Nota

1. 9/29/2011
DNA structure & function
• DNA is able to accomplish 2 very important things
DNA and RNA for an organism.
– DNA is used to pass genetic information on to the
next generation of organism.
The Molecular Basis of Heredity – Determine an organism’s characteristic by controlling
the synthesis of the protein.
Thus….to understand both of above process…we need
to know its chemical structure!
DNA structure Hydrogen
bond
• DNA is a polymer.
• The monomer units of DNA are nucleotides, and
the polymer is known as a "polynucleotide.“
• Each nucleotide is composed of:
- sugar molecule DNA nucleotide
- a phosphate group
- nitrogenous bases (Adenine, A; Guanine, G;
Cytosine, C; and Thymine, T)
DNA helix
3 hypothesis of DNA replication
• Conservative - old strand acts as a template.
- One daughter strand is the original template
while the other strand is composed entirely out of
new nucleotides.
• Dispersive Model
- Each strand of both daughter molecules
contains a mixture of old and newly synthesized
DNA parts
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2. 9/29/2011
• Semiconservative - old strand splits apart and
acts as a template.
-Both daughter strands are composed of one of
the old strands and one comprised out of new
nucleotides
- Watson & Crick
But, how DNA replication works? Ingredients to make a copy of DNA
• In DNA replication process…it involve 3 process • Template strand
- The DNA serves as a template to guide the
1- Initiation incoming nucleotides.
2- Elongation
3- Termination • DNA polymerase
- The enzyme that helps catalyze in the
polymerization of deoxyribonucleotides into a
DNA strand.
- "reads" an intact DNA strand as a template and
http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replic
uses it to synthesize the new strand.
ation/index.html
• Free 3’ hydrocyl (Primer) • DNA ligase
- DNA polymerase can add a nucleotide onto only - It can catalyze the formation of a
a preexisting 3'-OH group, and, therefore, needs phosphodiester bond given an unattached but
a primer at which it can add the first nucleotide. adjacent 3'OH and 5'phosphate.
• Helicase • Single-stranded binding proteins
- accomplishes unwinding of the original double - important to maintain the stability of the
strand, once supercoiling has been eliminated by replication fork.
the topoisomerase.
• Ribonuclease H  removes RNA primers.
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What happen during initiation process?
• An enzyme, helicase bind (breaking of hydrogen
bonds between bases of the two antiparallel
strands. ) to the DNA and separate the 2 strands
of DNA. Topoisomerase helps helicase!
• The initiation point where the splitting starts is
called "origin of replication“ which create
replication fork!.
• In prokaryotes, DNA replication begins at a
single, fixed location in this molecule, the
replication origin.
• The splitting happens in places of the chains
which are rich in A-T. Why?
• That is because there are only two hydrogen
bonds between Adenine and Thymine (there are
three hydrogen bonds between Cytosine and
Guanine).
• Single-stranded DNA binding protein (SSB)
attached to each strand  prevent re-annealing.
How can entire chromosome be
Now…the elongation process…
replicated during S phase?
DNA replication begins at many • One of the most important steps of DNA
specific sites –replication bubbles!
Replication is the binding of RNA Primase in the
Parental strand
Origin of replication
Daughter strand the initiation point of the 3'-5' parent chain.
• RNA Primase can attract RNA nucleotides which
bind to the DNA nucleotides of the 3'-5' strand
due to the hydrogen bonds between the bases.
• RNA primase build Primer which is strand of
nucleic acid that serves as a starting point for
Two daughter DNA molecules DNA synthesis.
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4. 9/29/2011
• 5’-3’'Template: The 5'-3' template cannot be
• The elongation process is different for the 5'-3'
"read" by DNA Polymerase. Why?
and 3'-5' template.
• DNA polymerase can add free nucleotides to only
• 3'-5' Template: The 5'-3' proceeding daughter
the 3' end of the newly forming strand
strand -that uses a 3'-5' template- is
called leading strand because DNA Polymerase
can "read" the template and continuously adds • The replication of this template is complicated
nucleotides (complementary to the nucleotides of and the new strand is called lagging strand.
the template, for example Adenine opposite to
Thymine etc). • In the lagging strand the RNA Primase adds more
RNA Primers.
Why we need primer?
• It then create a short molecule of single-stranded • DNA polymerase can only extend a nucleic acid
DNA at lagging strand  Okazaki fragment. chain but cannot start one from scratch.
• To give the DNA polymerase a place to start, an
RNA polymerase called primase first copies a
short stretch of the DNA strand.
• This creates a complementary RNA segment, up
to 60 nucleotides long that is called a primer.
What is DNA polymerase?
• DNA polymerases are a family of enzymes that
carry out all forms of DNA replication.
• DNA polymerase then synthesizes a new strand
of DNA by extending the 3' end of an existing
nucleotide chain, adding new nucleotides
matched to the template strand one at a time via
the creation of phosphodiester bonds.
• DNA polymerases are extremely accurate,
making less than one mistake for every
107 nucleotides added.
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5. 9/29/2011
At the end of the process…termination!
• In the lagging strand the DNA Pol I -
• The RNA Primers are necessary for DNA
exonuclease- reads the fragments and removes
Polymerase to bind nucleotides to the 3' end of
the RNA Primers.
them.
• Rnase H removes the RNA fragments used to
• The daughter strand is elongated with the binding
initiate replication by DNA Polymerase, and
of more DNA nucleotides.
another DNA Polymerase enters to fill the gaps.
• The gaps are closed with the action of DNA
Polymerase (adds complementary nucleotides to
the gaps) and DNA Ligase (adds phosphate in
the remaining gaps of the phosphate - sugar
backbone).
• The DNA replication process is completed when
the ligase enzyme joins the short DNA pieces
together into one continuous strand.
Summary of DNA replication
• Helicase unwind the DNA, producing a replication • RNA primase initiate the DNA replication at origin
fork. of replication with short RNA nucleotides called
Primer.
• Single-stranded binding protein (SSB) prevent the
single stranded of DNA from recombining. • DNA polymerase attached to the RNA primer and
begin elongation (adding the nucleotides to the
• Topoisomerase removes twist and knots in the DNA complement strand)
double stranded template as a result of the
unwinding induced by helicase. • The leading complementary strand is assembled
continuously.
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6. 9/29/2011
RNA structure and function
• The lagging complementary strand is assembled
in short Okazaki fragment, which are
subsequently joined by DNA ligase.
• The RNA primer are replaced by DNA nucleotide.
• Cells use DNA and RNA differently.
• DNA is the original source for information to make
proteins. • These various type of RNA all participate in
making protein, but their role are different.
• But, RNA is made by enzymes that read the
protein-coding information in DNA.
• RNA is single-stranded.
• Different type of RNA molecules are classified by
the way in which it used.
• RNA can be classified as mRNA, tRNA and rRNA
mRNA - messenger RNA - carries information from DNA of the structural gene to the ribosome
where the protein is made
rRNA - ribosomal RNA - major structural
component of the ribosome where protein
synthesis occurs
tRNA - transfer RNA - carries amino
acids to mRNA at the ribosome to
assembly the protein being made
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7. 9/29/2011
Protein synthesis Process of protein synthesis
DNA replication
• DNA and RNA are both important part of making
protein.
Transcription
• DNA ---> RNA ---> Protein Nucleus
• beginning with amino acid synthesis and
transcription of nuclear DNA into messenger
RNA, which is then used as input for translation.
Translation
cytoplasm
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hill.com/sites/0072507470/student_view0/chapter3/animation__mrna_synthesi
s__transcription___quiz_1_.html
DNA to mRNA (transcription)
• Component needed are:
• DNA Template - strand of DNA providing
directions for transcription of mRNA.
• RNA Polymerase - enzyme that helps to pull
apart DNA strands and link new mRNA
nucleotides.
• Promoter - sequence of DNA that signals where
(and on which strand) transcription should begin
Transcription of mRNA
• Termination Signal - sequence of DNA that Initiation
causes RNA Polymerase to detach with the newly
transcribed mRNA strand.
Elongation
Termination
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8. 9/29/2011
Transcription of mRNA: initiation Transcription of mRNA: Elongation
• In eukaryotes, RNA polymerase, and therefore • As transcription proceeds, RNA polymerase
the initiation of transcription, requires the traverses the template strand and uses base
presence of a promoter sequence in the DNA. pairing complementarily with the DNA template to
create an RNA copy.
• Promoters are regions of DNA that promote
transcription. RNA polymerase uses to find a • Unlike DNA replication, mRNA transcription can
protein-coding region of DNA and to identify involve multiple RNA polymerases on a single
which of the two DNA strand is the coding strand. DNA template and multiple rounds of transcription
(amplification of particular mRNA), so many
• Without these promoter sequence, RNA mRNA molecules can be rapidly produced from a
polymerase will note transcribe the gene. single copy of a gene.
Proofread… Transcription of mRNA: Termination
• Elongation also involves a proofreading • When the RNA polymerase reaches the
mechanism that can replace incorrectly termination sequence, the sequence cause the
incorporated bases. mRNA to fold back on itself.
• In eukaryotes, this may correspond with short • This prevent transcription from continuing and
pauses during transcription that allow appropriate both the RNA polymerase and mRNA strand fall
RNA editing factors to bind. off the DNA strand.
• http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html
Remember!!! Summary of RNA transcription
• The entire molecule of DNA is not expressed in • Initiation: RNA polymerase attached to the
transcription. RNAs are synthesized only for promoter region on the DNA & begin to
same selected regions of DNA. unzip/unwind the DNA into 2 strand.
• RNA polymerase differs from DNA polymerase in • Elongation: RNA polymerase unzips/unwinds the
two aspects. No primer is required for RNA DNA & assembles RNA nucleotides using one
polymerase and , further this enzyme does not strand of the DNA template.
possess end or exonuclease activity. Due to lack - Elongation occur in 5’  3’ direction.
of the latter function, RNA polymerase has no - Only 1 DNA strand is transcribed.
ability to repair the mistake in the RNA
synthesized.
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9. 9/29/2011
RNA processing
• Termination: when RNA polymerase reaches a • Transcribed mRNA must first be processed
special sequence of nucleotides that serves as before it can leave the nucleus for the cytoplasm.
termination point (termination sequences).
• Steps of mRNA Processing:
- In eukaryotes: termination sequence always at
DNA sequence AAAAAAA. - Guanine Cap - Several Guanine nucleotides are
added to the front end of the mRNA strand in
order to bind to the ribosome more effectively.
- Poly-Adenine Tail - Several Adenine nucleotides
are added to the tail end of the mRNA strand to
prevent destruction by RNases (enzymes which
break down RNA).
- Splicing - Introns are removed and Exons are
joined together
- Intron - segment of mRNA which does NOT
code for protein; therefore, it is removed.
- Exon - segment of mRNA which does code for
protein; therefore, it remains for expression in
protein
Protein, here we come …vocabulary
• Genetic code  “Genetic Alphabet” • Triplet Code - three nucleotides code for one
amino acid
• Genetic "Alphabets" - there are three alphabets
involved in the entire process of protein synthesis: - Codon - three nucleotides of mRNA determining
which amino acid is added to a protein
1) DNA - A, C, G and T
2) RNA - A, C, G and U - Sample Genetic Code
3) Protein - Twenty different amino acids • Example
1) mRNA Codon = AUG
2) Amino Acid = Methionine
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10. 9/29/2011
Code characteristics
• It is described in terms of the mRNA codons.
STOP codons - UAA, UAG, UGA - all three of
these codons signal the end of a polypeptide
chain
Universal - The genetic code is the same in all
living organisms, from bacteria to humans.
Degenerate - More than one codon is assigned
to each amino acid. This allows for possible
mutations to be less damaging.
AAA - Lysine
AAG - Lysine
UAA - Lysine
mRNA translation to protein
• Initiation
• Third Base is usually less specific than the first
two. This is also known as the "Wobble - begin when the small ribosomal subunit
Hypothesis" because often the third base can attaches to a special region near the 5’ end of the
change, but the amino acid remains the same. mRNA.
- A tRNA (with anticodon UAC) carrying the
amino acid methionine attaches to the mRNA with
the hydrogen bond (start codon is AUG).
- large ribosomal subunit attaches to the mRNA,
forming a complete with the tRNA occupying at P
site.
- The remaining tRNA moves from the A side to
• Elongation the P site. Now the A side is unoccupied and a
new codon is exposed. This is anologous to the
- begin when next tRNA bind to the A site of the ribsome moving over one codon.
ribosome. The methionine is removed from the
first tRNA & attached to the amino acid on the - A new tRNA carrying a new amino acid enter
newly arrived tRNA. the A side. The 2 amino acid on the tRNA in the P
site are transferred to the new amino acid,
- the first tRNA, which no longer carries an amino forming a chain of three amino acid.
acid, is released. After its released, the tRNA can
again bind with its specific amino acid, allowing - the tRNA in a P site is released, the process is
repeated deliveries to the mRNA during repeated, polypeptide chain still growing.
translation.
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11. 9/29/2011
• Termination • Once the polyeptide is completeed, interaction
among the amino acids give its secondary &
- occurs when the ribosome encounters one of tertiary structure.
the three “stop” codon.
• Subsequent processing by the endoplasmic
- At termination, the complete polypeptide, the reticulum or a Golgi apparatus may make final
last tRNA, and the two ribosomal subunits are modifications before the protein functions as
released. structural element or as an enzyme.
- The ribosomal subunits can now attach to the
same or another mRNA and repeat the process.
Summary of protein synthesis!!!
Applied Biotechnology
• Polymerase Chain Reaction!!
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