukaryotes have 3 RNAPs (I II & III)
W6
Translation: nuclear exosome looks for unwanted RNA and chews them up.
tRNA: 2 single strand areas (3 end and anticodon). Rest is double stranded e.g. D/T loop
10% nucleotide go under conformation.
* I is post transcriptional modification of Adenine
Small ribosomal unit provides structure for incoming mRNA. Large subunit includes peptidyl activity (peptidyl
transferase)
EF1/EF2 in eukaryotes, EFG and EFTY in prokaryotes. Elongation factors
3 places to proofread: EFTU escorts tRNA to ribosome and checks for the correct AA. Checking whether hydrogen
bonding can happen between ancicodon and mRNA (bent tRNA). If correct, GTP dissociates phophosate to release
EFTU. EFG improves processivity of the small ribosome (its slow and limiting factor). Hydrolysis of GTP on EFG
helps ribosome move faster to catch up to large ribosomal subunit. If correct, large ribosomal subunit hydrolizes
GTP, changing conformation of EfTU to leave and making tRNA not bent.
Small subunit rRNA forms hydrogen bond with anticodon-codon.
Role of elongation factors: Can perform synthesis w/o EFs, but very slow. Role: Improving speed & efficiency, and
error checking. Mediated by release of EFTU and EFG (associated with GTP hydrolysis). EFTU binds to tRNA
and EFG improves processivity).

Ribosome is a ribozyme (RNA that has ability to catalyze some reaction)

Initiation of translation (Euk): initiation factor(elF2) binds to GTP & initiation tRNA, elF4E looks for 5 cap, ElF4G
looks for poly A binding proteins. Its how the cell checks for a legit mRNA.
mRNA binds @ small ribosome subunit and looks for starting initiation AUG. (Consensus sequence ACC AUG G).
ATP hydrolysis for helicase to unwind secondary mRNA structure. Phosphorylation for large ribosomal subunit.
10% of the time the first AUG sequence is skipped. (Leaky initation)
Initiation of translation (prok): Shine-dargarno sequences (AGG AGGU)
Termination: Binding of release factor to A site followed by termination. Water attacks the peptidyl bond. Ribosome
shifts over, senses its over, dissociates, and ends.
Human translation release factor: a protein and similar charge association. (molecular mimicry)
Polysomes/polyribosomes
Protein folding: Protein folding right away as it leaves the ribosome. Most cannot completely fold until completely
out of the ribosome. Proteins need help folding. Molten globule (protein beginning to fold). Chaperones catalysis
help protein fold into the desired configuration (if not folded properly, will be sent to protease to be degraded). E.g.
of molecular chaperones are HSP60, heat shock protein 60/70.