D N A

s t r u c t u r e ,
r e p l i c a 0 o n a n d
p r o t e i n s y n t h e s i s
D r W e b b

DNA Structure a History

Frederick Grith (1928)

Oswald Avery (1944)

DNA Structure a History

Erwin Charga (1952)

DNA Structure a History

Wilkins & Franklin (1952)

Watson & Crick (1954)

DNA double helix

one
helical
turn
34

major
groove
12

minor
groove
6

The double helix has direc0onality

Other forms of DNA

Although B form DNA is the primary in
vivo conforma?on DNA RNA hybrids
adopt the A conforma?on and Z DNA
may arise in GC rich stretches of
chromosomal DNA.
A and B DNA are right handed helices
and Z DNA is a leG hand helix.
There are large dierences in the depths
of the grooves.
B DNA major and minor grooves similar
depths
A DNA deep major groove and shallow
minor groove
Z DNA deep minor groove and a major
groove into which the bases are
extruded

DNA Replica0on

It has not escaped our no0ce that

the specic pairing we have
postulated immediately suggests a
possible copying mechanism for the
gene0c material (Watson & Crick,
1953).

Models for DNA Replica0on

1958: Meselson & Stahls Experiment

Model of replica0on in E. coli

DNA polymerase
DNA polymerase I catalyzes forma0on of phosphodiester
bond between 3-OH of the deoxyribose (on the last
nucleo0de) and the 5-phosphate of the dNTP.
Energy for this reac0on is derived from the release of two
of the three phosphates of the dNTP.

A prokaryo0c replica0on Fork

The two parent strands are unwound by DNA helicase

SSB proteins aNach to the unwound strands, preven?ng them from winding back together
DNA polymerase III catalyses the elonga?on of the leading and lagging strands
RNA primers are needed repeatedly on the lagging strand to ini?ate synthesis of the Okazaki
fragments
The RNA primers are removed and the gaps are lled by DNA polymerase I
Finally DNA ligase covalently joins all the fragments together

A Eukaryo0c replica0on fork

Replica0on in Eukaryotes

Each eukaryo0c chromosome is one linear DNA double helix

Average chromosome ~108 base pairs long

With a replica0on rate of 2 kb/minute it would take ~35 days to replicate one
human chromosome

Solu0on ---> DNA replica0on ini0ates at many dierent sites simultaneously.

What about the ends (or telomeres) of

linear chromosomes?

DNA polymerase/ligase cannot ll gap at end of chromosome acer RNA primer is removed. this
gap is not lled, chromosomes would become shorter each round of replica0on!
Solu0on:
1.

Eukaryotes have tandemly repeated sequences at the ends of their chromosomes.

2.

Telomerase (composed of protein and RNA complementary to the telomere repeat) binds to
the terminal telomere repeat and catalyzes the addi0on of of new repeats.

3.

Compensates by lengthening the chromosome.

4.

Absence or muta0on of telomerase ac0vity results in chromosome shortening and limited

cell division.

Protein Synthesis

Transcrip0on
Transcrip0on is the DNA directed synthesis of RNA

Direc0on of Transcrip0on

Like DNA polymerase RNA polymerase can only synthesise nucleic acid in
the 5-3 direc0on while reading a DNA template in the 3-5 direc0on

Transcrip0on
Promoter

Terminator- Rho independant

Transcrip0on in Eukaryotes
Instead of one RNA polymerase,
there are three (RNA Polymerases I,
II, and III) involved in eukaryo?c
transcrip?on.
RNA polymerase I (localized to the
nucleolus) transcribes the rRNA
precursor molecules.
RNA polymerase II produces most
mRNAs and snRNAs.
RNA polymerase III is responbsible
for the produc?on of pre-tRNAs,
5SrRNA and other small RNAs.
The mitochondia and chloroplasts
have their own RNA polymerases.

Transcrip0on in Eukaryotes
All three of the nuclear RNA polymerases do
not bind directly to their promoters but to
proteins [transcrip0on factors] that are in
turn bound to specic DNA sequences that
cons?tute each promoter.

Example-transcrip?on by RNA polymerase II

Requires at least 5 basal transcrip?on
factors, TFIIB, TFIID, TFIIE, TFIIF, TFIIH.

Transcrip0on in Eukaryotes
The mRNAs are longer lived and the processes of
transcrip?on and transla?on are spa?ally and temporally
separated [i.e. transcrip?on occurs in the nucleus, transla?on
occurs in the cytoplasm]
The primary transcript must be modied by the addi?on of
a 5'CAP and a poly(A) tail.
The mature mRNA is 1/10 the size of the primary transcript
due to the removal of introns by RNA splicing.
Eukaryo?c mRNA is monocistronic

Transcrip0on to transla0on

Transla0on

RNA -- Protein
Change from nucleo?de language to amino acid
language
On ribosomes
Vectorial nature preserved
5 end of mRNA becomes amino terminus
of protein
Transla?on depends on gene?c code and is
mediated by tRNA molecules

Transla0on
Ribosome

The ribosome matches the base sequence on the mRNA

tRNA

Transla0on-amino acid ac0va0on

Transla0on -Ini0a0on

The small subunit of the ribosome

binds to a site (Shine-Dalgarno
sequence) upstream of the start of
the message.
It proceeds downstream (5' -> 3')
un?l it encounters the start codon
AUG. Here it is joined by the large
subunit and a special ini0ator tRNA.
The ini?ator tRNA binds to the P site
on the ribosome.

Transla0on -Elonga0on

An?codon of next tRNA binds

to mRNA codon at A site of
ribosome

A pep?de bond is formed

between the amino acid at A
site and previous amino acid at
the P site

The Ribosome then moves

along one codon and the next
tRNA binds at A site

Transla0on -Termina0on
Final codon on mRNA contains termina?on
signal
Releasing factors cleave polypep?de chain
from tRNA that carried nal amino acid.
mRNA released from ribosome and broken
down into nucleo?des.

Eukaryo0c transla0on-major dierences

Transla0on of mRNA is highly regulated in eukaryotes, eg. in rela?on to the cell cycle or in
response to cellular stresses. Mechanisms include regula?on by signal-ac?vated
phosphoryla0on or dephosphoryla0on of ini?a?on and elonga?on factors. In prokaryotes
regula?on occurs mainly at the level of transcrip?on.
Protein factors that mediate and control transla?on are more numerous in eukaryotes than
in prokaryotes. Eukaryo?c factors are designated with the prex "e".
Ribosomes. Eukaryo?c ribosomes are larger. They consist of a 60S large subunit and a 40S
small subunit, which come together to form an 80S par?cle having a mass of 4200 kd,
compared with 2700 kd for the prokaryo?c 70S ribosome.
Ribosome binding involves the 5r cap, rather than a Shine-Dalgarno sequence.
Ini0a0on of protein synthesis is much more complex in eukaryotes, and requires a large
number of protein factors. Some eukaryo?c ini?a?on factors (e.g., eIF3 and eIF4G) serve as
scaolds, with mul?ple domains that bind other proteins during assembly of large ini?a?on
complexes. Usually a pre-ini0a0on complex forms, including several ini?a?on factors along
with the small ribosomal subunit and the loaded ini?ator Met-tRNA which in eukaryotes is
not formylated.

In eukaryotes the process of replica0on, transcrip0on and transla0on occur

in the context of chroma0n

More on that in the next lecture!