Notes for BMB/Micrb 251 (lec 1-21)
(see Dr. Gilmour for lec 22-41)
Molecular & Cellular Biology I
Fall 2004
Textbook: Molecular Biology of the Cell, 4th Edition
Author: Alberts et al.
Instructor: Dr. B. Franklin Pugh
Meeting time: 12:20-1:10 MWF
Meeting place: 26 Hosler
CHAPTER 1 CELLS AND GENOMES
The
basic unit of life is the cell. Fig. 1-1
Most
forms of life are just a single cell.
Humans
have 1013 cells, all derived from a single cell!
Cells
must consume energy in order to grow and multiply.
Chemical Code (DNA)
DNA: A, T, C, G (each is called a nucleotide)
computer: 0, 1
All Cells Replicate Their Hereditary Information by Templated
Polymerization
Each
nucleotide is composed of three parts:
phosphate, sugar, and a base. Fig.
1-2
Only the base is different between A, T, C, G.
A polymer of nucleotides is called a polynucleotide.
The
unique arrangement of nucleotides forms the genetic code.
The genetic code provides all the information necessary to make
an organism.
When
cells grow and multiply, they must duplicate the genetic code.
The
polynucleotide provides a template for its own replication.
A only pairs with T
C only pairs with G
All Cells Transcribe Portions of Their Hereditary Information
into the
Same Intermediary Form (RNA)
In
order to use the information in the genetic code, the DNA must be ÔreadÕ. Fig. 1-4
Transcription is the process by which parts
of the DNA are ÔreadÕ.
Transcription
is similar to DNA replication, except that an RNA polynucleotide is made.
DNA: A, T, C, G
RNA: A, U, C, G (a ÔUÕ is used instead of a ÔTÕ).
Same pairing rules apply.
(RNA also has an extra ÐOH group on each sugar.)
RNA and DNA are two different kinds of polynucleotides.
Different
RNAs have different sequences of nucleotides.
Some
RNAs direct chemical reactions (more on this later). Fig.
1-6
Messenger
RNA (mRNA) codes for the production of proteins.
Translation is the process by which mRNA
is ÔreadÕ into protein. Fig. 1-4
Proteins
do almost all the work in the cell.
Proteins
make each cell different.
Proteins
are made up of a linear polymer of amino acids.
Proteins range in size (~100-1000 amino acids).
There are 20 different kinds of amino acids.
The arrangement of the amino acids is dictated by the DNA ˆ RNA nucleotide sequence.
A
protein is also called a polypeptide.
Polypeptides
fold up into very precise 3-dimensional structures. Fig.
1-7a
Proteins
that catalyze (facilitate) chemical reactions are called enzymes. Fig. 1-7b
Other
proteins also serve as signaling molecules and provide structure to the cell.
Three
nucleotides at a time are translated into one amino acid. Fig. 1-9
A group of three nucleotides that code for an amino acid is
called a codon.
There are 64 possible codons (4x4x4), that code for 20 amino
acids.
Codons
are translated by transfer RNAs (tRNA).
The
anticodon part of the tRNA pairs with the codon.
So, there are many different kinds of tRNAs
Each
kind of tRNA is attached to a particular amino acid.
The
ribosome
uses mRNA as a template to align the tRNAs, which then allows the amino acids
to be stitched together. Fig. 1-10
The ribosome is composed of mostly ribosomal RNA (rRNA) and
ribosomal protein.
Not
all of the DNA has genes.
Certain
stretches of DNA regulate the ÔexpressionÕ of genes.
When the cell needs to make a particular protein, it ÔreadÕ or ÔexpressesÕ the corresponding gene.
The entire sequence of DNA of an organism is called a genome.
Life Requires Free Energy
Cells
take energy (food) from its environment and use it to build more of itself.
Fig. 1-13
It
is composed of lipids (fats).
A Living Cell Can Exist with Fewer Than 500 Genes Fig. 1-14
Humans have over 30,000 genes.
Summary
The
cell is the minimal operational unit.
All
info to make a cell is stored in DNA.
Central
Dogma: DNA makes RNA makes protein.
Proteins
do much of the work in the cell.
Most
of life on earth is microorganisms (single-celled),
Cells Can Be Powered by a Variety of Free Energy Sources
Where
do cells get their energy to make more of themselves?
Inorganic chemicals
The sun
Other organisms
Some Cells Fix Nitrogen and Carbon Dioxide for Others
The Greatest Biochemical Diversity Is Seen Among Procaryotic
Cells
Prokaryotes
have no nucleus. Fig. 1-18
They live in a wide variety of habitats (hydrothermal vents,
Arctic, bogs, sea, dirt, other organisms)
Are microorganisms.
Eukaryotes
have a nucleus. Fig. 1-43b
A nucleus is an intracellular compartment that houses DNA.
Eukaryotes can be microorganisms or multicellular.
The Tree of Life Has Three Primary Branches (domains): Bacteria,
Archaea, and
Eucaryotes Fig. 1-21
Bacteria
and Archaea are prokaryotes.
Note
how diverse the prokaryotes are.
Note
that plants, animals, and fungi are highly related!
Some Genes Evolve Rapidly; Others Are Highly Conserved
When
DNA is replicated, mistakes are made (albeit very rarely).
Most
mistakes (mutations) have little effect on the organism.
Some
are detrimental to the organism. Fig. 1-22
Such mutant organisms are
eliminated by natural selection (ability/inability to thrive or
compete with other organisms for survival).
Therefore the mutation is also
lost.
Such regions of DNA are
therefore highly conserved and are indicative of important genetic information.
In
rare cases, the mistake changes the genetic code for a protein in a beneficial
way.
This is the core of evolution.
Natural selection favors those organisms that can reproduce the fastest.
Small size
Small genome
Specific environmental niche
New Genes Are Generated from Preexisting Genes Fig. 1-23
Mutation
Nucleotides within a gene can mutate.
Happens very frequently with HIV (as
an example).
Duplication
Duplicating a
gene allows one to mutate, while the other provides the essential function.
Segment shuffling
Two
or more genes can be broken up and pieced back together differently.
Horizontal transfer
A gene from one organism can be
transferred to a related or unrelated organism.
Humans have bacterial DNA in their
genomes!!
Gene Duplications Give Rise to Families of Related Genes Within
a Single Cell
Related
organisms have related genes. The
related genes are said to be orthologs.
When a gene is duplicated within the same cell, allowing them to evolve separately, then these genes are paralogs.
Homologs refer to both orthologs and
paralogs.
All
homologs form a gene family. Fig. 1-26
Genes Can Be Transferred Between Organisms, Both in the
Laboratory
and in Nature
Bacterial viruses (bacteriophages) are mobile genetic vehicles that allow genes to move horizontally.
Bacteria can also take up DNA from their environment.
This
and the rapid rate of replication, allow bacteria to evolve rapidly. Fig. 1-21
Think antibiotic resistance.
Horizontal Exchanges of Genetic Information Within a Species Are
Brought About by Sex
Primordial life may have extensively used
horizontal transfer.
Groups of genes might have moved together.
Bacteria and Archaea but not Eukaryotes have
similar metabolic genes
Metabolic genes are involved in
getting food
Archaea and Eukaryotes but not Bacteria have
similar genes that control information flow
DNA replication, transcription,
translation
Horizontal
gene transfer is essentially bacterial sex.
The Function of a Gene Can Often Be Deduced from Its Sequence
Genes
with similar sequence have similar function.
If
you know the function of one homolog, you then know the function of all
homologs.
More Than 200 Gene Families Are Common to All Three Primary
Branches of the Tree of Life Table 1-2
Mutations Reveal the Functions of Genes
How do we figure out what the function of any given gene is?
Isolate the protein coded for by a gene, and determine what
chemical reaction it carries out.
Determine
function through genetics.
Mutate the gene and see what effects (phenotype) it has on the
organism (e.g. growth rate).
Molecular Biologists Have Focused a Spotlight on Model
Organisms
Bacteria
Ð E. coli
Eukaryote
Ð Yeast, Arabidopsis (plant), fruit flies, mice, and more
Summary
All life requires energy, via inorganic
chemicals, sunlight, or other organisms.
Prokaryotes represent the bulk of lifeÕs
diversity and mass on earth.
There are three domains of life: bacteria,
archaea, and eukaryotes.
All life evolved through mutation, duplication,
shuffling, and horizontal transfer of genes.
Humans are eukaryotes, so we have an interest in
how eukaryotes work.
Eukaryotes are much more complex than prokaryotes.
But not more evolved!!
Eukaryotes have more complex genomes, cell
organization, and can be multi-cellular.
Eucaryotic Cells May Have Originated as Predators
A eukaryotic cell is ~1000x larger than a
prokaryotic cell. Fig.
1-31
Eukaryotes have a nucleus (nuclear membrane or
nuclear envelope) that compartmentalizes the DNA.
Eukaryotes also have other internal membrane
compartments.
Eukaryotes have a protein cytoskeleton that gives
shape to the cell. Fig.
1-32
Prokaryotes use a cell wall.
By rearranging the
cytoskeleton, eukaryotic cells can rapidly change shape.
Eukaryotic cells can engulf
bacteria (think immune system).
A primitive eukaryotic-like
cell might have eaten other bacteria.
Eucaryotic Cells Evolved from a Symbiosis
Eukaryotic cells have mitochondria. Fig. 1-34
Mitochondria are membrane compartments
(organelles) that convert food energy into usable chemical energy (respiration).
Mitochondria also have a small
genome.
Some eukaryotic cells (e.g., plants) have chloroplasts.
Chloroplasts are organelles and
have a genome.
Chloroplasts convert light
energy into usable chemical energy (photosynthesis).
Mitochondria and chloroplast were once free-living bacteria that were engulfed by primitive eukaryotes and formed a symbiotic relationship. Fig. 1-35
Is mitochondrial and chloroplast DNA more like
bacteria or eukaryotes?
Eucaryotes Have Hybrid Genomes
Eucaryotic Genomes Are Big
Fig. 1-38
~1000x longer than bacteria, but only about 20x
more genes.
99% of the eukaryotic genome does not code for
genes!
Probably just ÔjunkÕ
When was the last time you
removed unnecessary files from your 80 gig hard drive?
The Genome Defines the Program of Multicellular Development
Multi-cellular organisms have a diversity of cell
types, all derived from a single fertilized egg, and all having the same
genome.
Humans have skin cells, liver
cells, and brain cells (sometimes).
Plants have leave cells, flower
cells, and root cells.
Different cell types are made when different
subsets of genes are expressed.
When you are listening to music itÕs like having a different favorite play lists, depending on your mood or what your doing (e.g., breaking up w/ your boy/girlfriend vs. having a party Ð maybeÉ).
Cells are constantly sending signals to each
other.
Cells at different locations get different
signals.
Signals trigger the expression of particular sets
of genes (favorite play list). Fig. 1-40
No signal Ð no expression. Fig. 1-41
A Yeast Serves as a Minimal Model Eucaryote
Saccharomyces cerevisiae Ð bakers yeast and
brewers yeast. Fig.
1-43
Fungi
Advantages:
Small genome
Easy genetics and biochemistry
Rapidly grows and divides
Inexpensive
Most cellular functions highly
conserved with humans.
The Expression Levels of All The Genes of An Organism Can Be
Monitored Simultaneously
As scientists, we used to study one gene at a
time.
Now that the entire yeast genome has been
sequenced we can study the expression of all 6300 yeast genes at a time, using
DNA microarrays. Fig.
1-45
Each spot corresponds to the DNA of a particular
gene. 6300 spots for yeast.
mRNA from a particular gene will bind (hybridize)
to itÕs cognate spot.
If we color the mRNA first, the spot turns color.
To see how the yeast genetic program changes when they are hungry vs. when they are well-fed (glucose):
Isolate mRNA from hungry cells
and color it red.
Isolate mRNA from fed cells and
color it green.
Mix both together and hybridize to the DNA spots.
Red spots mean the gene was
turned on in hungry cells and off in fed cells.
What do the green, yellow and
black spots mean?
Arabidopsis Has Been Chosen Out of 300,000 Species
As a Model Plant
The World of Animal Cells Is Represented By a Worm, a Fly, a
Mouse,
and a Human
worm:
Caenorhabditis elegans
fly: Drosophila
melanogaster
mouse: Mus musculus
human:
Homo sapien
Studies in Drosophila Provide a Key to Vertebrate
Development
The Vertebrate Genome Is a Product of Repeated Duplication
Mutating a gene might knock out its function, but if another gene serves the same function, there will be no phenotype.
Gene duplication provides great opportunities for
the duplicated gene to evolve new functions. Fig.
1-51
The Mouse Serves as a Model for Mammals
Able to knock out specific genes.
We Are All Different in Detail
Mouse Ð Human 90%
identical
Chimp Ð Human 99%
Human Ð Human 99.9%
Summary
Eukaryotes are evolutionary less diverse than
prokaryotes, but are way more complex.
Eukaryotic cells arose by symbiosis with
prokaryotes.
ÒPlayingÓ different parts of the genome gives
rise to different cell types.
Model organisms are used as experimental
surrogates to humans.
The more complex the organism the harder it is
to work with (but may be a better proxy for humans).
CHAPTER 2 CELL CHEMISTRY AND BIOSYNTHESIS
Cells Are Made of Relatively Few Types of Atoms
>99% of living matter is composed of six
elements: P, S, C, O, H, N
(C,O,H,N) represent ~97%.
Other important elements: Cl, Ca, Mg, Si, Zn, Co,
Mn, Fe, Se and others.
The Outermost Electrons Determine How Atoms Interact
Ionic Bonds Form by the Gain and Loss of Electrons
Know how a covalent bond differs from an ionic bond. Fig.
2-5
Covalent bonds are very strong and stable, essentially riveting atoms together.
Ionic bonds a positive charge
interacting with a negative charge.
Ionic bonds are weak in water,
because water interacts with the positive and negative charges making it hard
for them to interact with each other. Fig. 2-14
Ions have gained or lost a charge (electron).
Cations are positively charged ions.
Anions are negatively charged ions.
Covalent bonds are strong.
Atoms form molecules through covalent bonds.
Covalent bonds also link
repeating units of a polymer together
Defined as a stable chemical link between two
atoms produced by sharing one or more pairs of electrons.
The amount of energy required to break a covalent
bond varies depending on atoms and environment. (ave. ~90 kcal/mol.)
Enzymes are required to break covalent bonds
(under normal physiological conditions).
There Are Different Types of Covalent Bonds
Polar vs. nonpolar
Different representation of molecules. Fig. 2-12
Water Is the Most Abundant Substance in Cells
~70% of a cell is water.
Some Polar Molecules Form Acids and Bases in Water
Four Types of Non-Covalent Interactions Help Bring Molecules
Together in Cells Table 2-2, and
Panels 2-2 and 2-3 on pp. 112-115
Ionic bonds
Weak; relative strength = 3
Cohesion between a positively
charged atom and a negatively charged atom. Fig. 2-14
Water and salts are polar or
charged and so can compete with these interactions, thereby weakening them.
By measuring the interactions between two molecules as a function of salt (NaCl, KCl, etc.) conc. one can get a quantitative handle on the extent of ionic interactions.
Examples:
DNA phosphates and protein
lysine side chains make ionic bonds.
Protein side chains glutamate
and arginine make ionic interactions.
Hydrogen bonds
Weak; relative strength = 1
Hydrogen atom with partial positive
charge interacts with two electronegative atoms. Fig. 2-15
Two electronegative atoms (such
as N and O) can share a hydrogen atom, even though a H atom can only form a
single covalent bond.
Water can compete with H-bonds,
thereby weakening them.
Example:
Protein secondary structure
DNA base-pairing
van der Waals interactions
Very weak; relative strength =
0.1
A large number of them can add
up to generate strong interactions.
Due to asymmetric electrical
charges, two atoms at very close distances will attract each other.
ÒHand-in-a-gloveÓ fit
Hydrophobic interactions
Water interacts with itself via
hydrogen bonds (surface tension).
Nonpolar groups cannot interact
with water and so are excluded (oil and water don't mix).
Water exclusion causes nonpolar
groups to self associate.
Example:
Interior of proteins
Two surfaces of proteins
A Cell Is Formed from Carbon Compounds
Carbon represents the core constituent of all life
because it can make a variety of strong covalent bonds with other elements.
Chemical groups (Functional groups) Panel 2-1 on page 111
Sugars Are Energy Sources for Cells and Subunits of
Polysaccharides Fig. 2-18,19,20
Panel 2-4, pp. 116-117
Fatty Acids Are Components of Cell Membranes Fig.
2-21,22
Panel 2-5, pp. 118-119
Amino Acids Are the Subunits of Proteins Fig.
2-23, 24
Nucleotides Are the Subunits of DNA and RNA Fig.
2-26, 27, 28
Panel 2-6, pp.120-121
The Chemistry of Cells is Dominated by Macromolecules with
Remarkable Properties
Noncovalent Bonds Specify Both the Precise Shape of a
Macromolecule and its Binding to Other Molecules Fig.
2-32
Summary
Cells are composed of primarily six elements:
PSCOHN
Covalent bonds stably connect atoms to form
molecules to form biopolymers.
Ionic, hydrogen-bonds, van der Waals, and
hydrophobic interactions drive dynamic interactions between and among
biopolymers and small molecules.
Specific interactions are provided through
precisely positioned functional groups on the 3-D structure of the
bio-molecules.
Sugars are the building blocks of
polysaccharides.
Fatty acids are the building blocks of
membranes.
Amino acids are the building blocks of proteins
Nucleotides are the building blocks of DNA and
RNA.
The
rest of this chapter will be covered in more detail in the second half of BMB
251. For now, just read the text
relevant to the figures.
Cell Metabolism Is Organized by Enzymes Fig. 2-34
Every enzyme has one particular function (division
of labor).
A cell can have many copies of the same enzyme.
Biological Order Is Made Possible by the Release of Heat Energy
from Cells
Photosynthetic Organisms Use Sunlight to Synthesize Organic
Molecules
Enzymes Lower the Barriers That Block Chemical Reactions Fig. 2-44, 46
(Fig. 2-46c doesnÕt make any sense).
Breaking covalent bonds requires activation
energy.
Enzymes lower the activation energy.
Enzymes bind to substrates and convert them to
products.
How Enzymes Find Their Substrates: The Importance of Rapid Diffusion
Life processes require molecules to interact.
They do so via random diffusion or active transport.
Diffusion is temperature dependent Fig. 2-48
The distance coverage only goes up by the square root of the allowed time.
Large molecules diffuse slower than small
molecules.
Long distances movements may involve active transport.
Active transport requires the input of energy.
Diffusion-limited reactions occur as fast as the
molecules collide.
More concentrated, the fast the reaction goes.
Why are many reactions not diffusion limited?
Nonproductive collisions
Conformational changes in the
protein
Catalytic steps.
Dissociation of a regulatory
subunit or molecule
The cell is very crowed with biopolymers and
other biomolecules. Fig.
2-49
The Free-Energy Change for a Reaction Determines Whether It Can
Occur
The Concentration of Reactants Influences DG
For Sequential Reactions, DG¡Values Are Additive
Activated Carrier Molecules are Essential for Biosynthesis
When cells take in food, they break down the
bonds, which releases energy.
Some of the energy dissipates as heat.
Some of the energy is coupled to the production of
activated carriers.
Activated carriers have high energy covalent bonds
that can be used to make an unfavorable reaction more favorable.
Important energy carriers: ATP, NADH (and NADPH).
Favorable Reaction Fig. 2-56
High energy covalent bonds are very unstable, and
thus their easy breakage can be coupled to the breakage of more stable covalent
bonds.
ATP Is the Most Widely Used Activated Carrier Molecule Fig. 2-57
Be able to recognize ATP, and distinguish it from
ADP.
Energy Stored in ATP Is Often Harnessed to Join Two Molecules
Together Fig. 2-59
ATP (adenosine triphosphate) is hydrolyzed to ADP (adenosine diphosphate).
Hydrolysis as its name implies means using water to break bonds.
Know the ATP hydrolysis reaction.
There Are Many Other Activated Carrier Molecules in Cells
The Synthesis of Biological Polymers Requires an Energy Input
Example of polynucleotide synthesis Fig. 2-67
Summary
Cellular reactions (e.g. synthesis of more cell
components) are carried out by enzymes.
Energetically unfavorable reactions can be
coupled to favorable ones.
ATP is the major energy carrier in the cell
(equivalent to $ in our economy).
Food Molecules Are Broken Down in Three Stages to Produce
ATP Fig. 2-70
1.
Enzymatic break down of food
Covalent bonds are broken such that biopolymers are broken down to monomer units (e.g. glucose).
This happens in the intestines, and is relevant to multi-cellular organisms.
Monomer units are absorbed into the cell.
Transport proteins help the molecules traverse the membrane.
The molecules end up in the cellÕs cytoplasm.
2. Glucose is cleaved into two molecules called pyruvate.
This occurs over many steps, with many chemical intermediates.
A series of enzymes catalyze these coupled reactions.
ATP input is used to help lower the activation energy.
Ultimately, more ATP produced.
The whole cascade of events is called glycolysis.
Pyruvate diffuses into mitochondria, where it becomes a substrate for
respiratory enzymes.
3.
Pyruvate is converted to an activated molecule called acetyl-coA
A series of very complex
enzymatic reactions couples the break down of acetyl-coA to the production of
ATP.
Glycolysis Is a Central ATP-producing Pathway
Fermentations Allow ATP to Be Produced in the Absence of Oxygen
Storage
The Citric Acid Cycle Generates NADH by Oxidizing Acetyl Groups
to CO2
Electron Transport Drives the Synthesis of the Majority of the
ATP
in Most Cells
Organisms Store Food Molecules in Special Reservoirs
Amino Acids and Nucleotides Are Part of the Nitrogen Cycle
Many Biosynthetic Pathways Begin with Glycolysis or the Citric
Acid
Cycle
Metabolism Is Organized and Regulated
Summary
Food is broken down into monomer units.
Breakage of covalent bonds release energy,
which is re-couped in the form of ATP (and other energy carriers).
CHAPTER 3 PROTEINS
The Shape of a Protein Is Specified by Its Amino Acid Sequence
A protein is made from a polymerized chain of amino acids.
The covalent bond that links amino acids is called
a peptide bond. Fig. 3-1
Small chains of amino acids
(<100) are called peptides.
Long chains are called polypeptides.
Polypeptides and proteins mean
the same thing, but often polypeptide refers to the unfolded chain.
There are 20 different amino acids. Fig. 3-2
The part that is similar among
all 20 is called the backbone.
The part that is unique is
called the side chain.
Be able to recognize amino acid
names.
Know that some have basic,
acidic, or nonpolar side chains.
Each type of amino acid serves
a purpose in the context of a protein.
Amino acids with similar side
chain properties have similar function.
If you are a serious BMB major, then you should memorize all 20 amino acid names and properties, and be able to identify their side chains. (ItÕll be needed to really understand 251 and related courses).
Fig. 3-3 and Panel 3-1.
A polypeptide chain folds back on itself giving
the protein a unique 3-D structure.
Four noncovalent forces direct the folding: Fig. 3-5, 3-6, 3-7
Ionic, hydrogen-bonding, van
der Waals, and hydrophobic
Proteins Fold into a Conformation of Lowest Energy Fig. 3-8
Although the sequence of amino acids dictate the
folding pathway and the final folded conformation, in the cell, other proteins
called chaperones, assist the folding process.
Proteins range in size from 50-2000 amino acids.
Large proteins have multiple independent folding domains. Fig.
3-12
Domains can be thought of as
different proteins strung together (more below).
The a Helix and the b Sheet Are Common
Folding Patterns Fig. 3-9
Primary structure is the linear arrangement of
amino acids.
Secondary structure involve a helices and b
sheets.
The protein can be partially unfolded and still have secondary structure.
Tertiary structure represents the full 3-D
structure of a protein. Fig. 3-12, 3-13
A protein can have one or more
independently folding domains.
Domains range from 5-350 amino
acids.
Quaternary structure refers to complexes of
multiple proteins.
So, quaternary interactions
involve more than one protein.
Tertiary structure of a multi-domain protein is
analogous to quaternary structure.
Few of the Many Possible Polypeptide
Chains Will Be Useful
Certain amino acids along the
polypeptide chain are more important than others in determining the structure
of a protein.
Proteins Can Be Classified into Many Families
Proteins of similar biochemical function are
likely to have similar structure. Fig. 3-14
However, the proteins may or may not have nearly
identical primary sequence. Fig. 3-15
Proteins Can Adopt a Limited Number of Different Protein Folds
Sequence Homology Searches Can Identify Close Relatives Fig. 3-17
Computers can be used to align the primary sequence of proteins, to determine if they are related.
into Known Protein Folds
Some Protein Domains, Called Modules, Form Parts of Many
Different
Proteins
Some proteins may be unrelated except in one
domain (or module). Fig. 3-18, 3-19, 3-21
Remember, genes can evolve by shuffling parts of the gene.
Since we might not know if a conserved region (as defined by comparing primary sequence) meets the definition of a domain (independently folding unit), we call them modules.
Some proteins may be related only by a motif.
A motif is a small sequence of
amino acids found in many proteins.
A motif is small than a domain, and probably does not fold independently of the rest of the protein.
Motifs typically represent an
interface Ð a section of the protein that binds something else (like another
protein or a small molecule.)
The Human Genome Encodes a Complex Set of Proteins, Revealing
Much That Remains Unknown
Larger Protein Molecules Often Contain More Than One Polypeptide
Chain
Proteins are generally thought of as having one
polypeptide.
However, many proteins have more than one
polypeptide, and can be thought of as protein complexes. Fig. 3-21, 3-22, 3-23, 3-24
Each polypeptide of a protein complex is called a subunit.
Some Proteins Form Long Helical Filaments Fig. 3-25, 3-26
A Protein Molecule Can Have an Elongated, Fibrous Shape
Extracellular Proteins Are Often Stabilized by Covalent
Cross-Linkages Fig. 3-28, 3-29
Disulfide bonds between cysteine side chains stabilize protein tertiary and quaternary structure.
Protein Molecules Often Serve as Subunits for the Assembly of
Large
Structures
Advantages of using repeating identical subunits
to build very large protein structures:
Less genetic information required.
Assembly and Disassembly can be easily controlled
since interactions are repeated.
Single-domain small subunits allow mis-folded
subunits not to be incorporated.
Some very large protein structures with repeated subunits: Fig. 3-30
Actin filaments
Tubulin filaments
Bacterial flagella
Some super-sized structures: Fig. 3-31, 3-32
Viral coats (capsids)
Many Structures in Cells Are Capable of Self-Assembly
The Formation of Complex Biological Structures Is Often Aided by
Assembly Factors
Summary
The sequence of amino acids dictates the
structure of a protein.
Proteins have substructure including a helices and b sheets.
a helices and b sheets fold into domains
Domains fits together to form the protein.
Certain proteins can coalesce to form complexes
or large structures.
Proteins with similar function will have
similar structure and may (or may not) have similar amino acid sequence.
All Proteins Bind to Other Molecules Fig. 3-37
Molecules that bind to proteins are called ligands.
Proteins are very selective toward the ligands
they bind.
Precise docking of the ligand provides specificity.
Driving forces: ionic, hydrogen-bonding, van der Waals, hydrophobic. Fig. 3-38, 3-43
The Details of a ProteinÕs Conformation Determine Its Chemistry
Amino acid side chains can be made to be very
reactive.
Good for catalyzing biochemical reactions.
Sequence Comparisons Between Protein Family Members Highlight
Crucial Ligand Binding Sites Fig. 3-40
Binding Strength Is Measured by the Equilibrium Constant Fig. 3-44
Enzymes do not get used up in the reaction.
Enzymes do not alter the equilibrium ratio of
substrate and product.
Enzymes speed up reaction rates.
Enzymes can catalyze the reverse reaction as well.
Substrate Binding Is the First Step in Enzyme Catalysis
The reaction that an enzyme catalyzes occurs in
the enzyme active site.
The interplay of amino acid side chains in the
active site can cause a side chain to be hyper reactive.
The first step in an enzyme-catalyzed reaction is the binding of a substrate to the enzyme's active site.
The second step is catalysis:
The third step in the reaction is the dissociation
of the products:
How fast an enzyme works depends on the
concentration of substrate.
KM is a measure of substrate affinity
(as well as its tendency to react).
Enzymes Speed Reactions by Selectively Stabilizing Transition
States
Enzymes Can Use Simultaneous Acid and Base Catalysis
Lysozyme Illustrates How an Enzyme Works
Tightly Bound Small Molecules Add Extra Functions to Proteins
Where amino acid side chains are insufficient,
coenzymes are employed.
Other small molecules add functionality
Multi-enzyme Complexes Help to Increase the Rate of Cell
Metabolism Fig. 3-54
Product of one reaction becomes the substrate for
the next.
Think of a hand-off in a relay race.
No need for diffusion!
The Catalytic Activities of Enzymes Are Regulated Fig. 3-55, 3-56
Feed back inhibition: Product of one reaction inhibits the production of the substrate for that reaction.
When you eat and get full, and donÕt feel like
eating anymore, this is like feed back inhibition.
Allosteric Enzymes Have Two or More Binding Sites That
Interact Fig. 3-57, 3-58
An allosteric site binds a ligand, changing the
conformation of the active site (an protein).
Each OtherÕs Binding
Symmetric Protein Assemblies Produce Cooperative Allosteric
Transitions Fig. 3-60
The Allosteric Transition in Aspartate Transcarbamoylase Is
Understood
in Atomic Detail
See movie 3.9 on Cell Biology Interactive to get a better feel for the dynamics of allosteric regulation.
Many Changes in Proteins Are Driven by Phosphorylation
Protein kinases add phosphates to proteins
(called phosphorylation). Fig. 6-63
Phosphate is derived from ATP
Amino acid side chains that can get phosphorylated have ÐOH groups:
Tyrosine
Serine
Threonine
Protein phosphatases remove phosphates from
proteins.
Done via hydrolysis.
A Eucaryotic Cell Contains a Large Collection of Protein Kinases
and
Protein Phosphatases
Cells contains hundreds of different kinds of
kinases and phosphatases.
Each is very specific for a set of proteins.
Phosphorylation/dephosphorylation serves as a
molecular switch, turning on and off the activity of a protein.
Phosphorylation/dephosphorylation events are
dynamic.
The Regulation of Cdk and Src Protein Kinases Shows How a
Protein
Can Function as a Microchip Fig. 3-66
See movie 15.8 on Cell Biology Interactive to get a better feel for this.
Get a sense of the major regulatory themes, and not specific names.
Proteins That Bind and Hydrolyze GTP Are Ubiquitous Cellular
Regulators Fig. 3-70
Some proteins are activated when they bind GTP
(e.g. Ras).
The GDP-bound protein is inactive.
Fig. 3-71
Guanine nucleotide exchange proteins induce GDP to
dissociate, thereby allowing the protein (e.g. Ras) to be reactivated.
See movie 15.9 on Cell Biology Interactive to get a better feel for this.
Get a sense of the major regulatory themes, and not specific names.
You must know the major intracellular signaling
pathways. Fig. 3-72
Motor Proteins Produce Large Movements in Cells Fig. 3-75, 3-76
ATP hydrolysis converts a random walk into a
directional walk.
ATP hydrolysis is coupled to conformational
changes in the protein that allow forward but not backward movement.
(Any backward movement must be coupled to ATP
synthesis, which is energetically unfavorable, but could be accomplished if the
ADP and phosphate concentrations were high enough and the ATP concentration low
enough.)
Examples of directional movement
Muscle contraction
Mitosis
Cell migration
Pumping ions (e.g. sodium) out
of a cell.
DNA polymerization
Protein synthesis
Membrane-bound Transporters Harness Energy to Pump Molecules
Through Membranes
Also the reverse is true: Movement of ions
(specifically hydrogen ions) through a membrane is coupled to ATP synthesis.
See movie 14.1 on Cell Biology Interactive to see an awesome example.
(See also 14.2, if you want to know what grad students do with their
spare time.)
Proteins Often Form Large Complexes That Function as Protein
Machines
We be seeing lots of this over the next several
weeks:
DNA replication
Genetic recombination
Transcription
Translation
RNA splicing
A Complex Network of Protein Interactions Underlies Cell
Function
Summary
Proteins are designed to bind other molecules
to elicit some biological change.
Enzymes are a type of protein that catalyze
biochemical reactions.
To catalyze biochemical reactions, enzymes bind
substrates, make or break covalent bonds, and release products.
Proteins are regulated by the binding of small
molecules, other proteins, or by phosphorylation. These molecules cause their protein targets to change shape,
making them more or less active.
Enzymes can use ATP hydrolysis to get work
done.
Part II Basic Genetic Mechanisms
CHAPTER 4 DNA AND CHROMOSOMES
A DNA Molecule Consists of Two Complementary Chains of
Nucleotides
A DNA double helix consists of two antiparallel
polynucleotide chains. Fig. 4-3
The two chains are held together by hydrogen
bonding of bases
A base pairs with T
C base pairs with G
A sugar + phosphate + base is
called a what?
Must know how to identify antiparallel strands.
Must know what 5Õ-to- 3Õ direction is.
The Structure of DNA Provides a Mechanism for Heredity
In Eucaryotes, DNA is Enclosed in a Cell Nucleus
Summary
Genetic information that defines an organism is
carried in a linear sequence of nucleotides.
DNA is double helix of two antiparallel strands
A pairs
with T, and C with G.
CHROMOSOMAL DNA AND ITS PACKAGING IN THE
CHROMATIN FIBER
Eucaryotic DNA Is Packaged into a Set of Chromosomes Fig. 4-10
One chromosome corresponds to one continuous
DNA double helix
Humans have 24 different chromosomes.
Two sets of 22 different
autosomal chromosomes.
Males have one each of sex
chromosomes: X and Y
Females have two X chromosomes.
So humans have a total of 46
chromosomes in every cell.
A chromosome can be as long as a hundred million
nucleotides.
Eukaryotic chromosomes are linear.
Bacterial chromosomes are circular (one end is connected to the other end).
Chromosomes Contain Long Strings of Genes
The Nucleotide Sequence of the Human Genome Shows How Genes
Are Arranged in Humans
Fig. 4-15
The human genome has been sequenced
A composite of seven ethnically diverse people.
It contains over 3 billion base pairs and over
30,000 genes.
There could be as many as 60,000 genes, but we
donÕt yet know how to recognize them.
Genes are arranged linearly along chromosomes.
Genes are split into pieces consisting of exons.
The DNA between exons is called introns.
Exons are about 5% of the length of introns.
Much of human DNA contains repetitive
elements Fig. 4-17
Less than 2% of the human genome codes for protein!
Comparisons Between the DNAs of Related Organisms Distinguish
Conserved and Nonconserved Regions of DNA Sequence
Since exons comprise <2% of the genome, and
since we do not know how the DNA sequence dictates where exons start and stop,
we donÕt really know what is coding and what is noncoding.
Introns are often considered to be junk since they
are of little importance and are generally not conserved.
By comparing (aligning) the human genome with the
mouse genome we should be able to determine what regions are conserved.
Conserved regions are likely to be important.
Human/mouse/other comparisons reveal that the
linear arrangement of genes along a chromosome is not static. Fig. 4-19
Large chunks move from chromosome to chromosome.
Chromosomes Exist in Different States
Throughout the Life of a Cell
Cells grow and divide. This repeated process is called the cell
cycle (more on
this later).
At the next stage (mitosis), the chromosomes get
compacted and packaged so that they can be delivered to both cells after cell
division.
These condensed chromosomes are what is normally shown in textbooks.
Normally, chromosomes are decondensed (and therefore barely visible by even the most powerful microscope). Fig. 4-21
Genes are expressed when the chromosomes are decondensed.
a Centromere, Two Telomeres, and Replication Origins
Replication origin Ð locations along the
chromosome where the replication machinery initiates chromosome duplication.
Centromere Ð a region of DNA the mitotic spindle
attaches so that it can drag the chromosome to the daughter cells (during cell
division).
Telomere Ð DNA sequences that act as ÔcapsÕ, protecting the ends of
chromosomes. Fig. 4-22
Sometimes chromosomes can get
damaged, like when they break.
The cell has machinery to
repair broken chromosomes.
Remember that normal
chromosomes are linear.
The ends of normal chromosomes
would be recognized as broken, if not for telomeres.
DNA Molecules Are Highly Condensed in Chromosomes
Parts of chromosomes are packaged to varying degrees.
The level of packaging is quite dynamic,
reflecting the need to access/sequester the genetic information.
Nucleosomes Are the Basic Unit of Eucaryotic Chromosome
Structure
Chromosomes are normally covered with many
different proteins, each having a different role in managing the genetic
information.
The DNA plus these proteins is generally referred
to as chromatin. Fig. 4-23
The major proteins that packages DNA are called histones.
Histone H2A, histone H2B,
histone H3, and histone H4
These are collectively referred
to as core histones
Several histones get together to form a protein
complex, in which 150-200 base pairs of DNA wrap around. Fig. 4-25
The histone complex plus the
DNA is called a nucleosome.
Actually 146 bp (base pairs)
are in contact with the histones.
~50 bp form a linker between adjacent nucleosomes.
The beginning part of each
histone polypeptide chain reach out like arms, and help regulate the
accessibility of the DNA.
Called amino-terminal tails. Fig. 4-32
Nucleosomes make chromosomal DNA look like beads
on a string.
The Structure of the Nucleosome Core Particle Reveals How DNA Is
Packaged Fig. 4-25
The Positioning of Nucleosomes on DNA Is Determined by Both
DNA Flexibility and Other DNA-bound Proteins
Nucleosomes Are Usually Packed Together into a Compact Chromatin
The Ôbeads-on-a-stringÕ 10 nm fiber are usually compacted further
by histone H1 to form a 30 nm fiber.
The chromatin fiber toggles between the 10 and 30
nm fiber, when it is generally decondensed (not in mitosis) and the genetic
info needs to be read or not read.
Fig. 4-31
The amino terminal tails of the core histones may
also contribute to formation of the 30 nm fiber. Fig.
4-32
ATP-driven Chromatin Remodeling Machines Change Nucleosome
Structure
To access the genetic information the histones must be moved or removed.
Chromatin remodeling complexes move and/or remove
histones. Fig. 4-33
Chromatin remodeling complexes control gene expression. Fig. 4-34
There are different kinds of chromatin remodeling complexes.
Some use the energy of ATP hydrolysis to move
histones around.
Covalent Modification of the Histone Tails Can Profoundly Affect
The core histone amino terminal tails are
subjected to covalent modification.
Covalent modification alters what the ÔtailsÕ can
do to the DNA, resulting in a different functional state of the chromatin
(i.e., accessible vs. not accessible).
Note that DNA is negatively
charged (phosphate backbone).
Histones are positively charged
(lysine and arginine side chains).
Modified histone tails lose
their positive charge.
What is the consequence of this
with regard to histone Ð DNA interactions?
Lysine side chains are acetylated by histone
acetyltransferases (called HATs).
HAT are enzymes that convert
lysine to acetyl lysine.
Enzymes that remove the acetyl
group are called histone deacetylases (HDACs).
Other enzymes can methylate the lysines
Others phosphorylate serines on the tail.
*Proteins bind to modified
histone tails!
Summary
Eukaryotic
DNA resides in a group of polynucleotide chains called chromosomes.
Genes
are scattered and fragmented through the chromosomes, covering <2%.
Chromosomal
DNA is packaged into chromatin by histones, forming nucleosomes.
Chromosomes
are continuously condensing (compacting) and decondensing for purpose of
packaging and transport (mitosis), and for accessing/sequestering genetic
information.
Accessibility
is regulated by enzymes that modify chromatin by physically moving histones or
by covalently modifying histones.
THE GLOBAL STRUCTURE OF CHROMOSOMES
Lampbrush Chromosomes Contain Loops of Decondensed Chromatin
Drosophila Polytene Chromosomes Are Arranged in
Alternating Bands
and Interbands
Both Bands and Interbands in Polytene Chromosomes Contain Genes
Individual Polytene Chromosome Bands Can Unfold and Refold as a
Unit
Heterochromatin Is Highly Organized and Usually
Resistant to
Gene Expression
Highly condensed chromatin that is generally devoid of genes.
However, heterochromatin does play important roles in chromosome maintenance.
Genes that are placed in heterochromatin (by
scientist) are generally in active.
The same gene place in normally
active euchromatin is active
Centromeres Are Also Packaged into Heterochromatin
Heterochromatin May Provide a Defense Mechanism Against Mobile
DNA Elements
Mitotic Chromosomes Are Formed from Chromatin in Its Most
Condensed State Fig. 4-55
Each Mitotic Chromosome Contains a Characteristic Pattern of Very
Large Domains
Nucleus Fig. 4-60
Summary
Chromatin structure is quite diverse and largely unknown
CHAPTER 5
DNA REPLICATION, REPAIR AND RECOMBINATION
THE MAINTENANCE OF DNA SEQUENCES
DNA encodes all the information necessary to make
an organism.
In order for an organism or cell to reproduce, it
must make a nearly exact copy of its DNA.
I said ÒnearlyÓ, since mistakes during DNA
replication are the essence of evolutionary change.
A permanent change in the DNA is called a mutation.
E. coli makes a permanent mistake (mutation) about
once every billion nucleotides of replicated DNA.
Many Mutations in Proteins Are Deleterious and Are Eliminated by
Natural Selection
Low Mutation Rates Are Necessary for Life as We Know It
Mutations at very low rates are essential for
evolution.
However, high mutation rates in germ cells are
detrimental to the species
Germ cells are sperm and egg, which go to make the next generation.
High mutation rates in somatic cells cause a
variety of diseases, including cancer.
Somatic cells are all non-germ cells (like, skin cells, liver cells, brain cells, etc.)
Cancer is an uncontrolled proliferation of cells.
Your body has built-in mechanisms to stop cells from dividing.
If it didnÕt, since human cells
take about a day to duplicate themselves, you would be as large as the entire
class and weight about 20 tons!
Mutations can inactivate those
growth control mechanisms, which would lead to cancer.
Summary
Mutations are rare and unhealthy for the
organism, but are the driving force behind evolution (adaptation to a changing
environment).
DNA REPLICATION MECHANISMS
DNA replication is fundamental to all organisms.
All organisms replicate their DNA the same way.
E. coli has been used as the model system.
Base-pairing Underlies DNA Replication and DNA Repair
One strand acts as a template for the other
strand. Fig. 5-2
Remember:
A pairs with T, and C with G.
Fig. 5-3
DNA polymerase is the name of the enzyme that replicates DNA. Fig. 5-4
The substrates for DNA polymerase are:
dATP (deoxy ATP)
dTTP
dCTP
dGTP
Collectively, these four
nucleotide substrates are called dNTPs.
WhatÕs the difference between
dATP and ATP?
How many phosphates does dGTP
have?
Also
need are a template and a 3Õ ÐOH from the growing polynucleotide chain.
The DNA Replication Fork Is Asymmetrical
Replication forks are the point at which DNA
replication is occurring. Fig. 5-6
Replication proceeds in the 5Õ (five prime) to 3Õ (three prime) direction, NOT in the 3Õto 5Õ direction! Fig. 5-7
Therefore the replication fork must be
asymmetric. Fig. 5-8
Remember that the each strand in the DNA double helix is antiparallel.
Continuous polymerization in the same direction as
the fork is called leading strand synthesis.
Polymerization in the direction opposite to the
fork has to be discontinuous, and is called lagging strand synthesis (I guess
because it takes so long).
Why must lagging strand synthesis be discontinuous (i.e., synthesized in short stretches)?
What the heck are Okazaki fragments?
What was the name of the scientist who discovered these fragments?
You canÕt just leave them there as fragments.
How do Okazaki fragments get stitched together?
Mechanisms
Normally base-pairing
interactions (What kind? Hydrogen bonding Ð good) between the incoming
nucleotide base and the template dictate which of the four possible nucleotides
is accepted.
Random nucleotides of all sorts
are constantly diffusing in and out of the active site. Fig. 5-9
Two mechanisms keep the correct
nucleotide in the active site long enough to react with the 3Õ end of the
growing polynucleotide chain;
1. A and G have
big bases; C and T have small bases.
Only a big and small one can occupy the tight quarters of the active
site at the same time.
2. Proper
base-pairing keeps the correct nucleotide there long enough to react.
Believe it or not, the nucleotide bases can morph into other chemical structures.
This happens very rarely.
Called tautomerization
The morphed structures can fool DNA polymerase.
For example, C can morph into something that pairs with A instead of G.
After it gets incorporated into
the growing polynucleotide chain, it can morph back.
This happens about once for every 100,000 nucleotides incorporated.
If left uncorrected, this mutation frequency would be lethal to the cell.
Before moving on and incorporating the next
nucleotide, DNA polymerase looks back and checks to see whether the newly
incorporated nucleotide is correctly base-paired.
In reality, its hard for the growing 3Õ end to stay in the enzyme active site if it is not properly base-paired (and thus positioned) with the template strand.
An unpaired nucleotide at the 3Õ end is Ôflapping
around in the breezeÕ. Fig. 5-10
This nucleotide will ÔflapÕ into a different
active site located next door to the polymerization active site.
This adjacent active site cuts
off the unpaired nucleotide.
This enzyme activity is called
a 3Õ-to-5Õ proofreading exonuclease.
About one out of every 100
misincorporations is missed by this proofreading activity.
The result is continued DNA
polymerization, with an incorporated mutation.
Fortunately, there is one final check. Another enzyme scans the DNA looking
for mis-matches.
If it finds one, it chops out the mismatched nucleotide (more on this later).
This Ômismatch repairÕ is able to find 99 out of
100 mismatches.
If you put together all the proofreading
mechanisms, nucleotide misincorporation occurs about once every billion
incorporation events. Table 5-1
Only DNA Replication in the 5Õ-to-3Õ Direction Allows Efficient
Primer Molecules on the Lagging Strand
DNA polymerase can only
polymerize off of a 3Õ end.
Remember, it needs an ÐOH to attack the phosphate on the incoming nucleotide triphosphate.
Why? I donÕt know. Perhaps DNA polymerase canÕt proofread
as well on the first nucleotide??
Anyway, the cell initiates DNA replication by first polymerizing RNA on the template instead of DNA. Fig. 5-12
This RNA is called a primer, because it
primes DNA synthesis (i.e., gets it going).
The enzyme is called DNA
primase, and is a kind of RNA polymerase.
The RNA is about 10 nucleotides
long.
The RNA/DNA double helix looks about the same as a DNA double helix except for those two chemical properties unique to RNA. Remember what those two properties are?
On the lagging strand, each Okazaki fragment
begins with an RNA primer.
Synthesis of Okazaki fragments initiates at intervals of about every 200 nucleotides along the template.
How many RNA primers are required for the leading strand?
Once the RNA primer is made, DNA polymerase
displaces primase and begins polymerizing DNA. Fig.
5-13
The RNA is then erased (removed) by an RNase
RNases hydrolyze an RNA polymer into individual nucleotide monophosphates.
On the lagging strand, DNA polymerase polymerizes until it runs into the prior Okazaki fragment.
The fragments are joined by a DNA ligase. Fig.
5-14
of the Replication Fork
The DNA double helix is very stable, meaning that
it is very hard to pull apart the two strands.
What kind of interactions prevent strand separation?
Separation of the two strands is called denaturation.
In order for DNA polymerase to insert new
nucleotides, the strands have to be separated, so that the bases can pair with
the correct nucleotide.
A DNA helicase separates the DNA
strands. Fig. 5-15, 5-16
This requires a lot of energy.
Guess where this energy comes
from?
But the two strands can re-anneal (or renature, same thing) to form the double helix again.
Another problem with single stranded DNA is that it tends to form base-pairs with itself, particularly if a complementary sequence is nearby. Fig. 5-17
These intramolecular interactions are called hairpins.
Want to guess why they are called hairpins?
So what else might be
important to keep the strands separated?
by a Sliding Ring
After DNA polymerase adds a
nucleotide to the growing polynucleotide chain, one of two things can happen:
1.
DNA polymerase can step forward and add another nucleotide.
2.
Or it can dissociate from the template, and diffuse away.
When would it want to step forward?
When would it want to dissociate?
DNA
polymerase has the natural tendency to dissociate, so a sliding clamp holds it on the DNA. Fig. 5-19
The sliding clamp forms a ring around the DNA.
You can see why, on a very long piece of DNA how the sliding
clamp cannot fall off the DNA.
So, how does the clamp get on the DNA?
A clamp loader separates the ring, allowing it to
encircle the DNA.
Do you think energy is required
to do this?
How is that energy supplied?
When DNA polymerase runs into the next Okazaki
fragment, it dissociates. Fig. 5-20
The Proteins at a Replication Fork Cooperate to Form a Replication
Machine
So lets review what we have so far:
|
Protein |
Function |
|
DNA
helicase |
Separates
DNA strands |
|
SSB |
Keeps
single-stranded DNA from re-annealing |
|
Primase |
Lays
down the RNA primer |
|
Clamp
loader |
Loads
the sliding clamp onto DNA |
|
Sliding
clamp |
Holds
the DNA polymerase on the template |
|
DNA
polymerase |
Makes
the DNA |
|
RNase |
Removes
the RNA primer |
|
DNA
ligase |
Joins
together Okazaki fragments |
Actually there are a lot more proteins involved
but we wonÕt get in to them.
Many of these proteins work closely together and
so they form complexes.
For example, the primase and helicase form a complex called the primosome.
Actually, the whole enchilada is called a
replisome. Fig. 5-22
A Strand-directed Mismatch Repair System Removes Replication
Errors That Escape from the Replication Machine
A protein complex scans newly replicated
DNA for bulges in the DNA helix. Fig. 5-23
You can imagine how a protein
can sense a bulging DNA.
These bulges correspond to
mismatches, due to incorrect nucleotide incorporation (after fooling DNA
polymerases proofreading activity).
The scanning must happen very
soon after the new strand is made
The complex is called the mismatch repair complex.
The mismatch repair complex, cuts out the
mismatched nucleotide (and a bunch of surrounding nucleotides as well).
DNA polymerase then comes along a fills in the
gap.
Big, big problem: How does the mismatch repair complex know which of the two
opposing mismatched nucleotides is the wrongly incorporated (the other being
the parental template)?
Solution: Have the parental strand marked in some way so that the mismatch repair complex know who the parent was.
How to mark?
In bacteria, methylate the ÔAÕ
of a GATC nucleotide sequence of the parental strand.
In eukaryotes, make a break (called a nick) in the
newly synthesized strand.
Long before replication is
initiated a DNA methylase goes along marking the bacterial DNA.
The newly synthesized strand is
unmethylated.
So the mismatch repair complex finds the unmethylated strand and cleaves it. No problem!
So there is a race between the mismatch repair
complex and the DNA methylase.
Without the mismatch repair complex you will get more mutations in you DNA and be more susceptible to certain cancers.
DNA Topoisomerases Prevent DNA Tangling During Replication
Strand separation by the DNA helicases causes a
topological problem. Fig. 5-24
The DNA helix ahead of the replication fork get
twisted upon itself.
.
Some topoisomerases break (nick) on of the strands
of the double helix, allowing the other strand to swivel. Fig. 5-25
Is
energy required for this?
DNA Replication Is Similar in Eucaryotes and Bacteria
Summary
DNA replication begins by separating the DNA
strands.
Next, an RNA primer laid down.
DNA polymerase initiates off the primer.
A sliding clamp keeps the DNA polymerase on the
template.
DNA polymerase proceeds continuously on one
strand and discontinuously on the other.
Topoisomerases keep down the supercoiling.
DNA polymerase proofreads the DNA to make sure
it put in the correct nucleotide.
A mismatch repair complex further proofreads
the DNA immediately after DNA polymerase has done its thing.
THE INITIATION AND COMPLETION OF DNA
REPLICATION IN CHROMOSOMES
DNA Synthesis Begins at Replication Origins
DNA replication begins at very precise locations. Fig. 5-29
The replication machinery does
not begin at the end of the chromosome, or at random locations.
Why?
In bacteria, there are no
chromsomal ends.
Eukaryotic chromosomes are too
long to do end-to-end replication.
The replication machinery may
physically interfere with gene expression.
Head-on collision of DNA
polymerase and RNA polymerase Ð ouch!
Co-ordination of the two
polymerases could alleviate this problem.
Certain regions of chromosomes
need to replication before other regions.
A specific sequence of nucleotides, comprising the
replication origin, binds to proteins that specialize in recruiting the
replication machinery.
In bacteria, the origin recognition protein is called dnaA protein (generically called Ôinitiator proteinsÕ in the text).
In eukaryotes, the origin is
recognized by the Origin Recognition Complex (ORC).
Another feature of replication origins is that part of the sequence has a lot of ÔAÕ and ÔTÕ nucleotides. Why?
Hint 1: A-T base pairs are bonded by two hydrogen bonds (G-C, by three).
Hint 2: DNA replication requires strand
separation.
Bacterial DNA replication is regulated at the
point of initiation. Fig. 5-31
Once initiation begins, it continues until the whole chromosome is duplicated.
How do you prevent reinitiation? Fig. 5-32
Mark the parental strands, and only assemble dnaA if both strands are parental.
DNA methylases chemically
modify (ÔmarkÕ) the DNA.
Eucaryotic Chromosomes Contain Multiple Origins of Replication
Eukaryotic chromosomes are large, so there is a
need for multiple origins of replication.
A bacterial chromosome has a few
million base pairs.
A eukaryotic chromosome has hundreds
of millions of base pairs.
In Eucaryotes DNA Replication Takes Place During Only One Part
of the Cell Cycle
There are four phases to the eukaryotic cell cycle (growth and duplication of a cell):
Fig. 5-34
á
G1
á
S
á
G2
á
M
DNA synthesis occurs during S phase.
Different Regions on the Same Chromosome Replicate at Distinct
Highly Condensed Chromatin Replicates Late, While Genes in Less
Condensed Chromatin Tend to Replicate Early
Well-defined DNA Sequences Serve as Replication Origins in a
Simple
Eucaryote, the Budding Yeast
A replication origin in yeast is called an ARS
(autonomously replicating sequence).
An ARS is a DNA sequence of about 150 base pairs
in length, and binds multiple protein complexes.
How might you use the power of genetics to isolate
an ARS? Fig. 5-36
Sequencing of the entire yeast genome has revealed
the location of ARSs Fig. 5-37
An ARS has binding sites for multiple protein complexes. Fig. 5-38
of Replication
This protein complex is called an ORC (origin recognition complex).
An ORC binds to a portion of an ARS.
An ORC is bound to the chromosome throughout the
cell cycle.
During S-phase the ORC is phosphorylated, allowing
it to recruit other initiation factors (helicase, etc.)
Replication Have Been Difficult to Identify
How does the replication machinery move through
nucleosomes? Fig. 5-41
Remember chromatin remodelling
complexes?
Where do the nucleosomes go after the replication fork has passed?
How do additional nucleosomes get assembled?
Chromatin assembly factors (CAFs) help assemble
histones and DNA into a nucleosome.
Telomerase Replicates the Ends of Chromosomes
How do eukaryotes replicate the ends of their
linear chromosomes? Fig. 5-43
Remember, the RNA primer is
laid down, then removed.
How do you replicate that part
of the chromosome?
If you donÕt, repeated cell
divisions will lead to progressively shortened telomeres.
Telomerase extends the chromosomal end using an RNA template.
< :p>
Since telomerase repeatedly
extends the chromosomal ends,
eukaryotic chromosomes have repeating sequences at their telomeres.
A DNA polymerase Ð primase protein complex then performs lagging strand synthesis.
*Note: The problem of replicating the actual chromosomal end never actually gets resolved.
Telomeres are packaged into different protein complexes and have different structures than the rest of the chromosome.
Telomere Length Is Regulated by Cells and Organisms
Somatic cells are initially formed with the full
complement of telomeric repeats.
Telomerase is not made in somatic cells.
Somatic cells can undergo only a limited number of
cell division due to progressive telomere shortening.
In every cell division the
telomere gets a bit shorter.
After a number of cell
division, the telomeric repeats are gone, and subsequent cell divisions lead to
progressive loss of coding information (genes) located near the telomeres.
Cells without these genes die
(called replicative cell senescence).
This might explain in part why we stop growing.
It also might provide a mechanism to prevent
uncontrolled cell growth (cancer).
Summary
DNA replication begins at precise locations on
the chromosome called origins.
Bacteria have one origin, eukaryotes have
multiple origins.
An origin recognition complex binds to origins
and recruits the replication machinery.
In eukaryotes, DNA synthesis occurs during S-phase
of the cell cycle.
Telomerase is used to maintain the ends of
eukaryotic chromosomes.
Telomerase is a reverse transcriptase that uses
RNA to extend chromosomal ends.
Telomerase is turned off in somatic cells and
turned on in cancer cells.
DNA REPAIR
Genetic variability allows a species to evolve and
adapt to a changing environment.
Genetic stability is important for the functioning
of an organism.
Genetic instability leads to cancer, aging, death, and other not-so-fun things. Table 5-2
Your body is constantly bombarded by solar
radiation (nice tan!), environmental toxins (smoke that cigarette), and
metabolic by-products (have a big lunch).
This stuff damages (mutates) your DNA.
Mutate means a chemical change in a nucleotide, which might alter its coding information.
Also this stuff can actually break your chromosomes.
Each cell of your body acquires thousands of mutations a day.
Fortunately for you, you have several potent DNA
repair
machines.
<1 mutation in a thousand escapes these repair
machines.
Without DNA Repair, Spontaneous DNA Damage Would Rapidly
Mutations can occur at a variety of
locations. Fig. 5-46
Thymidine dimers Fig.
5-48
Depurination
Fig. 5-47
Deamination
Fig. 5-52
DNA replication prior to DNA repair propagates the
mutation. Fig. 5-49
The DNA Double Helix Is Readily Repaired
The beauty of the DNA double helix is that each
strand provides an information ÔbackupÕ for the other strand.
Damage one strand and information on the complimentary strand can be used to repair the damage.
Each and every cell contains many copies of a
variety of DNA repair machines.
DNA Damage Can Be Removed by More Than One Pathway
Common features:
Damage is cut out.
Nondamaged strand is used as a template to restore
the correct nucleotide sequence.
What enzyme would do this?
Base excision repair Fig.
5-50A
Initially just the damage base is removed
The enzyme that does this is
called a DNA glycosylase
Every kind of base mutation has
a particular kind of DNA glycosylase designed for its removal.
The enzyme scans the DNA,
Ôflipping outÕ each base and checking it for damage. Fig. 5-51
Then the sugar phosphate is removed by a different
enzyme
AP endonuclease
Nucleotide excision repair Fig.
5-50B
Targets large bulky mutations
Thymine dimers form UV light.
Carcinogens in tobacco smoke
covalently attach to DNA.
A long patch of damage DNA is excised (12
nucleotides stretch).
The Chemistry of the DNA Bases Facilitates Damage Detection
The four bases (GATC) were selected during evolution in part because, deamination does not lead to interconversion to another base.
Instead they are recognized as non-natural and are removed.
Double-Strand Breaks are Efficiently Repaired
Some types of DNA damaging agents break both
strand of DNA. Fig. 5-53
If left unrepaired the chromosomes would fragment.
A DNA ligase can rejoin the fragments. Called nonhomologous end-joining
Typically there is a loss of a
base.
The vast majority of the mammalian genome is noncoding, so losing a nucleotide is no problem.
Homologous chromosomes can be used for
repair. Called homologous
end-joining.
Remember that we get one set of
chromosomes from Mom and the other from Dad.
Cells use the information from
one of the chromosomes to repair the other chromosome.
If the double strand break
occurs during the G2 phase of the cell cycle, sister chromosomes can be used.
Cells Can Produce DNA Repair Enzymes in Response to DNA
Damage
When cells are bombarded by DNA and/or protein damaging agents they undergo a stress response.
Called heat shock response in
the textbook, since the response pathway was first characterized by a response
to high temperatures.
Stress proteins (or heat shock
proteins) are produce that help stabilize the cell against damage.
SOS response occurs in response to DNA damage.
Single-stranded DNA is one indicator of DNA
damage.
Results from UV light.
SOS response is initiated when the recA protein binds to the single-stranded
DNA.
This leads the expression of a number of DNA
repair genes.
One of the SOS induced proteins is an error-prone
DNA polymerase.
It is used when there is so
much DNA damage that the template strand cannot be used to restore the genetic
info.
Because it is damaged too.
Better to put in any nucleotide
and take your chances, rather than leave a lethal gap in the DNA.
DNA Damage Delays Progression of the Cell Cycle
When you have damaged DNA, the last thing you want
to do is duplicate your chromosomes.
Mutations would get replicated.