DNA as Genetic

Material
Searching for Genetic Material

 Mendel: modes of heredity in pea plants (1865)

 Morgan: genes located on chromosomes
(1910)
 Griffith: bacterial work; transformation:
change in genotype and phenotype due to
assimilation of external substance (DNA) by a
cell (1928)
 Avery et al.: transformation agent was DNA
(1944)
 Hershey & Chase: proving once and for all that
DNA is the genetic material (1952)
The discovery of transformation: Griffith’s experiment (1928)
1944 - Avery, MacLeod, and McCarty expand Griffith’s work
• Mixed R strain with DNA from S strain and isolated S bacteria
• Added DNase which broke down DNA and prevented R bacteria from
transforming to S bacteria
• Proteases (broke down proteins) did not inhibit transformation
• DNA was determined to be the “transforming principle”
 1952 - Hershey and Chase
 Used T2 bacteriophages (phages), a virus that infects bacteria
 Radiolabeled the bacteriophage with S35 (Protein) and P32 (DNA)
 Bacterial cells were infected and put in a blender to remove phage particles
√ DNA, not protein, is the hereditary material
√ Expt: sulfur(S) is in protein, phosphorus (P) is in DNA; only P was found in
host cell
DNA Structure
1953 James Watson and Francis Crick determined the structure of DNA
with the help of Rosalind Franklin and Maurice Wilkins as well as Erwin
Chargaff
• Rosalind Franklin and MauriceWilkins
provided X-ray diffraction data
• Erwin Chargaff determined the ratios
of nitrogen bases in DNA
• DNA replication model - 1953
• DNA bases made up of purine and
pyrimidine
• Nobel Prize - 1962
Structure of nucleotides
Nucleotides have three characteristic components:

A nitrogenous base
A phosphate group (pyrimidines or purine)

A pentose sugar
Pyrimidine & Purine
• The bases are
abbreviated by their
first letters (A, G, C, T,
U).
• The purines (A, G) occur
in both RNA and DNA

• Among the
pyrimidines, C occurs
in both RNA and DNA,
but
• T occurs in DNA, and
• U occurs in RNA
Deoxyribonucleotides
2'-deoxyribose sugar
with a base (here, a purine,
adenine or guanine)
attached to the C-1'
position is a
deoxyribonucleoside (here
deoxyadenosine and
deoxyguanosine).

Phosphorylate the 5' position

and you have a nucleotide(here,
deoxyadenylate or
deoxyguanylate)

Deoxyribonucleotides are abbreviated (for example) A, or dA

(deoxyA), or dAMP (deoxyadenosine monophosphate)
The major deoxyribonucleotides
DNA
Nucleotide monomers
can be linked together via a
phosphodiester linkage
formed between the 3' -OH
of a nucleotide
and the phosphate of the
next nucleotide.
Two ends of the resulting poly-
or oligonucleotide are defined:
The 5' end lacks a nucleotide at
the 5' position,
and the 3' end lacks a nucleotide
at the 3' end position.
 DNA structure
• DNA consists of two helical
chains wound around the
same axis in a right-handed
fashion aligned in an
antiparallel fashion.
• There are 10.5 base pairs, or
36 Å, per turn of the helix.
• Alternating deoxyribose and
phosphate groups on the
backbone form the outside
of the helix.
• The planar purine and
pyrimidine bases of both
strands are stacked inside
the helix.
DNA structure
Interstrand H-bonding between DNA bases

Watson-Crick base pairing

DNA strands
• The antiparallel strands of DNA are
not identical, but are complementary.
• This means that they are positioned
to align complementary base pairs: C
with G, and A with T.
• So you can predict the sequence of
one strand given the sequence of its
complement.
• Useful for information storage and
transfer!
• Note sequence conventionally is given
from the 5' to 3' end
 Ribonucleotides
• The ribose sugar with a base
(here, a pyrimidine, uracil or
cytosine) attached to the
ribose C-1' position is a
ribonucleoside (here, uridine
or cytidine).
• Phosphorylate the 5'
position and you have a
ribonucleotide (here,
uridylate or cytidylate)

• Ribonucleotides are abbreviated (for example) U, or UMP

(uridine monophosphate)
The major ribonucleotides
Nucleotide nomenclature
Nucleotide nomenclature
RNA
Nucleotide monomers
can be linked together via a
phosphodiester linkage
formed between the 3' -OH
of a nucleotide
and the phosphate of the
next nucleotide.
Two ends of the resulting poly-
or oligonucleotide are defined:
The 5' end lacks a nucleotide at
the 5' position,
and the 3' end lacks a nucleotide
at the 3' end position.
Central Dogma of Biology
All cells have common cycles

 Born, eat, replicate, and die

DNA, RNA, and the Flow of
Information
Replication

Transcription Translation
Overview of DNA to RNA to Protein

 A gene is expressed in two steps

1) Transcription: RNA synthesis
2) Translation: Protein synthesis
 DNA Replication: a synopsis
 Occurs in the S phase of the cell cycle
 Replication is semiconservative, with each
DNA strand serving as template for synthesis
of the complementary strand
 DNA synthesis occurs in replicons consisting of
an origin of replication and two diverging
replication forks (bidirectional)
 Each replication fork contains a complex of
enzymes, including DNA polymerase
– Other enzymes include primase, helicase,
topoisomerase
DNA replication is semiconservative
Replication is Fork movement
semiconservative,
with each DNA strand R
e
serving as template p
for synthesis of the l
i
complementary strand c
a
t
This is true for all i
o
eukaryotes, prokaryotes, n
viruses and f
o
bacteriophage r
k
Meselson-Stahl Experiment
Meselson-Stahl Experiment Data

Density after 1 generation = ?

Density after 2 generations = ?

 DNA Synthesis
DNA chain growth
Replication fork
5’ 3’
growth

3’ 5’
DNA chain growth
 Leading and Lagging Strands
 Limitation is imposed by synthesis of
DNA in a 5’ to 3’ direction only
 The two DNA strands are used
differently at replication fork
– leading strand is used for continuous DNA
synthesis
– lagging strand is used in discontinuous
synthesis
• forms Okazaki fragments leading
• fragments joined by DNA ligase

lagging
Lagging strand synthesis
Must supply a primer
(i.e. 3’-OH) to start
DNA synthesis
This is the function of
primase which makes
RNA primers

Must ‘seal’ the DNA

fragments made on the
lagging strand template
This is the function of
DNA ligase
Replication Machine

Role of primase? Role of helicase?

Role of topoisomerase? Role of single-stranded DNA binding proteins?
 Origins of DNA Replication
 DNA replication begins from specific nucleotide
sequences called origins of replication
– recognized by origin recognition proteins that open
the helix and recruit the replication machinery
 DNA synthesis proceeds in both directions
outward from the origin
– replicated double helices being produced ultimately
join each other
– when complete, there are two identical daughter
molecules
Bidirectional DNA Replication
DNA synthesis occurs in replicons consisting
of an origin of replication and two diverging
replication forks (bidirectional)

Fork movement Fork movement

 Transcription
 RNA polymerase catalyzes RNA synthesis
– uses one DNA strand as template
• always the same strand for a given gene
– locally unwinds DNA
– adds free nucleotides to growing RNA strand at 3’
end
• 5’ to 3’ RNA synthesis
• template read 3’ to 5’
• uses rules of base pairing to synthesize complementary RNA
molecule
• starts RNA chain de novo
 Transcript is identical in sequence to
nontemplate strand, except T’s replaced by U’s
Transcription is asymmetric – only one strand of the DNA is
transcribed into RNA; the template strand
The RNA transcript has the same sequence as the
nontemplate strand

RNA is synthesized in a 5’ to 3’ direction only

The template strand is read in the 3’ to 5’ direction
Either strand of the DNA can be used as the template strand
for transcription
However, in any one gene only one strand of the DNA serves as
the template for transcription

DNA
Multiple RNA polymerases
can transcribe a gene
simultaneously creating a
train of RNA polymerases
 RNA polymerases
 Prokaryotes: single RNA polymerase
– Transcribes mRNA, rRNA and tRNA
– Transcription and translation are coupled
 Eukaryotes: three RNA polymerases
– RNA polymerase I transcribes rRNA genes
– RNA polymerase II transcribes protein-encoding genes;
i.e. makes mRNA
• primary transcript will be processed
– RNA polymerase III transcribes tRNA genes and 5S rRNA
genes
 Transcription and translation occur in separate
compartments of the eukaryotic cell
– In organelles they occur in the same compartment
 Translation (protein synthesis)

 Information in mRNA
translated into primary
sequence of a protein in 4
steps:
– ACTIVATION
– INITIATION
– ELONGATION
– TERMINATION
 Translation (protein synthesis)

 ACTIVATION
– Each amino acid
activated by
reacting with ATP
– tRNA synthetase
enzyme attaches
activated amino
acid to own
particular tRNA
Protein synthesis: Translation initiation
Protein synthesis:
elongation (2)
Protein translation:
termination (3)
Protein translation: summary

Elongation

Initiation

Termination
Cell Information: Instruction Book of Life

 DNA, RNA, and

Proteins are examples
of strings written in
either the four-letter
nucleotide alphabet of
DNA and RNA (A C G
T/U)
 or the twenty-letter
amino acid alphabet of
proteins. Each amino
acid is coded by 3
nucleotides called
codon. (Leu, Arg, Met,
etc.)
End