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Chapter 4 Notes: Part 1
Biochemistry 461
Fall 2010
CHAPTER 4, Part 1: LECTURE TOPICS:
DNA and RNA - MOLECULES OF HEREDITY
1) DNA/RNA structures, nomenclature, shorthand conventions
2) DNA and RNA as genetic material
3) General properties of DNA Double Helix
4) Basic mechanism of DNA replication
5) Important physical-chemical properties of DNA
Reminder from Chapter 25 Lectures: Nucleic acids (DNA, RNA): are polymers
consisting of pentose sugars, phosphate, and bases
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RNA and DNA are chains of ribo (or dexyribo-)nucleosides connected by 5' to 3'
phosphodiester bonds. They are polynucleotides.
** The chains have polarity, and are always written and read from 5' to 3'
(left to right) as in the examples of DNA and RNA trinucleotides. (Fig.5.3)
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Nucleic Acid Shorthand Notations:
Trinucleotide shorthand example (Fig.5.7): pACG
(see also Powerpoint version)
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DNA and RNA as genetic material:
Proofs
!
Transformation with pure DNA (Pneumococcus, 1928, 1944)
!
Bacteriophage T2 DNA not Protein (1952)
BACTERIOPHAGE VIRUS LIFE CYCLE
Scenario:
attach to cell
inject DNA
reproduce (new DNA and proteins)
kill cells
release progeny (new) virus
infect more cells
new
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!
TMV - RNA as genetic information(1955)
!
Retroviruses: RNA to DNA (1970's)
(Fig.5.23)
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!
PRIONS - Proteins (not DNA or RNA are transmissible agents (1970's to present)
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DNA (chromosomes) are long as seen by electron microsocopy of E. coli DNA
(Fig.5.8)
DNA molecules are doble-helices - deduced from x-ray diffraction patterns of B-DNA
in 1938. The data showed that there had to be regularly spaced units at 3.4 Angstroms
along the helix. (Fig.5.10)
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DNA Structure: Watson-Crick Double Helix (1953)
!
B-DNA was the form upon which Watson and Crick derived their model. DNA
occurs primarily in this form in vivo. The important features of this model of DNA
are:
!
Two helical polynucleotide chains coil around a common axis. The chains are
antiparallel in polarity.
!
The purine and pyrimidine bases are inside the helix, whereas the phosphate
and deoxyribose units are on the outside.
!
The planes of the bases are perpendicular to the helix axis.
!
The planes of the sugars are nearly at right angles to those of the bases.
!
The helix diameter is 20Å.
!
Adjacent bases are spaced 3.4Å along the helix axis. Adjacent bases are related
by a rotation of 36 degrees. Hence, the helical structure repeats after each ten
residues on each chain (360 degrees, and at intervals of 34Å).
!
The two chains are held together by hydrogen bonds between pairs of bases.
Adenine always pairs with thymine. Guanine always pairs with cytosine. [The
specificity of the pairing of bases is the most important aspect of the DNA double helix. Watson
and Crick deduced - while building the model - that adenine must pair with thymine, and guanine
with cytosine, because of steric and hydrogen-bonding features. Chemical analysis of DNA
(Chargaff, 1950) showed that A=T and G=C, as also predicted by the Watson-Crick structure.]
!
Any sequence of bases may occur along a polynucleotide chain. The precise
sequence of bases carries the genetic information.
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DNA helix - flat version shows polarity
and base pairing:
DNA Helical ladder as
In real B-DNA
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Space-filling version of Watson and Crick B-DNA model: (Fig.5.11a)
Top View,
(Figs.5.1.b and 5.13)
looking down helical axis:
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A-T and G-C base pairs: (Fig 5.12)
A-T base pair
G-C base pair
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A three base pair stack of DNA showing:
!
antiparallel polarity (3'-5' and 5'-3')
!
bases parallel to each other and perpendicular to helix axis
!
dexyribose perpendicular to bases
!
phosphates on outside of helix
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REPLICATION OF DNA: Watson and Crick deduced that the complementary chains of
a double helix are templates for each other in replication. The base sequence of each
chain determines that of daughter DNA molecules.
!
DNA replication is semiconservative: Shown by the Meselson-Stahl (1958)
experiment [Figs. 4-13, Fig 14, 15 ].
!
DNA strands with 15N and 14N containing bases were analyzed through several
generations of E. coli cell division (and DNA replication).
!
Changes in buoyant density (see physicochemical
properties of DNA below) of old (15N) vs new (14N)-DNA
after each generation proved that parent DNA strands
were conserved during replication.
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DNA Replication: Primer - Template relationships
Primer (free 3'-OH)
(DNA) n+1
(DNA) n+2
Template (base-paired to primer)
DNA Replication by DNA polymerase requires:
!
!
!
All four dNTPs (dATP, dGTP, dCTP, and dTTP)
!
A primer chain with a free 3'-OH end
!
A template strand to which the primer is base-paired
!
Double-stranded DNA that is fully intact and lacking a free 3'-OH end will
not be replicated (ex: intact circular DNA)
Mg++
[***HINT: Draw your own Template-Primer complex to see how it works]
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DNA Replication: Phosphodiester bond formation by DNA polymerase
Summary of the basic mechanism of DNA replication:
!
Replication is semiconservative
!
DNA polymerase requires a template-primer complex
!
dNTPs are the substrates for DNA synthesis
!
PPi breakdown to 2 moles of Pi (catalyzed by pyrophosphatase) drives
DNA synthesis reaction
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Some important physical-chemical properties of DNA:
!
Reversible separation-reassociation of DNA strands
[Denature/renature/melting temperature (Tm) proportional to %G-C bp]
!
Buoyant density analysis of DNA shows it is proportional to %G-C bp.
!
Enormous range of lengths - :m to cm lengths
!
Conformation of DNA can be linear, circles (open/supercoiled), Single(SS) or double-stranded (DS).
Reversible strand separation of DNA by heat (and other denaturants)
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!
DNA UV-light absorbance by
single and double-stranded
DNA
!
Denaturation - A-T
vs G-C base pairs
!
Melting temperature of DNA proportional to %G-C base pairs
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Enormous size ranges of DNA molecules in nature:
!
From about 2 microns (virus DNA) to 2.1cm (Drosophila largest chromosome) to
1.6 - 8.2 cm (human chromosome)
!
Note: Useful conversion factors: [1 kb DNA = 10 3 base pairs = 0.34 x 10-6
meters]
!
Most have double-stranded DNA, but some viruses (X174) have single stranded
DNA in the virus. [These DNA molecules replicate in host cells in a double-stranded replicative
form (RF), which gives rise to new single-stranded viral DNA that is packaged into new virus
particles].
!
Some DNAs are circular (E. coli chromosome, mitochondrial DNA [Fig.5.18],
plasmids), some are linear (T7 DNA, lambda phage DNA)
!
Circular DNA molecules can be supercoiled or relaxed. Supercoiling is necessary
for "packaging" in cells. [Fig.5.18]
Definitions:
1 kb (kilobase) of DNA or RNA = 1,000 bases long (single or double-stranded).
1 kb double-stranded DNA = 0.34 :m long
1 kb 1 kb double-stranded DNA = 660 kd
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SUMMARY: DNA and RNA - Molecules of Heredity
1) DNA/RNA structures
!
Know nomenclature + shorthand conventions
2) DNA and RNA as genetic material
!
Transformation with DNA
!
T2 DNA not Protein
!
TMV RNA
!
RNA to DNA (Retroviruses)
!
PRIONS (Proteins as transmissible agent?)
3) General properties of Watson-Crick DNA Double Helix
!
Antiparallel strands
!
Right-handed helix
!
10 bp/helix turn; 3.4Angstroms/bp
!
bases on inside and parallel
!
bases perpendicular to deoxyribose-P chain
!
A-(double bond)-T and G-(triple bond)-C base pairs
4) Basic mechanism of DNA replication
!
Semiconservative
!
DNA polymerase I (template-primer complex)
!
PPi --> 2Pi (pyrophosphatase) drives DNA synthesis
5) Some important physical-chemical properties of DNA
!
Buoyant density (proportional to %G-C)
!
Denature-renature - melting (T, proportional to %G-C)
!
:m to cm lengths
!
linear, circles (open/supercoiled), SS, DS
!
RNA molecules usually exist as partially double-stranded structures