Organization and Control of Eukaryotic Genomes
(Chapter 19 Old ; 18 New)
I. Problem:
**2x1013 meters of DNA present in one adult human =
(length 1 base pair) x (# base pairs per cell) x (# cells in body) =
(0.34x10-9m) x (6x109) x (1013)
(Equivalent to the distance from the Earth to the Sun and back)
**20-30x103 genes in the human genome
Tangled Mess? How to package? How to move during mitosis? How to replicate?
How to “find” genes and express them?
II. -Eukaryotic Chromatin Structure:
A. Levels of Packing of DNA (Fig. 19.1)
1. nucleosomes – “beads on a string”, 4 histones (H2A, H2B, H3, H4), 2
molecules each.
2. chromatin fiber – coiled nucleosomes due to phosphorylated histone H1.
3. looped domains – chromatin fiber forms on a protein scaffold.
4. chromosomes – looped domains folds and compacts.
B. Terms
1. Histones: + charged proteins (arginine/lysine) attract to – PO4
2. Nucleosomes: 4 histone protein structure around which is wrapped DNA
with linker DNA between; linker variable-average 200 nucleotide pair
repeat, 146 in nucleosome.
III. Dynamics of chromatin:
A. Chromatin: the cell’s way of packaging and handling its library of
information in DNA
B. Nucleosomes remain essentially intact through out the cell cycle. They
undergo structural changes during replication and transcription to allow
polymerases to pass.
1. During mitosis:
a. Metaphase: chromatin condenses into chromosome;
usually no transcription occurs.
b. Interphase: chromatin fibers attach to inside of
nuclear envelope. Site-specific transcription occurs
in nucleus.
Euchromatin-less compacted DNA, not necessarily
transcribed (Active chromatin=transcribed)
Heterochromatin-compacted DNA, generally not
transcribed.
IV. Genome Organization:
A. Non-coding DNA (large part of total DNA, 97%):
1. Regulatory seguences- ex. Promoters
2. Introns
3. Repetitive DNA:
a. Tandemly repetitive DNA – up to several hundred
thousand repeats of 1-10 bp, 10-15% of genome.
Found in telomeres, centromeres
Related diseases:
Fragile x syndrome: A major cause of mental
retardation. Due to long stretches of tandemly repeated
“CGG” nucleotide triplets 5’ of the fragile x gene. 30
repeats is normal, hundreds of repeats causes that site to
be fragile. Number of repeats increase through
generations.
Huntington’s: Affects the nervous system. Due to long
stretches of tandemly repeated “CAG” triplets.
Translation of these triplets yield proteins with long
strings of glutamines. The number of triplet repeats
correlate with the severity of the disease.
b. interspersed repetitive DNA – hundreds to thousand
bp repeats of similar but not identical sequence, 2540% of genome, includes transposons.
B. multigene families:
collection of identical or similar genes, likely derived during
evolution from a single ancestral gene, clustered or dispersed,
some function, others pseudogenes
1. Examples:
rRNAs (identical) (Fig. 19.2)
globin genes (divergent) (Fig19.3)
C. gene amplification: rRNA genes, tandem repeats; some eggs have
extrachromosomal DNA (frogs, ~1000 DNA circles of tandem repeat
rRNA in that many nucleoli)
D. gene loss: chromosomes or parts thereof lost in somatic cells of some
insects, worms
E. rearrangements:
1. transposons, retrotransposons (Fig. 19.5): jumping genes
2. immunoglobin genes: recombination in differentiating B lymphocytes
(Fig 19.6); recombinases bringing about are active only in B (and T) cell
differentiation
V. Control of Gene Expression (Fig. 19.7)
A. Chromatin Modifications: crude control
1. DNA methylation –CH3 added to bases, usually cytosine in –CGsequences, after synthesis of DNA (methylation of opposite strand)
tends to repress expression long term inactivation during cell
differentiation
2. Acetylation of Histones –COCH3 added, change confirmation, allow
transcription; acetylation/deacetylation often assoc. with
transcription complex
B. Control of Initiation of Transcription: DNA – protein-protein interactions rule
fine specificity of expression
1. Overview of Gene-Transcript Organization (Fig. 19.8)
2. Control regions: (Fig. 19.8-19.9)
a. Promoter: region of DNA assembling transcription initiation
complex
b. Enhancers: distal control regions, bind activators
3. Transcription factors (Fig. 19.10) DNA: binding domains
a. Helix turn helix
b. Zinc finger
c. Leucine zippers
d. All have protein binding domains
C. RNA processing:
1. alternative splicing (see Fig. 17.9, 17.10)
a. introns/exons
b. snRNPs (small nulear ribonucleoproteins-snRNA)
-spliceosomes
c. recognize sequences on ends of introns
2. capping 5’ end, 3’ poly (A) tail (Fig. 17.8)
D. RNA Degradation:
1. mRNA lifetimes vary from minutes to days or longer
2. regulated by 3’ untranslated region (3’UTR)
a. enzymatic shortening of poly A tail (AU rich seq.) 5’ cap
removal, 5’ degradation
b. cleaved by endonuclease (repeat seq. in 3’UTR)
E. Translational Control:
1. 5’ leader sequence binds factors initiating translation
2. poly A tail: affects translation as well as mRNA stability
3. global control in eggs-activation of translation at fertilization
4. light activation in plants
F. Protein processing:
1. Transport: targeted to cell membrane domain (mistarget of chloride
channel in cystic fibrosis), or secretion, or cytoplasmic
2. Glycosylation: add sugars (membrane proteins)
a. Activation: phosphorylation/dephosphorylation
b. Proteolytic processing: pro-versus mature forms
G. Protein degradation:
1. Ubiquitin-small protein attached by ubiquitin conjugation enzymes
-recognizing delfects/targets
2. Proteasome recognizes- “flushes” them
DNA STRUCTURE - The Double Helix
- Backbone - 2 antiparallel polynucleotide strands
deoxyribose sugar, phosphate
Two strands in DNA run antiparallel
- one 5’ to 3’ (Watson strand)
- one 3’ to 5’ (Crick strand)
Steps – nitrogen bases
Purines (2 ring structure)
Adenine (A) and Guanine (G)
Pyrimidines (1 ring structure)
Thymine (T) and Cytosine (C)
Bases pair by hydrogen bonds according to Chargaff’s Rules:
A-T
2 H+ bonds
G-C
3 H+ bonds
Double Helix facts
1.) 2 nm wide
2.) bases stacked 0.35 nm apart
3.) makes one turn every 3.4 nm along its length
4.) about 10 base pairs per turn of helix
5.) has major and minor groove
Forms of DNA
1.) B-DNA
- long, thin right handed helix
- most common in cells
2.) A-DNA
- short, wide, right-handed helix
- dehydrated DNA
3.) Z-DNA
- long, then left-handed helix
- discovered in-vitro
4.) C-DNA
- copied/constructed from RNA using reversetranscriptase
Properties of DNA
1.) Bending
- DNA is threadlike and bendable–
2.) Solubility
- soluble in aqueous solutions (water)
- insoluble in alcohol
3.) Melting Temperature
- temp at which double stranded DNA comes apart into 2 single stranded
polynucleotide chains (denatures)
G-C rich DNA needs higher temps due to more H+ bonds
The Search for Genetic Material – The Experiments
Frederick Griffith
- provided evidence that genetic material is a specific molecule
- used 2 strains of S. pneumonia R and S
- transformed R strain into S strain
Transformation
- change in phenotype due to the assimilation of external DNA by a cell
Alfred Hershey & Martha Chase
- provided evidence that DNA was the heredity material
- used “tagged” bacteriophages, virus that infects bacteria
http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio21.swf::Hershey and Chase Experiment
Erwin Chargaff
- # of adenines approx. = # of thymines
- # of guanines approx. = # of cytosines
- Chargaff’s Rules
Watson and Crick
- Discovered the double helix structure of DNA
Meselson and Stahl
 Demonstrated semiconservative replication of DNA leading to each identical
strand being composed of 1 backbone of old and 1 backbone of new material.
http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio22.swf::Meselson%20and%20Stahl%20Experiment
DNA Replication – Copying DNA
When does DNA replication take place?
 S-phase of cell cycle
 An exact copy is made
1.) Complex
- Helical molecule must untwist while it copies 2 antiparallel strands simultaneously
- Requires cooperation of over a dozen enzymes and
other proteins
2.) Extremely Rapid
- Up to 500 nucleotides added per second
- Takes only a few hours to copy 6 billion bases of a
single human
3.) Accurate
- Only about 1 in a billion nucleotides incorrectly paired
Requires:
1.) Starting point
- Origin of replication
2.) Unwinding the helix
- Helicase
3.) Minimum strain on the DNA
- topoisomerase
4.) copying and proof-reading new DNA
- DNA polymerase
Process
http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio23.swf::How Nucleotides are Added in DNA Replication
http://www.courses.fas.harvard.edu/~biotext/animations/replication1.swf
****Replication is continuous for leading strand but discontinuous for the lagging
strand****
Leading Strand
1.) Helicase binds at the origin of replication
- Synthesis proceeds in both directions
- Replication “bubbles” fuse where they meet
- Helicase binds at the replication fork and unwinds the “parental” helix
2.) Topoisomerase nick and unwinds strands to release stress of unwinding
3.) Single stranded binding proteins stabilizes the unwound parental helix and keeps
the separated strands apart
4.) DNA polymerase adds on to the primer and finishes copying the entire piece of
DNA
- Polymerase links the new nucleotides to the growing strand
- Strands grow in the 5’ to 3’ direction
- New nucleotides are added only to the 3’ end
Lagging Strand
1.) Primase makes a short RNA primer
2.) DNA polymerase replaces the RNA primer with DNA to produce Okazaki
fragments
3.) DNA ligase joins the Okazaki fragments
Proof-reading
1.) DNA polymerase proofreads each mewly added nucleotide against the
template
- Error detected, polymerase removes and replaces it before continuing
(mismatch repair)
2.) Damage in existing DNA can be repaired by “cutting” out the damaged
segment and filling in the gap
- DNA polymerase and DNA ligase fill in the gap