| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
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Techniques | Exams |
Outline:
We now consider the Structure of Genomes, leading to the Function of Genomes.
We have seen that all Genomes are composed
of Chromosomes and that the Genetic Material is
Nucleic Acid found within these Chromosomes, generally
double-stranded DNA molecules. We have also reviewed the structure
of Nucleic Acid at two of its three levels:
1. Primary structure -
all Covalent Bond Structure: Nucleotides, Phosphodiester bonds
2. Secondary structure
- major Hydrogen Bond Structure: interstrand structure
forming B-form dsDNA, intrastrand stem-helix structure
forming A-form RNA.
Chromosome Structure is more complex than just that of these two levels of nucleic acid, varying in complexity from single Nucleic Acid molecules to complex Nucleic Acid - Protein complexes. Here we examine the structure of Prokaryotic Chromosomes and then subsequently the structure of Eukaryotic Chromosomes.
The third level of Nucleic
Acid structure is Tertiary Structure.
This is any structure other than Primary (covalent bonds)
or Secondary (helical), and is found mainly in DNA molecules.
1. Common Examples in double-stranded DNA (dsDNA):
Linear: break in backbone of both strands,
opposite each other or close
If close, then have short region of single-stranded DNA (ssDNA)
Open circular: break in one strand, other strand is closed
circular ... ocDNA
Closed circular: both strands are closed, no breaks
also called Covalently Closed Circular DNA (cccDNA or ccDNA)
Supercoiled DNA: ... [Brown, Fig 6.11]
covalently closed circular DNA contains helical structure IN ADDITION
to the Watson-Crick double helical structure ...
Other examples of Tertiary Structure:
Bends in DNA ... Kinks in DNA ...
DNA replication forks ... [Brown, Fig 12.15, 12.16, etc]
Recombination intermediate forms, eg Halliday junctions
... [Brown, Fig 13.24, Fig 13.27 ... see two animations here and here]
DNA damage distortions, eg Thymine dimers ... [Brown, Fig 13.20]
Bacterial Nucleoid structure ... [Brown, Fig 6.12]
Such structures can be induced in DNA by binding of DNA-binding
Proteins,
eg Restriction enzyme EcoRI ... Phage Lambda repressors CI and
Cro ... [Brown, Fig 7.20]
Binding of Zinc finger proteins ...
2. Topology of Supercoiled
DNA: see Voet-Voet,
pgs 873-879
Described by three parameters:
Linking Number L:
the number of Complete Revolutions of one DNA strand about the
other
Twisting Number T:
1. the total number of turns about the dsDNA Helical Axis
2. in other words: the total number of turns of the DNA duplex
itself
T is a property of the double helical structure by itself ...
independent of tertiary structure ...
Writhing Number W:
total number of turns about the superhelical axis
Lewin: "turning of the axis of the duplex in space"
Lodish: "describes the pathway of the DNA backbone in space"
V-V: "the number of turns the duplex axis makes about the
superhelical axis"
A measure of the "superhelicity" of the DNA molecule
Relation between the 3 parameters: L = T + W
Thus, L represents the TOTAL number of turns of one DNA strand
about the other
and is the SUM of the turns due to the Watson-Crick helix, T,
and the turns due to the superhelical content of the DNA, W.
V-V 3: 28-36 shows interconversion between these ...
Supercoils in Biological DNA:
All native DNA which is closed circular shows superhelical content
...
and nearly all show the SAME superhelical content:
One negative supercoil per 20 Watson-Crick turns
Thus, W = -1 for every T = 20
This leads to a SuperHelical Density, defined as W / T,
of:
-1 / 20 = - 0.05 supercoils / turn
of the helix
(note the minus sign ... this indicates negative supercoils
in native supercoiled dsDNA)
Since there are 10 basepairs
(bp) per turn of the helix in B DNA, this gives:
1 negative supercoil per 200 bp
Problem: Given a cccDNA with 4000 bp, how many
negative supercoils will this DNA have assuming that it has the
normal superhelical density?
Answer: 4000 bp => 4000/10 = 400 turns (approximately
... close enough) ... thus: T = 400
Superhelical Density: W/T = -0.05 = -1/20 ... Thus: W = -T/20
= -400/20 = -20
In other words, this DNA has 20 negative supercoils ...
Also: L = T + W = 400 + (-20) = 400 - 20 = 380 ... Linking Number:
total turns for this DNA
Another example: animal virus SV40 dsDNA
Taken from Lodish et
al, "Molecular Cell Biology", Figure 4-15, with corrections
made.
Can change Superhelical content via intercalating agents such
as Ethidium Bromide:
As EtBr is added to cccDNA, L remains constant and T decreases
...
W must then increase ... from Negative
number ... through 0 ... to Positive numbers
Function of Supercoiled DNA:
1. More easily open
DNA during DNA unwinding, eg during DNA replication
2. Protect DNA from nuclease attack and other damage
3. Provide intermediate, condensed structure for higher structure
forms, eg
nucleoid structure, chromosome structure
3. Enzymatic Interconversion
between Topological Forms of DNA:
... see Voet-Voet,
pgs 879-882
a. DNases - DeoxyRiboNucleases - Nucleases:
Nick in dsDNA: break in backbone of ONE DNA Strand
cccDNA --> ocDNA ... Supercoiled DNA is relaxed ...
W --> 0, L and T can change
ocDNA and linear DNA remain ocDNA and linear DNA when nicked
Chop in dsDNA: break in backbone of BOTH DNA Strands
Both cccDNA and ocDNA --> linear DNA
W = 0, L = T ... no Supercoiling ...
b. TopoIsomerases - Enzymes which alter the Topology of DNA...
[Brown, Fig 12.4]
Convert closed circular DNA from one Isomer (W is changed)
to another
These enzymes do not work on Open Circular or Linear DNA
Two types: Topoisomerase I and Topoisomerase II
Topoisomerase I: ... [Brown, Fig 12.4]
relaxes Negatively supercoiled DNA with change in W of 1 ...
W increases by 1: W change = + 1
Mechanism: Voet-Voet, Figure 28-41
transient break introduced into one DNA strand
the uncut strand passes through the break in the other strand
the break is resealed in the cut DNA strand
Negative superhelical content is decreased by one:
W change = + 1, T change = 0, L change = + 1
Enzyme Example: E. coli Topo I (Omega protein) ...
topA gene ... V-V, Fig 28-42
Enzyme covalently attached (Tyr residue) to 5'-P at break in the
cut DNA strand
Also: E. coli Topo III is a Type I topoisomerase
Eukaryotic Type I topoisomerases do the same to dsDNA as do prokaryotic Type I topos, but the details of the mechanism are different.
DNA Example:
For a cccDNA with W
= -20, TopoI would change W to -19, then to -18, etc, all the
way to zero, thereby completely relaxing the negatively
supercoiled DNA substrate.
Topoisomerase II: ... [Brown, Fig 12.4]
changes superhelical content of cccDNA by units of two ... W change
= + or - 2
W change = - 2 ... negative supercoils are INTRODUCED
into cccDNA: DNA gyrase
W change = + 2 ... negative supercoils are REMOVED
from cccDNA:
DNA gyrase and other Topo II enzymes
Mechanism:
Enzyme folds DNA over itself forming Positive and Negative nodes
Enzyme holds the Positive Node in place
Breaks BOTH strands of one DNA segment at Negative node
The unbroken DNA segment is passed through the double-strand break
in
the broken DNA segment
Both strands in the broken DNA segment are resealed
Thus: changes occur in 2's ...
2 strands are broken
2 strands pass through the break
2 negative or positive supercoils are introduced: W change = +
2 or W change = - 2
ATP required for introduction of supercoils
Enzyme Example: E. coli DNA gyrase ... 2 subunits
gyrA (nalA) 105 kd 2 of each subunit: tetramer protein
gyrB (cou) 95 kd ..
DNA structure, including Tertiary Structure, accounts for essentially all of the structure found in simple Replicons such as prokaryotic Plasmids.
However, additional Chromosome Structure considerations are found in bacterial and higher chromosomes, due to Protein and RNA interactions.
1. Nucloids
Superhelical domains ... 40-80 kb in size ... [Brown, Fig 6.12; Research Brief 6.1]
separated from each other by RNA or Protein "locks"
Possible Proteins:
1. Expect small basic Proteins ... ala Histones in Eukaryotes
2. HU: hupA, hupB genes ... 2 subunits
HU: basic protein ... binds DNA in Sequence-independent manner ... has Stimulatory effect in DNA replication
BUT deletions in hupA or hupB
have only partial effect on cell growth
(cold-sensitive phenotype for one hupA mutant)
3. Other protein candidates:
family of HU-like proteins ... could provide 'back-up' functions
IHF - Integration Host Factor, discovered via phage lambda integration
FIS - Filament Inducing Substance ...
Summary of Structures of the Bacterial Chromosome:
In the bacterial cell: a Nucleoid region, no Nuclear Membrane, DNA is highly condensed (> 10 mg/ml) in region less than 1 µm in diameter.
When isolated in the presence of high salt (1 M NaCl), the bacterial Chromosome is isolated as the bacterial Nucleoid or Folded Chromosome ... [Brown, Fig 6.12; Nucleoid observed using Electron Microscopy]
When isolated from cells very gently in the absence of high salt, proteins are removed from the nucleoid and the chromosome has then been "seen" using tritium Autoradioaugraphy as a circle with two forks (Replication Forks) ... [Brown, Fig 12.6]
When isolated from cells using typical isolation
procedures, proteins are removed from the nucleoid and the chromosome
(a single large circular DNA molecule of 4.5 million bp, diameter
~ 1 millimeter) is broken into linear fragments of size
5-500 kb.
Definition - Packing Ratio:
DNA-Protein complexes that keep DNA in vivo within well-defined volume
Highly condensed, viscous complexes ... 10 mg/ml in bacteria, up to 100 mg/ml in eukaryotes ... Gel
Packing Ratio (PR): length of DNA /length of unit containing it
Topological problems: how do
proteins find their binding sites?
How does DNA function in replication,
transcription, recombination, repair when present in vivo in such highly viscous complexes
of high Packing Ratios?
C. Eukaryotic Organelle Genomes ... [Brown, Section 6.1.2]
These are plasmid-type replicons found in organelles
other than the Nucleus in Eukaryotic cells.
All Mitochondrial and Chloroplasts contain such DNA molecules.
1. Structure of Organelle Genomes
Most Organelle Genomes are circular dsDNA molecules, although can exist as linear molecules and some protists have mitochondrial genomes that only exist as linear dsDNA molecules.
Sizes of Organelle Genomes ... [Brown, Table 6.3]
Mitochondrial dsDNA molecules (mtDNA) vary from about 15 kb to
several hundred kb.
Examples: ... [Brown, Fig 6.9]
Chloroplast dsDNA sizes vary less, and are
around 150 kb +/- 25 kb.
Example: ... [Brown, Fig 6.10]
2. Genetic Content of Organelle Genomes ... [Brown, Table 6.4]
Mitochondrial Genomes have genes that tend
to encode mainly proteins involved in:
1. oxidative phosphorylation and electron transport levels of
respiration
2. ribosome structure and function of Protein Biosynthesis
Consonant with encoding Protein Biosynthesis proteins, Mitochondrial Proteins also encode structural RNA genes for Ribosome structure and function: rRNA and tRNA genes
Mitochondria execute their own Protein Biosynthesis in the Expression of mtDNA genes, and the protein synthesis mechanisms and Ribosome Structures found in Mitochondria are much more similar to Prokaryotes than to Eukaryotes.
3. Origins of Organelle Genomes
This Prokaryotic nature of Protein Synthesis in Eukaryotic Organelles, coupled with the observation that Protein Sequences encoded by Organellar Genes more closely resemble Bacterial Homologs than they do Eukaryotic cognate Proteins, has led to the Endosymbiont Theory for Origins of these types of Eukaryotic Organelles
Endosymbiont Theory:
these Organelle Types arose via a symbiotic relationship between
Eukaryotic Cells and free-living Bacteria. During evolution, the
two organisms became completely interdependent, each losing its
ability to live in the absence of the other. The Bacteria further
evolved the unique structures and functions found in specific
Organelles such as Mitochondria and Chloroplasts, at the expense
of losing much original genetic information and proteins expressed
by these lost genes. Some of these genes "lost" by the
Organelle are found in the Nucleus of the Eukaryotic Cell, perhaps
having been 'transferred' in some way during evolution from the
ingested Prokaryotic Cell.
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith
