2.

7 DNA Replication, Transcription and

Translation
Essential Idea: Genetic information in DNA can be accurately copied and can be
translated to make the proteins needed by the cell.
(Nature of science: Obtaining evidence for scientific theories Meselson and Stahl
obtained evidence for the semi-conservative replication of DNA.)
Watson and Crick, Nature 1953:
'It has not escaped our notice that the specific pairing we have postulated immediately
suggests a possible copying mechanism for the genetic material'.
-

DNA replication begins with a partial unwinding of the double helix at an area known as
the replication fork
unwinding is accomplished by an enzyme known as DNA helicase
(single strand binding proteins bind to the strands to keep them apart)
as the two DNA strands separate or unzip, bases are exposed and DNA polymerase
(enzyme) moves into position at the point where synthesis will begin
short segment of RNA known as an RNA primer "primes" or tells DNA polymerase to
start synthesis at certain points
the primer is "laid down" complementary to the DNA template by an enzyme known as
RNA polymerase or primase

DNA polymerase uses the DNA strand as a template to add nucleotides one by one in
a complementary manner (A to T and G to C)
template strand is ALWAYS read in the 3' to 5' direction (that is, starting from the 3'
end of the template and reading the nucleotides in order toward the 5' end of the
template)
new DNA strand (since it is complementary) MUST BE SYNTHESIZED in the 5' to 3'
direction
DNA polymerase catalyzes the formation of the hydrogen bonds between each arriving
nucleotide and the nucleotides on the template strand
because the original DNA strands are complementary and run antiparallel, only one
new strand can begin at the 3' end of the template DNA and grow continuously as the
point of replication (the replication fork) moves along the template DNA

this strand is known as the leading strand (ie. 5' to 3')

the other strand is known as the lagging strand (ie. 3' to 5')
with the lagging strand as a template, the new strand must grow in the opposite
direction because it is complementary
but DNA polymerase only works in one direction so in order to make this new strand,
replication is carried out in small sections at a time (discontiguous) - producing of a
series of short pieces of new DNA called Okazaki fragments
to make sure that this new strand of short segments is made into a continuous strand,
the sections are joined by the action of an enzyme called DNA ligase
last step is for an enzyme to come along and remove the existing RNA primers (you
don't want RNA in your DNA now that the primers have served their purpose, do you?)
and then fill in the gaps with DNA
done by another type of DNA polymerase which has the ability to chew up the primers
(dismantle them) and replace them with DNA
human genome estimated to be approximately 3 billion base pairs DNA replication
takes several hours
many replication forks, forming replication bubbles produce many segments of DNA
strands that eventually meet up together and are joined to form the newly synthesized
double helix
incredible how few mistakes are made in this process - error occurs only about once in
each 10-100 billion bases!
above process ensures DNA molecule copied precisely from one generation to next
e.g.(1a)ATG CTC ATT TTA GGG CCC ATA CTC
= 24 bases thus we can write the complementary sequence of the other helix as:
12 (2a)TAC GAG TAA AAT CCC GGG TAT GAG

- in DNA replication (1a) will act as the template for a new complementary sequence of
bases (1b):
Copy 1:
(1a)ATG CTC ATT TTA GGG CCC ATA CTC
(1b)TAC GAG TAA AAT CCC GGG TAT GAG

and (2a) will act as a template for the other new complementary sequence (2b)
Copy 2:
(2a)TAC GAG TAA AAT CCC GGG TAT GAG
(2b)ATG CTC ATT TTA GGG CCC ATA CTC

therefore after replication, the sequence of copy 1 (1a)(1b) and copy 2 (2a) (2b) are
identical to each other, and importantly they are also identical in base sequence to the
original base sequence of (1a)(2a)

the DNA base sequence of the double helix is conserved from one replication to
another

as cells divide the DNA is copied so that each new cell possesses a copy of each of the
original DNA molecules

since each new strand is complementary to its old template strand, two identical new
copies of the DNA double helix are produced during replication

in each new helix, one strand is the old template and the other is newly synthesized, a
result described by saying that the replication is semi-conservative

Crick described the DNA replication process and the fitting together of two DNA strands
as being like a hand in a glove - the hand and glove separate, a new hand forms inside
the old glove, and a new glove forms around the old hand as a result, two identical
copies now exist!

Application: the use of Taq polymerase in polymerase chain reaction (PCR)

- process invented by Kary Mullis (won Nobel prize in 1993)
- PCR is used to make multiple copies of DNA from a very small sample
- recall first step in replication requires helicase to unzip DNA molecule
- can use high temp. to break the H-bonds holding the two DNA strands together
- so process of PCR involves changing temp. of sample to achieve replication:
- Step 1. Heat sample to 95C for 15 seconds this separates the DNA strands
- Step 2. Rapidly decrease temp. to 54 C this would allow strands to reanneal
(pair up), but
included in the sample are large numbers of single stranded DNA primers
that bind to the specific section of DNA that we want to replicate (this
takes care of need for RNA primers and also prevent DNA strand from
reannealing)
- Step 3. Replicaton of DNA using parent strands with primers attached as
templates
Taq polymerase comes from a bacteria, Thermus aquaticus, found in hot
springs need this particular polymerase because it doesnt denature at
high temp.
Taq polymerase functions best at 72 C, so raise temp. of mixture Taq
polymerase adds approx. 1000 nucleotides/sec! - allow enough time for
desired DNA sequence to be replicated
- Step 4. Start the PCR cycle all over again by heating sample to 95 C, etc.
After 30 cycles, approx.
billion copies of desired sequence produced (less than one hour)!
Transcription & Translation

transcription formation of an RNA strand (messenger RNA or mRNA) that is

complementary to a DNA strand

process of transcription:
- occurs in nucleus
- initiation RNA polymerase (enzyme) unwinds & unzips DNA helix at promoter region
of gene marks
where transcription should begin
- one of DNA strands forms template for transcription resulting mRNA is
complementary to this
strand template strand in known as the anti-sense strand
- elongation transcription moves in 5 to 3 direction
- RNA polymerase uses free nucleoside triphosphates to extend the growing
mRNA molecule
- as nucleosides linked on, 2 phosphates removed, forming RNA nucleotides 5
end of nucleotides added to 3 end of mRNA strand

- termination RNA polymerase reaches termination region of gene marks end of

coding sequence
- now have mRNA strand that is complementary to template DNA except that
thymine is replaced with uracil
- non-template strand same base sequence as mRNA (except for uracils)
known as sense strand
- mRNA strand detaches from DNA template, travels out of nucleus to cytoplasm
for protein synthesis
- DNA helix reforms

in the genetic code, each of the 4 bases are arranged in triplets called codons
- 43 = 64 possible triplets of DNA
- these codons then get translated into mRNA which codes for 20 amino acids and also
start and stop codons or signals
- genetic code is said to be degenerate more than one codon can code for the same
amino acid

translation an amino acid sequence is created using the mRNA as a template

- occurs in the cytoplasm
- after mRNA is made in the nucleus gets processed before moves out into
cytoplasm
- introns segments of DNA that are not part of the gene but get transcribed
these corresponding
segments in the mRNA need to be removed or spliced before protein
synthesis occurs
- exons actual portions of the gene (get expressed) part that gets translated
into actual amino
acid sequence

- translation carried out by free ribosomes (synthesize polypeptides for use

inside cell) or bound ribosomes on RER (make polypeptides for secretion)

- structure of ribosomes made up of proteins & rRNA (ribosomal RNA) (recall

rRNA is produced
in nucleolus) - 2 subunits large & small
- has binding sites for mRNA and tRNA (2 tRNA can bind at a time)

- structure of tRNA single strand of RNA, but contains sections where base
pairing creates loops
- at 3 end of molecule binding site for amino acid
- at loop across from amino acid binding site anticodon complementary
to specific codon on mRNA
- at least one tRNA for every amino acid, twenty different tRNA activating
enzymes join amino acid to appropriate tRNA according to anticodon
present requires ATP to form bond, when amino acid released from tRNA,
resulting energy from breaking this bond will be used to form peptide
bond between amino acid growing polypeptide chain

process of translation:
- initiation small subunit of ribosome binds to mRNA where start codon AUG is located
- tRNA with complementary anticodon (UAC) on loop binds to the AUG (tRNA is
carrying the corresponding amino acid to AUG methionine (met)) called
initiator tRNA
- large ribosomal subunit joins small subunit large subunit has two binding sites
initiator tRNA occupies first binding site, site next to it is open and ready for
tRNA that has anticodon complementary to next codon on mRNA
- translation occurs 5 to 3 on the mRNA molecule

- elongation tRNA with complementary anticodon to next mRNA codon binds to empty
site next to
initiator tRNA
- corresponding amino acid is attached to this tRNA and peptide bond forms
between first amino acid and new one requires enzyme and ATP
- first tRNA no longer has amino acid attached it has been transferred to tRNA
in site beside it, so the first tRNA exits ribosome
- ribosome moves over to next codon, attached tRNA now shifted over to first
binding site, makes room for incoming tRNA that has complementary anticodon

- termination process continues until ribosome reaches a stop codon (UAA, UGA, UAG)
stop codon has no complementary tRNA or corresponding amino acid
- release factor protein cuts polypeptide chain from last tRNA
- polypeptide chain, tRNA and mRNA released from ribosome, ribosomal subunits
separate

Application: The production of human insulin in bacteria is an example of the universality of

the genetic code
- diabetes can result from the malfunction or destruction of insulin producing cells in
the pancreas
- treatment involves insulin injections
- in the past, insulin was extracted from the pancreases of pigs and cattle porcine
insulin is almost a direct
match to human insulin (only one amino acid difference)
- however some diabetics developed allergies to these sources of insulin
- 1982 human insulin became available produced by genetically modified E. coli
human insulin gene
inserted into DNA of bacteria, resulting in transcription into mRNA and then
translation into insulin

- yeast cells and safflower plants have also been recently similarly modified
- this ease of gene transfer shows universality of genetic code from human (animal)
to prokaryote, fungus and plant!