Chapter 1 - Introduction to the cell and genomes

DNA is the basic heredity material

-Consists of 4 nucleotides: A , T, C, G
-A binds to T, C binds to G
-Nucleotide contains a sugar, with a phosphate group attached to it
-Nucleotides linked by the phosphate group
-this gives DNA a double stranded, double helixed strand
DNA is copied by templated polymerization

DNA expresses itself:
-Process begins with transcription: DNA segments used as templates for synthesis
-Translation creates the proteins


RNA:
-U is used in place of T
-Slightly different sugar (only ribose)
-DNA used repeatedly, RNA is disposable
-mRNA are the transcripts
-The folding of RNA due to complimentary base sequences within a strand can cause a shape that could
catalyze certain enzymes

All cells use proteins as Catalysts
-Proteins are polymers of amino acids.
-there are 20 amino acids
-Proteins act as enzymes.

All cells translate RNA the same way:
-Codon consists of three nucleotides
-There are 64 (4x4x4) codons, but only 20 amino acids
-tRNAs read the codons from the mRNA, they have a complimentary anti-codon
-This process of reading occurs on ribosome made of 2 strangs of rRNAs

One gene = One protein
-DNA can be transcribed in more than one way
A gene is KA:the segment of DNA sequences corresponding to a single protein or set of alternative
protein variants
-Genes are expressed at a regulated rate by stretches of regulatory DNA
Other DNA could be.. Punctuation for when a protein begins/ends

The genome of a cell is the total of its genetic information as embodied in its complete DNA sequence
dictates the cells proteins and when/where they are made

Life requires free energy
-A cell must take in energu and raw materials from environment
-Free energy is required for the propagation of information
-Free energy is needed to ensure the correct bonds are made
All cells funcition as biochemical factories with the same basic molecular building blocks
eg: ATP is used as a building block for DNA and RNA
Every cell is enclosed by a plasma membrane
-acts as a selective barrier
-It is an amphiphilic bilayer: Has both a hydrophilic(exposed in the bi-layer) and hydrophobic(hidden in the
bilayer) parts to it
-Hydrophobic tails are usually (CH2)-
n
polymers
This amphiphilicity follows the following important property of cells:
Cells produce molecules whose chemical properties cause them to self assemble into structures
that the cell needs
Since cells still need to transport specific molecules across the membrane, there are membrane transport
proteins.
The Mycoplasma genitalium has smallest known genome of about 480 genes
-580,070 nucleotide pairs, representing approximately 145,018 bytes of information.
-there are 60 genes in the core set shared by all living species without any known exception

The diversity of genomes and the tree of life
Microorganisms make up most of the mass of living matter on our planet
Cells can be powered by a variety of free energy sources
-animals, fungi and some bacteria get energy by feeding on other living things or the organic chemicals
they produce these are known as organotrophic
-Two classes of bacteria that derive energy from abiotic world:
1.Phototrophic organisms that rely on sunlight
-Oxygen on the earths atmosphere is a byproduct of the activities of these organisms
2.Litotrophic organisms that rely on energy-rich systems of inorganic chemicals
-microscopic, usually live in inhospitable envs like earths crust
- There are two lithotrophs: Aerobic (using oxygen) and Anearobic (no oxygen)
-Can be found in hydrothermal vents: downward percolating water is driven up
-These contain a bunch of chemicals incloding H2S H2 CO, etc which microorganisms
feed off
Organotrophs could not exist with either of these two primary energy converters (most abundant)
Some cells fix Nitrogen and Carbon Dioxide for others
-DNA, RNA and proteins are composed of six elements:
-H,C, N, O, S, and P
-Atmostpheric N2 and CO2 Are very unreactive and thus require a lot of energy
-Many living cells lack ability to fixate carbon and thus, organisms like us depends on plants for out
Nitrogen and CO2
-Plants in turn need bacteria to fixate nitrogen for them
The Greatest biochemical diversity exists among prokaryotic cells
There are two basic groups of cells:
Eukaryotes
-Membrane enclosed DNA

Prokaryotes
-no membrane enclosed enclosed DNA
-Usually small and simple, and has a cell wall, few micrometers in linear dimension
-fit many ecological niches, organotrophs eat.. anything, phototrophs harvest light in many ways
-aproximately 99% of prokaryotic species remain uncharacterized
The tree of life has three primary branches: Bacteria, Archae, and eucaryotes
-since DNA changes over time, a direct objective quantitative indication of the evolutionary distance
between them can be determined
-both bacteria and archaebacteria are two branches of prokaryotes.
Archaebacteria:
-found in environments we avoid (sewage, ocean depths, acid)
-handle energy like bacteria, handle Cjamges die DNA like eucaryotes

Some genes evolve rapidly; others are highly conserved
-Mutations are random exxcidents in the storage and copying of genetic info
-rarely, mutations are good, usually the error will cause damage
-There may also be selectively neutral changes that may be perpetuated or not
-Through natural selection and mutation, species evolve
-When important segments of DNA (parts that code for proteins) face a deleterious mutation, the faulty
cells are almost always eliminated; these proteins are known as highly conserved

Most bacteria and archae have 1000-6000 genes
-Small size means large SA:V ratio and thus maximizes uptake of nutrients
-The small pacxked genes of prokaryotic genes makes it easy to sequence them

New genes are generated from preexisting genes
-There is no natural mechanism for making long stretches of new random sequence
-Thus there is no new genes, some form of innovating genes:
1. Intragenic mutation: an existing gene can be modified by changes in its DNA through errors
2. Gene duplication: an existing gene is duplicated, these two may diverge by evolution
3.Segment shuffling: two or more existing genes are broken and rejoined making hybrids
4. Horizontal (intercellular) transfer: a piece of DNA can be transferred from the genome of one
cell to another and at times even to other species vertical transfer is from parents to progeny

Gene duplications give rise to families of related genes within a cell
-Sometimes accidents result in the duplication of just a part of the genome with retention of original and
duplicate segments in a single cell one of these copies may mutate to give rise to a different purpose
-this may give rise to a family of genes within a genome
-There are also sister genes that arise from evolution after speciation (example chimp and human
evolved DNA of a certain protein)
-Orthologs are the genes that are derived from the same ancestral gene in separate species
-paralogs are genes from duplication within a genome
-homologs are genes related by either way of descent

Procaryotes can be transferred between organisms both in the laboratory and in nature
-Viruses are not living cells but can act as vectors for gene transfer
-They replicate in one cell emery from it with protextion and infect other cells and use them
-Most of the times the virus kill, but when it sticks around, it can either stay as a separate intracellular
fragment of DNA known as a plasmid or in the genome of the host
-sometimes they take some host dna with them and may insert them (horizontal) into other cells
-More horizontal transfer occurs by just take up of theDNA from their surroundings
-horizontal gene transfer is responsible for penicillin resistance being spread around

Sex results in horizontal exchanges of genetic info within a species
-sex results in horizontal transfer of genetic info from two separate lineages (mother and father)
-sex results in offsprings being slightly related to one parent more than the other
-sex is more common in eukaryotes
The function of a gene can often be deduced from its sequence by comparing it to a database of genes
with similar sequences

More than 2000 gene families are common to all three primary branches of the tree of life
-It is hard to look at ancestral genes because they could have:
-moved around by horizontal transfer
-been lost by some species
-evolved past a recognizable point
-out of 4873 protein coding gene families, only 63 are ubiquitous in all three branches
-Most of these genes are components of translation and transcription
-There are however 264 ancient conserved gene families that are constant in the three families

Mutations reveal the functions of Genes
-Sequence comparisons show that there are subregions of a gene that are unchanged over evolution ,
probably the most important function wise

Molecular biologists have focused a spotlight on E. Coli
-A lot of biologists are dedicated to studying different aspects of the same model organisms
-E Coli is populatr ATM between many scientists
-Genetic mechanisms are seen both in us and E. coli. and are thus highly conserved evoluationary

Genetic information in eucaryotes
Eucaryotic cells may have originated as predators
-Keep DNA in the nuclear envelope, usually 10 times linearly larger, 1000 times larger in volume
-have a cytoskeleton (system of protein filaments crisscrossing the cytoplasm and forming together that
allows mechanical strength and controls its shape and drives and guides its movements)
-Possible theory is that eucaryotes arose by a cell that engulfed others after developing phagocytosis and
lived by eating other cells

Modern eukaryotic cells evolved from a symbiosis
-Predation also explains why all cells contain mitochondria
-mitochondria same size as bacteria and have their own DNA; receives shelter and nourishment in return
for the power generation
-Plants and algae also contain chloroplasts that perform photosynthesis (also have their own genome)
-Plants lost ability to move around and catch prey, instead they made a shield for themselves (cell wall)

Eucaryotes have hybrid genomes
-mitocondrial and chloroplast DNA are cut down versions of the corresponding bacterial genomes and
lack genes for many essential functions
Eucarytotic genes are big
-eucaryotes have vastly more DNA that does not code for protein or any other functional product
molecule

Eucaryotic genes are rich in regulatory DNA
-These regulate the expression of adjacent genes
-control when and where a gene is brought into play

The genome definges the program of multicellular development
-Different cues they get from their surroundings in the embryo results in the way in which cells make
selective use of their genetic instructions
-The genome in each cell is big enough to accommodate the info that speciefies an entire multicellular
organisms but any indiv cell only part of that info is used
-A large fraction of genes code for gene regulatory proteins which bind to the regulatory DNA adjacent to
genes that are being controlled

Many eukaryotes Live as solitary cells: The protists
-single celled eukaryotes
-protists are much more diverse than the other three major branches (animals plants and fungi

A yeast seves as a minimal model eukaryote
-Saccharomyces cerevisiae is used as a model unicellular yeast
-It can reproduce by cell division or sexually
-it has a small genome

The expression levels of all the genes of an organism can be monitored simultaneously

Arabidopsis has been chosen out of 300 k species as a model plant
-The different species of flowering palnts separated by about 150 million years
-Arabidopsis thaliana chosen as model, produces 1000s of offspring after 8-10 weeks has a 140 million
nucleotide pair genome and all of it is known

The world of animal cells is represented by a worm, a fly, a mouse, and a human
-C elegans is the model worm made of 959 body cells
-genome of 97 million nucleotide pairs codes for 19000 proteins

Studies in Drosophila provide a key to vertebrate development
-Has giant chromosomes that are visible in some of its cells
-Drosophila takes only 9 days to go from fertilized egg to adult
-Cheap and easy to breed

The vertebrate genome is a product of repeated duplication
-frogs from genus Xenopus hav e multiple duplications or triplications of the whole genome

Genetic redundancy is a problem for geneticists but it creates opportunities for evolving organisms
-genetic redundancy is where a biochemical function is encoded by more than 1 gene

The mouse serves as a model for mammals
-comparing differences of a certain protein sequence: human - elephant = 85%
-HUman bird about 70%


Chapter 2
Pages 51-55

An atom often behaves as if it has a fixed radius
-Space filling models show an accurate representation (big looking balls)
-van deer waals say that as atoms get closer to each other, repulsion increases steeply, while at further
distances, might slightly attract each other
-this results in a distance which repulsive and attractive forces balance and produce energy min

Water is the most abundant substance in cells
-accounts for 70% of a cells weight
-life hinges on the properties of water
-The two H2O bonds are highly polar because of Os EN
-unqeual distribution of e-s towards the O
-Hydrogen bonds result in linking water molecules and making it a liquid at room temperature
-other polar molecules are hydrophillic
-non polar molecules are hydrophobic
-hydrocarbons abused in cells for being hydrophobic

Some polar molecules are acids and bases
-Hydronium (H3O) generated when a hydrogen has no electrons (thiefed by its fellow elements) and
reacts with a nearby water molecule (H2O + H2O H3O+ + OH-
-Substances taht release protopns and form H3O in water are acids
-Interior of a cell is kept close to neutrall 7 ph by buffers
-A base accepts protons and raises the amount of hydroxide (OH-)

Four types of noncovalent attractions help bring molecules together in cells
-covalent bonds 10-100 times stronger than other attractive forces in aqueous solutions
Here are the other types of important attractions:
-Eelctrostatic attractions
-Oppositely charged atoms dipole attractions(ionic bonds is the greastest form)
-H-bonds
-Hydrogen bonding with something strongly electronegative
-van der waals attracions
-electron cloud flickers resulting in momentary dipoles
-hydrophobic force is not necessarily a bond but it pushes nonpolar surfaces out of H bond water network

A cell is formed from carbon compounds
-carbon is good because 4 bonds bla bla bla
-combinations with functional groups occur repeatedly in organic molecules

Cells contain four major fmailies of small organic molecules
-Monomers are used to construct giant macromolecules

page 59-64
Amino acids are the subunits of proteins
-Amino acids used to make proteins by forming peptide bonds and forming long chains known as
polypeptides
-we dont know why these are the chosen 20 of lyfee
-they exist as optical isomers known as D and L, only L forms found in proteins

Nucleotides are the subunits of DNA and RNA
-nucleotide has a nitroden base, 5 carbon sugar and a phosphate group
-Bases: pyrimidines: Cytosine, Thymine, and Uracil
purines: guanine, adenine
-Nucleotideds can act as short term carriers of chemical energy, particularly ATP ribonucleotide
-three phosphates in ATP linked by two phosphoanhydride bonds
-Nucleotides are the subunits of nucleic acids, linked by phosphodiester bonds








***DNA AND RNA
ARE LINEAR








*Thymine not thymidine
The chemistry of cells is dominated by macromolecules with remarkable properties
-Proteins serve as enzymes
-others useed to build structural components
-each polymer grows by addition of a monomer to a polymer chain by a condensation reaction ( uses a
molecule of water for each monomer added

Noncovalent bonds specify both the precise shape of a macromolecule and its binding to other molecules
-macromolecules have different conformations
-given enough covalent bonds, many proteins and most RNA molecules will hold on tightly to a certain
conformation

Chapter 3 (pages 125-152)
The shape and structure of a protein

The shape of a protein is specified by its amino acid sequence
-There are 20 amino acids in proteins, each with different chemical properties
-a protein is made of a long chain of AAs and is AKA polypeptide
-There is a repeating sequence of atoms along the core of the polypeptide known as polypeptide
backbone, the side chains, give an AA its properties
-amino acid sequence is always read in N to C direction
-this long polypeptide backbone and side chains fold as a
result of the three noncovalent bonds
-One by itself is weak but many bonds acting in parallel
can hold two regions of a polypetide together
-Hydrophobic are forced together in aqueous
environment in order to minimize their disruptive effect on
the H bond network of water
-Thus the hydrophobic(red) parts fold in while the
hydrophillic (green) parts fold out



Proteins fold into a conformation of lowest energy
-The final folded structure( conformation) of a polypeptide is one that minimizes its free energy
-unfolding of the protein is known as denaturing
-when denaturing solvent is removed, protein refolds (renatures) into its original confimration
-protein changes slightly when it interacts with other moleules in the cell, crucial for function of protein
-Molecular chaperones often assist in protein folding by binding to a partly folded polypeptide and help
them progress along the most energetically favorable folding
-proteins usually between 50-2000 AAs long that contain several protein domains (structural units that
fold independently of each other

The alpha helix and beta sheet are common folding patterns
-both of these are common because they result from H bonds between the NH and C=O groups in the
polypeptide backbone
-There are two types of beta sheets.. The one shown above where there are two chains fgoing in
opposite structure (antiparallel chain) and ones that run in the same orientation (parallel chains)
-The alpha helix is generated when a single polypeptide folds and forms a rigid cylinder using an hbond
between every fourth peptide bond and the C=O to the N-H of another
http://content.answcdn.com/main/content/img/oxford/Oxford_Chemistry/0192801015.alpha-helix.1.jpg
-alpha helixes wrap around each other known as coiled coil

-This happens when the alpha helices have most of their non-polar side chains on one side, so that they
can twist around each other with these particular side chains facing inwards, towards each other

Protein domains are modular units from which larger proteins are built
There are four levels of organization in the structure of a protein:
Primary structure:
-the amino acid sequence
Secondary structure:
-the alpha helices and beta sheets that are formed
Tertiary structure
-the full 3-D organization of a polypeptide chain
Quaternary structure
-a protein as a result of one or more polypeptide chains
-There is also the protein domain which is a substructure produced by a part of a polypeptide chain and
can fold independently into a compact stable structure (usually between 40 and 350 amino acids)

Few of the many possible polypeptide chains will be useful to cells
-there are 20^n possible different polypeptide chains that are n AAs long.
-only a small fraction of these would adopt a single stable 3-D conformation (about less than 1 in a billion)
-natural seledction eliminated naughty proteins

Proteins can be classified into many families
-THere are families as a result of duplicate genes that evolve independently with similiar amino acid
sequences and 3-D conformations
-in general, the structure of members within a family is more conserved than the amino acid sequence
-Many examples show that two proteins with more than 25% identity in their amino acid sequences
usually share the same overall structure
-there is a limited amount of ways (estimated 2000) that aprotein domain can fold up

Sequence searches can identify close relatives
-techniology makes it easier to see similiarities in genes
-30% identity in sequence is required to consider two proteins a match
-fingerprint sequences with certain functions are known and used to find more distant relationships

Some protein domains form parts of many different proteins
-In domain shuffling, many large proteins have evolved through the joining of pre existing domains in new
combinations and thus creating new genes
-protein modules are subsets of protein domains that have been especially mobile during evolution
-each domain has stable core structure formed from strands of B sheet
-loops form binding sites for other molecules
-can be easily integrated into other proteins (have n and c terminals at opposite ends)
-bc of this they can be linked with other in line domains

Certain pairs of domains are found together in many proteins
-more than 60% of proteins have two or more domains
-same pairs of domain occur repeatedly in the same relative arrangement
-half of domain families are common in all organisms
-5% of 2 domain combos are similarly shared

The human genome encodes a complex set of proteins, much is unknown
-we have a measly 25k genes same as some worms
-most of our proteins on avg more complex, twice as long, and many more domain combinations
-we dont know what 10k of our genes do

Larger protein molecules often contain more than one polypeptide chain
-any region on a proteins surface that can interact with another molecule is called a binding site
-binding sites rom diff proteins can come in contact with each other to form larger proteinsm each protein
becomes a subunit


Some proteins form from long helical filaments
-helixes form with repeating subunits because it minimizes free energy
-these repeating subunits are usually connected the same way to each other

Many proteins have elongated fibruous shapes
-so far weve discussed globular proteins (most have rounded shape)
-fibruous proteins are long and simple proteins eg: keratin
-these can coil to make intermediate fillings
-fibruous proteins are abundant out of cell, make up the gel-like extra-cellular matrix
-the most common extracellular protein is collagen

Many proteins have large amount of unstructured polypeptide chain
-this unstructuredness allows elasticity of fibers to stretch and recoil without tearing
-contains hydrophobic and very few bulky AAs
-composed highly of Gln, Ser, Pro, Glu, Cys

Covalent cross-linkages often stabilize extracellular proteins
-cross linkages provide stability: most common is disulfide bridges
-These do not change conformation, instead they only stabilize the polypeptide

Protein molecules often serve as subunits for the Assembly of large structures
-large objects like enzymes and filaments are made from non covalent assembly of several separately
manufactured molecules
Advantages:
-requires less genetic info
-assembly/disassembly readily controlled, multiple low energy bonds
-more correction mechanisms needed when there are more steps
-rings/tubes and spheres provide additional stability

Many structures are capable of self assembly




Page 202-218 (excluding 207)
Eucaryotic DNA is packaged into a set of chromosomes
-The human genome (3.2x10^9) nucleotides, is distributed over 24 different chromosomes
-The complex of DNA and protein is called chromatin
-Bacterial chromosomes (usually 1) do not have the same structure as eukaryote chromosomes
-Each human cell contains two copies of each chromosome, one from mom and one from dad, together,
called homologous chromosomes (homologs)
-Only non homologs are XY and XX chromosomes
-DNA hybridization and colour bands are methods to distinguish the chromosomes
-Display of 46 human chromosomes at mitosis is called the human karyotype.

Chromosomes contain long strings of genes
-Humans have a lot of junk DNA that doesnt carry any information, but may help expression of other
genes
-There is no simple relationshup between chromosome number, species complexity and total genome
size

The nucleotide sequence of the human genome shows how our genes are arranged
-bla lba bla human genome project
-DNA is made up of short mobile pieces of DNA that have gradually inserted themselves in the
chromosome
-the average gene size is over 27000 BP, generally large
-only 1300 BP required to encode for an average human proteins
-The 27,000 includes introns (non coding) and exons (coding) segments of DNA
-There is also regulatory DNA sequences which ensure the turning on and off of the gene at a
proper time and proper level

Chromosomes exist in different states throughout the life of a cell
-The cell cycle is the series of stages that lead to its duplication
-during interphase, chromosomes are replicated
-during mitosis mitosis, they become highly condensed and are separated into the two daughter nuclei
-These highly condensed chromosomes are called mitotic chromosomes
-interphase chromosomes are extended and entangled in nucleus (unrecognizable)

Each DNA molecule that forms a linear chromosome must contain a centromere, two telomeres, and
replication origins
-Three sequences that help replicate and segregrate chromosomes in DNA
1.Replication origin:
-the location at which duplication of the DNA begins
-Eucaryotes have many replication origins to replicate the chromosome more rapidly
2.Centromere
-allows one copy of each duplicated and condensed chromosome to be pulled into each daughter
cell when it divides
-A kinetochore forms and attaches a chromosome to the mitotic spindle (to pull them apart)
3.Telomere
-repeated nucleotide sequences at the ends of chromosomes
-allows chromosomes tgo be efficiently replicated
-DNA forms structures that protect the end of a chromosome from being mistaken as a broken
DNA molecule


DNA molecules are highly condensed in chromosomes
-chromosome 2 (example), would be 1.5 cm if just a long straight double helix, but the chromosome is
about 2 micrometers meaning a 10,000 fold compaction ratio
-interphase chromosomes are still packed to about a 500 fold ratio
-specific regions decondense when needed to gain access to specific DNA sequences

Nucleosomes are a basic unit of eucaryotic chromosome structure
-Two classes of DNA-binding proteins: the histones and nonhistone chromosomal proteins
-Chromatin includes the complex of both clases of proteins with DNA

Histones:
-DNA wraps around histones
-contains a complex of 8 histone proteins 2x(H2A, H2B, H3 and H4)
-147 BP wrapped around a histone complex
-klnown as a histone
octamer
-linked by a segment
of DNA known as
linker DNA
-also known as a
nucleosome
-Nucleosomes repeat
about every 200 BP
(147 BP + linker
DNA)

The structure of the
nucleosome core
particle reveals how
DNA is packaged
-The high res
structure showed
that DNA was tightly
wrapped at 1.7 turns/
coil
-the four of the
histones that make up
core are small (102-135
AAs), and have similar
structure KA histone fold
-Form in this order:










-142 H bonds are formed B/w DNA and the histone core in each nucleosome (half by phosphodiester
backbone)
-1/5th of AAs of histones are either Lysine or Arginine (Basic) and thus neutralize negatively charged
DNA
-Each of the core histone also has an N-terminal AA tail
-The AA sequence of histone H4 from pea and cow only differ at 2/102 positions; changes = lethal

Nucleosomes have a dynamic structure and are frequently subjected to changes catalyzed by ATP-
dependent chromatin remodlling complexes
-DNA in an isolated nucleosome enwraps at a rate of 4 times/second (10-50 ms each time)
-ATP dependent chromatin remodeling complexes binds to protein and to DNA and, through repeated
ATP hydrolysis catalyzing nucelosome sliding, makes the nucleosomal DNA available to other proteins
(loosens the grip of the nucleosome), some complexes can even remove it completely
-Cells contain dozens of these complexes most have 10+ subunits
-Prescence of other tightly bound proteins on the DNA also infleunces nucleosome positioning
-some of these proteins favour a certain formation of a nucleosome adjacent to them, others create
obstacles that force nucleosomes to move to positions between them

Nucleosomes are usually packed together into a compact chromatin fiber
-nucleosomes rarely just look like beads on string, nucleosomes packed on top of one another
geneerating chromatin in a fiber form with a 30 nm diameter
-the structure of this is proposed to be a zigzag
http://www.nature.com/nrm/journal/v13/n7/images/nrm3382-f4.jpg
-nucleome tails (most strongly the H4 tail) help to tighly pack it into chromatn fiber
-An additional histone (H1, larger than the rest)
http://news.illinois.edu/WebsandThumbs/mizzen,craig/Nucleosome_b.jpg
-in charge of binding both DNA and proteins in the fiber

Heterochromatin is highly organized and unusually resistant to gene expression
-two types of chromatin in interphase nuclei:
Heterochromatin (highly condensed)
-most prevalent in locations near centromeres and telomeres
-aacounts for over 10% of in a mammalian cell of genome packaging
Euchromatin (less condensed)
-When a euchromatin gene is experimentally relocated into a geterochrmatin section, it ceases to be
expressed and is thus silenced
this is an example of a position effect( activitvty of gene depends on its position

239-240
Chromatin loops decondense when the genes within them are expressed

Cjhromatin can move to specific sites within the nucleus to alter gene expression
-each of the 46 interphase chromosomes in a human cell occupies its own discrete territory within the
nucleus
-a genes position in the nucleus changes when it becomes highly expressed




Chapter 5 (263-276)
Maintenance of DNA sequences
When a DNA maintenance processes fail resulting in a permanent change, it is known as a mutation

Mutation rates are extremely low
-Mutation rate is the rate at which observable changfes occur in DNA sequences
-e coli divides once every 40 minutes in lab conditions 1 cell can make a colony of a bil in a day
-Many mutations are silent, these are corrected when calculating :
-a single gene for an avg sized protein get a mutation about once in 10^6 bacterial generations (in ecoli)
-Stated diff: 1 nucleotide change/10^9 nucleotides every cell generation in bacteria
-In the progenitor worm, there atre on average two new mutations in the haploid genome each generation
and thus 1 per cell division
-differennce of AAs in a sequence of a gene in 2 diverged species can tell us the average number of
years required to produce a s ingle stable mutation in the gene
-hhowever many of these mutations will spol the function of the protein and vanish it from the population
-The sequence of fibrinopeptides has a very high AA mutation tolerance since all it does it form fibrin
during blood clotting used to calculate time required to change : approximately 1/400 AA of a protein
will change once every 200 000 years

Low mutation rates are necessary for life as we know it
-If not for mutation, there wouldnt be anything more complicated than a fruit fly
-two types of cells in a sexually reproducing organism:
Germ cells:
-transmit genetic info from parent to offspring
-Protected agaisnt high levels of mutation to protect offspring
Somatic cells
-protected agaisnt mutation to protect the indicidual
-when uncontrolled, results in cancer
-cancer due to accumulated changes in DNA sequence of somatic cells

DNA replication
-DNA templating is the mechanism that uses the copy of the nucleotide sequence of one DNA strand
(template strand) into a complementary DNA seuqence
-Done by polymerizing enzye, DNA polymerase
-Creates a replication fork : http://en.citizendium.org/images/f/f9/Bidirectionalrep2.jpg
-DNA polymerase only works in 5 to 3 direction
-http://www.youtube.com/watch?v=TEQMeP9GG6M explains leading and lagging strand

The high fidelity of DNA replication requires several proofreading mechanisms
-theres 1 error every 10^9 nucleotides copied
-rare tautomeric forms occur when, for example, C pairs with A instead of G
-DNA polymerase performs first prooofread before a new nucleotide is added
-The correct nucleotide has a higher affinity (more energetically favourable) for the moving polymerase
than does an incorrect nucleotide
-Double chjecking occurs when hand-like polymerase conformationally tightens its fingers around the
active site, this occurs more readily when the correct nucleotide is put it
-exonucleolytic proofreading takes place immedaitely after those instances
--DNA polymerase ONLY likes a 3 OH end of a primer strang to ADD nucleotides to
-It changes anythijng other than 3 OH using another catalytic site known as proofreading exonuclease
-its not possible for an enzyme to start over (de novo)
-RNA errors occur 1 in 10^4, a rate that is 100 000 times greater than DNA

Only DNA replication in the 5 to 3 direction allows efficient error correction
-Since the 5 chain end would terminate DNA synthesis, mistakes in polymerization could not be
hydrolyzed away in a theoretical 3 to 5 synthesis
-It is only possible to correct a mismateched base only on the 3 end of a DNA chain (figure 5-10 pg 271)

A special nucleotide-polymerizing enzyme synthesizes short RNA primer molecules on the lagging strand
-DNA primase uses ribonucleoside triphosphates to synthesize RNA primers on the lagging strand
-Primers are 10 nucleotides long, and made at intervals of 100-200 nucleotides on the lagging strand
-theres an OH at the 3 end where DNA polymerase can begin elongation
-The synthesis of each ookazaki fragment ends when this DNA polymerase runs into the 5 end of the
previous primer
-a DNA repair system erases the RNA primer and replaces it quickly with DNA
-DNA ligase joins the 3 end of the new DNA fragment to the 5 of the precious one to complete the
process

Special proteins help to open up the DNA double helix in front of replication fork
-DNA is pretty strongly bonded (H bonds)
-DNA helicase hydrolyzes ATP, this makes the protein cyclical and allows it to propel along DNA
unzipping at rates of up to 1000 nucleotide pairs per second
-Both types of helicases exist (5 to 3 and 3 to 5)
-Single-strand DNA-binding (SSB) proteins AKA helix destabilizing proteins, bind to exposed DNA strands
w/o covering bases (leaves open for templating)
-prevents DNA from folding and bonding with itself (hairpin helices)

A sliding ring holds a moving DNA polymerase onto the DNA
-http://www.youtube.com/watch?v=-mtLXpgjHL0
-The sliding clamp (that holds DNA polynerase in place) uses ATP hydrolysis by a special protein
complex KA the clamp loader
-leading strand clamp is always clamped, lagging strand clamp loosens (VIDEO)

The proteins at a replication fork cooperate to form a replication Machine
-Most of the proteins are held together in a giant multienzyme clusterfuck that replicates DNA

pg 281-287
The initiation complection of DNA replication in chromosomes

DNA synthesis begins at replication origins
-IT is begun by special initiator proteins that bind to DNA and pry the strands apart at replication origins
-A-T RICH segments of DNA are easier to pull apart and tehse are usually replication origin

Bacterial chromosomes typically have a single origin of DNA replication
-E colis genome begins at a single origin of replication and goes in a circle to meet halfway
-The only point at which E. Coli can control DNA replication is initiation
-Begins by initiation proteins binding and attracting helicase bound to a helix loader (also keeps helicase
inactive)
-Initation protein is highly regualted and only activated when there is sufficient proteins
-The origin of replication also experiences a refractory period caused by a delay in the methylation of
newly synthesized A nucleotides

Eucaryotic chromosomes
-autoradiographic experiments described that many forks are moving simultaneously on each eucaryotic
chromosome
-1. Replication origins are activated in clusters, 20-80 origins at a time called replication units
-2. New replication units activated at different times in the cell cycle until all DNA is duplicated
-3. Within a unit, replicatio origins are spaced at intervals: 30k to 250k BP
-4.replication forks are formed as well, helicases stop when they collide or reach chromosome end

In the eukaryotic DNA replication takes place during only one part of the cell cycle
-DNA replication only takes place in teh DNA synthesis phase ( S phase)
-usually lasts for 8 hours in mammals, short as 40 mins for some yeast

Different regions on the same chromosome replicated at distinct times in S phase
-Replication origins replicated at different times in the 8 hour

Autonomously replicating seuquences:

Genes can be selectively amplified by PCR
-bla bla bla polymerase chain reaction


Page (276-281)
A strand directed mismatch repair system removes replication errors that escape from the replication
machine
-Mutator genes greatly increase the rate of spontaneous mutation by making a mutant form of the
proofreading exonuclease (3 to 5 in DNA polymerase)
-there is a strand directed mismatch repair system which detects distortion in the DNA helix and repairs it
based on that.
-this mechanism needs to distinguish the newly synthesized strand because if it didnt it would only
correctly correct half the time (other half would change correct DNA on original strand)
-strand distinguised by selected A residues in GATC sequences need to be methylated
-people that inherit these ghetto proofreading mechanisms are more prone to certain cancers
-most of us inherit two good copies of each gene
-Some yeast and Drosophila do not methylate their DNA and thus they contain nicks in their DNA

Telomere length is regulated by cells and organisms
-replicative cell senescence happens when the level of telomerase is turned down in some cells
(enzzymes cant keep up with all the duplication)
http://www.youtube.com/watch?v=AJNoTmWsE0s
-these cells lose 100-200 nucleotides each time they divide and thus, daughter cells, after many
generations, inherit defective genes
-Human fibroblasts proliferate for about 60 cell divisions before undergoing replicative senescence
-this tested by removing telomerase from mice and first gen mice develop normalle, however mice in later
gens have more defects, progressively.

DNA repair
-Fewer than 1/1000 accidental base changes in DNA result in a permanent mutation, rest are eliminated
by DNA repair
Without DNA repair, spontaneous DNA damage would rapidly change DNA sequences
-Each humn loses 5000 purine bases (A and G) per day because N-glycsyl linkages hydrolize
spontaenously (depurination)
-Deamination of C to U occurs bout 100 bses per day
-Chemicals or UV radtiation also damage bases

The DNA double helix is readily repaired
-The complimentary strand of the damaged strand is used to repair the damanged strand
-Very few organisms have single stranded DNA or RNA for genomes

DNA damage can be removed by more than one pathway
-Two general pathways for repair:
1. Base excision repair:
-Uses DNA glycosylases (at least 6 types of these)
-Used to remove deaminated Cs, As, alkylated or oxidized, or open ring bases
-enzymes flip the suspect base to look for damage, dmg recognized by AP endonuclease which
cuts the phosphodiester backbone, later fixed by DNA polymerase
2. Nucleotide excision repair
-fixes any large changes in the structure of DNA
-A large multienzyme complex scans the DNA for distortation
-When bulky lesion is found, backbone is cleaved and is peeled away by DNA helicase
-DNA polymerase and DNA ligase come again to replace the missing BP

Coupling DNA repair to transcription ensures taht the cells most important DNA is efficiently repaired
-RNA polymerase is used to guide repair mechanisms to sites of DNA lesions
-When this occurs, RNA polymerase disassociates from DNA, and gene is transcribed from the beggining
after the repair
-Known as transcription-coupled repair

The chemistry of the DNA bases facilitates Damage detection
-Distinction between damaged and undamaged bases is very clear
-RNA was possible genetic material before DNA , than why DNA no contain U?
-If DNA had U, it would be difficult to determine a deanimated C from a U

SPecial DNA polymerases are used in emergencies to repair DNA
-riskier DNA polymerases(more than 10) are used for heavier damage
-Some recognize the type of damage, others make food guesses on extensively damanged template
strands
-Backup polymerases lack exonucleolytic proofreading activity

Double strand breaks are efficiently repaired
-when both strands of double helix are borken = bad, result of ionizing radiation, replplication errors,
oxidizing agents and other metabolites
-Two mechanisms to fix this damage:
1.Nonhomologous end-joining
-broken ends are brought together and rejoined by DNA ligation (loss of 1 or more nucleotides)
-quick and dirty solution, acceptable in mammals because little DNA actually useful
-A human aged 70, has about 2000 of these scars
2. Homologous recombination
-uses sister chromatid in recently replicated DNA to fix the cuts

DNA damage delays progression of the cell cycle
-Delaying the progression of cell cycle until DNA repair is complete maximizes DNA effectiveness
-maintained through checkpoints that ensure the completion of one step before the next

Chapter 6
A lot of things go on in DNA that we dont know about like splicing, or RNA is sometimes final form of
some proteins without translation

From DNA to RNA

Portions of DNA sequence are transcribed into RNA
-Process begins transcription, turning DNA into RNA
-Differences between RNA and DNA *
-In RNA G occasionally pairs with U

Transcription produces RNA complementary to one strand of DNA
-RNA strand does not remain H bonded to DNA template, They are only a few thousand BP long
-RNA polymerases perform transcription, can start without a primer (primers used for accuracy)
-RNA polymerase moves stepwise and unwinds the helix just a step ahead of the active site, RNA chain
is extended one nucleotide at a time, powered by nucleoside triphosphates
-RNA copies can be made from same gene in a relatively short time (a thousand/hour from 1 gene)
-RNA makes mistakes every 10^4 nucleotides (10^7 in DNA polymerase)
-If incorrect NT is added, it can back up and fix it
-DNA and RNA polymerase are virtually unrelated, however both have Mg2+ ion at active site

Cells produce several types of RNA
-mRNA are the molecules that are copied (messenger RNA) accounts for 3-5% of total RNA
-Small nuclear mRNAs direct splicing of pre-mRNA
-microRNAs (miRNA) regulate eucaryotic gene expression
-a transcribed segment of DNA is called a transcription unit
-rRNA makes up bulk of cell RNA

Signals encoded in DNA tell RNA polymerase where to start and stop
-A detachable subunit in bacteria called sigma factor tell RNA polymerase where to begin transcribing
-RNA polynmerase holoenzyme is the sigma factor + RNA polymerase complex
-when a polymerase holoenzyme slides onto a promoter, (sequence of starting nucleotides), it binds
tightly and opens up the double helix, exposing a short stretch of NTs
-bond with sigma factor weakens and elongation begins at about 50nt/Sec
-elongation halts when a terminator sequence is reached
-finds another sigma factor and holoenzyme can begin again
-termination signal is a string of A-T that makes the transcribed RNA do a hairpin fold
-Formation of hairpin helps pull the RNA transcript from the active site

Transcription start and stop signals are heterogenous in nucleotide sequence
-A concensus nucleotide sequence is derived by comparing many sequences with the same basic
function and tallying up the most common nucleotide found at each position



Transcription initiation in eucaryotes requires many proteins
-Eucaryotes have 3 kinds of RNA polymerases: I,II and III
-I and III transcribe genes encoding transfer RNA, rRNAs and other small RNAs
-II transcribes most genes including all those that encode proteins.
Two big Diffs b/w eucaryotic and bacterial RNA polymerases
1. Eucaryotes require many additional proteins for transcription initiation to occur, collectively called the
general transcription factors
2.Eucaryotic transcription initiation deals with packing of DNA into nucleosomes and higher order forms of
chromatin

RNA polymerase II requires general transcription factors
-General (required at all promoters) transcription factors:
-help position eucaryotic RNA polymerase correctly at the promoter
-aid the pulling apart of the two strands of DNA (allowing transcription to begin)
-Releases RNA polymerase from the promoter into elongation when it does begin
-Sets of interacting proteins designated: TFII (transcription factor for polymerase II); TFIIB, TFIID etc
-Carry out functions similar to the signma factor in bacteria
-Assemble beegins when TFIID binds to DNA (with a lot of As and Ts KA the TATA sequence/TATA
box)
-TATA binding protein (TBP) is the subunit of TFIID that reconizes the TATA box
http://www.qiagen.com/geneglobe/static/images/Pathways/Assembly%20of%20RNA%20Polymerase-
II%20Initiation%20Complex.jpg
-TATA box is Usually 25 NTS upstream from transcription start site.
-TFIID causes a large distortion in the DNA (thought to be a landmark for location of promoter)
-Transcription initation complex : http://www.youtube.com/watch?v=sQIbfJdjR_s
-TFIIH in the initiation complex is most complicated (9 subunits,including helicase, as large as RNAP)
-TFIIH hydrolyses ATP, to unwind DNA and make is available to RNAP
-after conformational changes, RNAP II moves away from the promoter and begins elongation
-One step is the addition of phosphate groups to the tail of the RNA polymerase known as the
CTD which is ( in humans) a repeat of 52 tandem repeats of a 7 AA sequence
-The serine located on the fifth position in the repeat sequence is phosphorylated by TFIIH
-This allows the polymerase to disengage from the general transcription factors, and tighten its
hold
on DNA

Polymerase II also requires activator, mediator, and chromatin modifying proteins
-Transcriptional activators bind to specific DNA sequences and help attract RNA polymerase II
-Mediator also required; allows the activator proteins to communicate with the RNAP II and the general
transcription factors
-Finally, chromatin modifying enzymes also required
-include remodelling complexes, and histone modifying enzymes
-allow easier access to the DNAm and facilitate assembly of the transcription initiation machinery

Transcription elongation produces superhelical tension in DNA
-RNAP jerks and pauses at some sequences and speeds through other
-RNAPs are associated with elongation factors: proteins that decrease the likelihood that RNAp will
dissociate before it reaches the end of a gene
-ATP-dependent chromatin remodelling complexes aid Euca..RNAp with chromatin structure
-May move with RNAp or find and rescue
-Some elongation factors help RNAp facilitate trancripition through nucleosomes w/o energey
-These proteins can dislodge H2A and H2B dimers

-DNA supercoiling: reppresents a conformation that DNA adopts in response to superhelical tension
(tension from the loops and coils)
-There are 10 NTTs for every helical turn in a double helix.
-DNA supercoil will form to compensate for each 10 NT pairs that are opened
-a moving polymerase generates positive superhelical tension inn the DNA in front of it and negative
helical tension behind it
-this tension facilitates the unwrapping of DNA in nucleosomes
-DNA topoisomerase rapidly removes this super helical tension
-whenever a region of the helix opens, it removes the negative supervoils from bacterial DNA
-DNA gyrase makes the opening of DNA helix energetically favorable





































PG 366-390

From RNA to protein
coding problem - how is info on a linear sequence translated into proteins?

an mRNA is decoded in sets of three nucleotides
-Translation is the conversion of RNA info to proteins
-Genetic code the rules (triplets) of mRNA that are translated into proteins
-4x4x4 (four letters, three per code) = 64 possible combinations, only 20 AAs
-The triplets are known as Codons
-Mitochondria have their own transcription and protein synthesis systems that operate independently of
the cell
-Three reading frames: http://www.cs.wustl.edu/~cytron/cse131/Modules/3/Extensions/frame.jpg

tRNA molecules match AAs to codons in mRNA
-tRNAs (80 NTs in length), serve as adaptors that mRNA recognizes and binds to, rRNAs carry AAs
-has a cloverleaf structure, after primary folding, and L-shaped after more folding
http://www.biologyreference.com/images/biol_04_img0444.jpg
-Two regions of unpaired NTs
1. Anticodon: three NTs that pairs with a complementary codon
2. 3 end where the AA that matches the codon is attached to tRNA
-Some AAs have more than 1 tRNA
-some tRNAs have a wobble position (third base pair is not exact, first 2 more important)
-humans have 48 anticodons, for 500 tRNA genes

tRNAs are covalently modified before they exit from the nucleus
-tRNAs synthesized by RNAP III, also contain introns that must be spliced
-splicing will not occur on misfolded tRNA, this could serve as quality control

Specific enzymes couple each AA to its appropriate tRNA molecule
-aminoacyl-tRNA synthetases covalently couple AAs to approriate set of RNA molecules
-usually 1 synthetase per AA so 20 synthetases
-some bacteria have less than 20 synthetases, same synthetase used for coupling more than one AA
-a second enzyme is used to modify incorrectly attached AAs
- AA added to 3 end of tRNA , produces high energy bond between tRNA and the AA
-two checks that right AA is used