- DNA is made of deoxyribonucleotides consisting of a base, a deoxyribose
sugar, and a phosphate group, in equal proportions. RNA differs only in using
ribose, instead of deoxyribose.
- The DNA molecule is the carrier of genetic information in all procaryotic
and eucaryotic organisms. RNA or DNA can be the genetic material of viruses.
- DNA molecules can be very long. They are threadlike (linear) or circular
(supercoiled, usually). RNA is linear, usually.
- The sequence of the bases along a DNA strand (polynucleotide)
determines its genetic function.
- DNA is generally double-stranded, with each strand being a complement
of the other---this property suggests that each may be a template for the
other when DNA replicates.
- Using a DNA strand as a template, DNA polymerase I replicates DNA with an
error frequency of about 1x10-8.
DNA(RNA) COMPOSITION AND DNA
STRUCTURE
- Four bases (adenine, guanine, cytosine and thymine) are found
in DNA. In RNA, uracil replaces thymine.
- Bases and sugars (deoxyribose or ribose) in DNA and RNA are joined
in glycosidic bonds between C1 of sugar and N1 of
pyrimidine bases (C, T, U) and N9 of purine bases.
- deoxyribonucleoside triphosphates (ex: dATP) are precursors of
DNA synthesis (for RNA, use ribonucleotides - ATP, for example)
- Polynucleotides (DNA and RNA): nucleosides
are joined by phosphate groups in phosphodiester
links between 3'-OH of one nucleoside's sugar and the 5'-OH of the adjacent
nucleoside's sugar. [Fig. 5-3,
p. 119]
- DNA (and RNA) chains have polarity.
One end is the 5'-end (which is not attached to another
nucleotide) and the other end is a 3'-end (which is also not
attached to another nucleotide). [Fig.
5-3, p. 119]
- Nomenclature
and shorthand conventions
for DNA and RNA: The polarity (by convention) is with
the 5'-end at the left and the 3'-end at the right, as in (5') ACG
(3'). A more detailed schematic
can be drawn with the phosphates indicated. Always draw a base sequence
in the 5'3' direction (especially on quiz or test answers!) If you don't
specify the ends, we will read according to convention. One should
still know the structure
involved in these shorthand conventions.
DNA AND RNA AS GENETIC INFORMATION
Proof that genetic information
resides in DNA (and RNA) came from several important experiments:
- Transformation of pneumococcus bacteria
by cell extracts and "purified" DNA [Illustrations p.13a,
p.13b]
a. Griffith (1928) - transformation of nonpathogenic (nonencapsulated,
R colonies) to pathogenic (encapsulated, S colonies) by a cell-free
extract of heat-killed S colonies.
b. Avery, et al (1944) - transformation of R (nonpathogenic) to
S (pathogenic) by purified DNA.
- Transfer
of DNA only as genetic information of T2 bacteriophage - (1952,
Hershey and Chase experiment). 32P-DNA (but not 35S-protein)
of T2 enters E. coli cells and is transferred to newly
synthesized T2 bacteriophage DNA. [Exp.
Results, Bacteriophage
Life Cycle]
- Also suggestive is that DNA content of cells is constant. Diploid cells
have twice the DNA of haploid cells.
- Fraenkel-Conrat & Williams (1955) -tobacco
mosaic virus RNA (not protein) contains the genetic information
of the virus [electron microsopy].
This is a simple system. The experiment used mixed reconstituted infectious
virus to solve the problem.
- RNA tumor viruses (retroviruses) like
Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) have RNA
as genetic information. But the virus has an enzyme called reverse transcriptase,
which copies the RNA into a DNA. First, a DNA strand complementary to
the viral RNA strand is synthesized; this is then used as a template for
synthesis of its complement, resulting in a double stranded DNA.
This DNA is integrated into a chromosome of the host. Its genetic information
is expressed by synthesizing a RNA (viral RNA) strand and proteins needed
for new virus particles. Retroviruses
got their name from this flow of genetic information from RNA to DNA.
Prions?
- Is a protein the transmissible agent (?) of slow brain
diseases like kuru, scrapie, mad cow disease -see August 1990 Scientific
American, pp.24 - 26 about possible transmission of scrapie from sheep to
man as Creutzfeldt-Jacob disease(kuru-like) in Czechoslovakia.
WATSON-CRICK DNA DOUBLE HELIX
In 1953, James Watson and Francis Crick deduced the three-dimensional structure
of DNA and immediately suggested a mechanism of replication from analysis
of x-ray diffraction photographs
of DNA fibers taken by Rosalind Franklin and Maurice Wilkins. The important
features of their model of DNA are:
- 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. [Fig.
5-11]
- The diameter of the helix is 20Å. Adjacent bases are separated
by 3.4Å along the helix axis and related by a rotation of 36 degrees.
Hence, the helical structure repeats after ten residues on each chain; that
is, at intervals of 34Å. [Fig.
5-11]
- The two chains are held together by hydrogen bonds between pairs of bases.
Adenine always pairs with thymine. Guanine always pairs with cytosine. [Fig.
5-12 ]
- Any sequence of bases may occur along a polynucleotide chain. The
precise sequence of bases carries the genetic information.
- The most important aspect of the DNA double helix is the specificity
of the pairing of bases. Watson and Crick deduced 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
predicted by the Watson-Crick structure.
- B-DNA was the form upon which Watson and Crick derived their model. DNA
occurs primarily in this form in vivo. [Fig.
5-10]
REPLICATION OF DNA
- 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
as shown by the Meselson-Stahl (1958) experiment [Fig.
5-14, Fig. 5-15 ]. DNA
strands with 15N and 14N nucleotides were followed
through several generations of E. coli cell division (and
DNA replication). Changes in buoyant
density of old (15N) vs new (14N) DNA after each
generation (see physicochemical properties of DNA below) proved that parent
DNA strands were conserved during replication.
- DNA
polymerases (such as E. coli DNA polymerase I) use a DNA
template strand and dNTPs to synthesize, one base at a time, a complementary
strand of DNA from the 5' to 3'-end of the newly synthesized strand.
PPi as a product of the reaction drives DNA syntheses [Fig.
5-22, Fig. 5-21] . The
error rate is 10-8 per base pair. Correct base-pairing is a necessity
for each step of the polymerase reaction.
DNA polymerase I requires: [***HINT:
Draw your own Template-Primer complex to see how it works]
(a) All four dNTPs (dATP, dGTP, dCTP, and dTTP)
(b) Mg++
(c) A primer chain with a free 3'-OH end
(d) A template strand to which the primer is base-paired
(e) Double-stranded DNA that is fully intact and lacking a free 3'-OH end
will not be replicated (ex: intact circular DNA)
- DNA polymerase chain-elongation mechanism: a nucleophilic attack of the
free 3'-OH terminus of the primer on the -P of the dNTP substrate. [Fig.
5-25]
PHYSICOCHEMICAL
PROPERTIES OF DNA
- Single-stranded DNA absorbs more light at 260nm than double-stranded
DNA [Fig. 5-17]
- DNA can be reversibly melted
(strands separated and reannealed - helix/coil transition)
-
DNA molecules
range
in length from about 2 microns (virus) to 2.1cm (
Drosophila
largest chromosome) to 1.6 - 8.2 cm (human chromosome)
Useful
conversion factors: [1 kb DNA = 10
3 base pairs =
0.34 x 10
-6 meters]
- Some DNA molecules are circular (E. coli chromosome, mitochondrial
DNA, plasmids) and 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]
- Some viruses (X174) have single stranded DNA in the virus. These DNA
molecules replicate in their host cells in a double-stranded replicative
form (RF), which also gives rise to new single-stranded viral DNA that is
packaged into virus particles.
DNA/RNA structures
· Know nomenclature
+ shorthand conventions
DNA and RNA as genetic material
· Transformation with DNA
· T2
DNA not Protein
· TMV RNA
· RNA to DNA (Retroviruses)
· ??? PRIONS ??? (Proteins as transmissible
agent?)
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
Basic mechanism of DNA replication
· Semiconservative
· DNA polymerase I (template-primer complex)
· PPi --> 2Pi (pyrophosphatase) drives DNA synthesis
Some important physical-chemical
properties of DNA
· Buoyant density (proportional to %G-C)
· Denature-renature - melting (T, proportional to %G-C)
· µm to cm lengths
· linear, circles (open/supercoiled), SS, DS
Last Updated on 9/4//2002 by Dr.
Don P. Bourque
Email: dbourque@u.arizona.edu
![[electron microsopy]](GRAPHICS/Chapter5-1/Slide14.JPG){kind=link}
