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DNA and RNA

The Molecular Basis of Heredity

DNA structure & function

• DNA is able to accomplish 2 very important things

for an organism.

– DNA is used to pass genetic information on to the

next generation of organism.

– 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

• 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

- a phosphate group

- nitrogenous bases (Adenine, A; Guanine, G;

Cytosine, C; and Thymine, T)

DNA nucleotide

DNA helix

Hydrogen

bond

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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• 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?

• In DNA replication process…it involve 3 process

1- Initiation

2- Elongation

3- Termination

http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replic

ation/index.html

Ingredients to make a copy of DNA

• Template strand

- The DNA serves as a template to guide the

incoming nucleotides.

• 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

uses it to synthesize the new strand.

• Free 3’ hydrocyl (Primer)

- DNA polymerase can add a nucleotide onto only

a preexisting 3'-OH group, and, therefore, needs

a primer at which it can add the first nucleotide.

• Helicase

- accomplishes unwinding of the original double

strand, once supercoiling has been eliminated by

the topoisomerase.

• DNA ligase

- It can catalyze the formation of a

phosphodiester bond given an unattached but

adjacent 3'OH and 5'phosphate.

• Single-stranded binding proteins

- important to maintain the stability of the

replication fork.

• 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

replicated during S phase?

Parental strandOrigin of replication

Two daughter DNA molecules

Daughter strand

DNA replication begins at many

specific sites –replication bubbles!

Now…the elongation process…

• One of the most important steps of DNA

Replication is the binding of RNA Primase in the

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

DNA synthesis.

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• The elongation process is different for the 5'-3'

and 3'-5' template.

• 3'-5' Template: The 5'-3' proceeding daughter

strand -that uses a 3'-5' template- is

called leading strand because DNA Polymerase

can "read" the template and continuously adds

nucleotides (complementary to the nucleotides of

the template, for example Adenine opposite to

Thymine etc).

• 5’-3’'Template: The 5'-3' template cannot be

"read" by DNA Polymerase. Why?

• DNA polymerase can add free nucleotides to only

the 3' end of the newly forming strand

• The replication of this template is complicated

and the new strand is called lagging strand.

• In the lagging strand the RNA Primase adds more

RNA Primers.

• It then create a short molecule of single-stranded

DNA at lagging strand Okazaki fragment.

Why we need primer?

• DNA polymerase can only extend a nucleic acid

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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• The RNA Primers are necessary for DNA

Polymerase to bind nucleotides to the 3' end of

them.

• The daughter strand is elongated with the binding

of more DNA nucleotides.

At the end of the process…termination!

• In the lagging strand the DNA Pol I -

exonuclease- reads the fragments and removes

the RNA Primers.

• Rnase H removes the RNA fragments used to

initiate replication by DNA Polymerase, and

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

fork.

• Single-stranded binding protein (SSB) prevent the

single stranded of DNA from recombining.

• Topoisomerase removes twist and knots in the

double stranded template as a result of the

unwinding induced by helicase.

• RNA primase initiate the DNA replication at origin

of replication with short RNA nucleotides called

Primer.

• DNA polymerase attached to the RNA primer and

begin elongation (adding the nucleotides to the

DNA complement strand)

• The leading complementary strand is assembled

continuously.

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• 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.

RNA structure and function

• Cells use DNA and RNA differently.

• DNA is the original source for information to make

proteins.

• 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

• These various type of RNA all participate in

making protein, but their role are different.

mRNA - messenger RNA - carries information from DNA of the structural gene to the ribosome

where the protein is made

tRNA - transfer RNA - carries amino

acids to mRNA at the ribosome to

assembly the protein being made

rRNA - ribosomal RNA - major structural

component of the ribosome where protein

synthesis occurs

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Protein synthesis

• DNA and RNA are both important part of making

protein.

• DNA ---> RNA ---> Protein

• beginning with amino acid synthesis and

transcription of nuclear DNA into messenger

RNA, which is then used as input for translation.

http://highered.mcgraw-

hill.com/sites/0072507470/student_view0/chapter3/animation__mrna_synthesi

s__transcription___quiz_1_.html

Process of protein synthesis

Translation

Transcription

DNA replication

Nucleus

cytoplasm

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

• Termination Signal - sequence of DNA that

causes RNA Polymerase to detach with the newly

transcribed mRNA strand.

Transcription of mRNA

Initiation

Elongation

Termination

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Transcription of mRNA: initiation

• In eukaryotes, RNA polymerase, and therefore

the initiation of transcription, requires the

presence of a promoter sequence in the DNA.

• Promoters are regions of DNA that promote

transcription. RNA polymerase uses to find a

protein-coding region of DNA and to identify

which of the two DNA strand is the coding strand.

• Without these promoter sequence, RNA

polymerase will note transcribe the gene.

Transcription of mRNA: Elongation

• As transcription proceeds, RNA polymerase

traverses the template strand and uses base

pairing complementarily with the DNA template to

create an RNA copy.

• Unlike DNA replication, mRNA transcription can

involve multiple RNA polymerases on a single

DNA template and multiple rounds of transcription

(amplification of particular mRNA), so many

mRNA molecules can be rapidly produced from a

single copy of a gene.

Proofread…

• Elongation also involves a proofreading

mechanism that can replace incorrectly

incorporated bases.

• In eukaryotes, this may correspond with short

pauses during transcription that allow appropriate

RNA editing factors to bind.

Transcription of mRNA: Termination

• When the RNA polymerase reaches the

termination sequence, the sequence cause the

mRNA to fold back on itself.

• This prevent transcription from continuing and

both the RNA polymerase and mRNA strand fall

off the DNA strand.

• http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html

Remember!!!

• The entire molecule of DNA is not expressed in

transcription. RNAs are synthesized only for

same selected regions of DNA.

• RNA polymerase differs from DNA polymerase in

two aspects. No primer is required for RNA

polymerase and , further this enzyme does not

possess end or exonuclease activity. Due to lack

of the latter function, RNA polymerase has no

ability to repair the mistake in the RNA

synthesized.

Summary of RNA transcription

• Initiation: RNA polymerase attached to the

promoter region on the DNA & begin to

unzip/unwind the DNA into 2 strand.

• Elongation: RNA polymerase unzips/unwinds the

DNA & assembles RNA nucleotides using one

strand of the DNA template.

- Elongation occur in 5’ 3’ direction.

- Only 1 DNA strand is transcribed.

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• Termination: when RNA polymerase reaches a

special sequence of nucleotides that serves as

termination point (termination sequences).

- In eukaryotes: termination sequence always at

DNA sequence AAAAAAA.

RNA processing

• Transcribed mRNA must first be processed

before it can leave the nucleus for the cytoplasm.

• Steps of mRNA Processing:

- 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”

• Genetic "Alphabets" - there are three alphabets

involved in the entire process of protein synthesis:

1) DNA - A, C, G and T

2) RNA - A, C, G and U

3) Protein - Twenty different amino acids

• Triplet Code - three nucleotides code for one

amino acid

- Codon - three nucleotides of mRNA determining

which amino acid is added to a protein

- Sample Genetic Code

• Example

1) mRNA Codon = AUG

2) Amino Acid = Methionine

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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

• Third Base is usually less specific than the first

two. This is also known as the "Wobble

Hypothesis" because often the third base can

change, but the amino acid remains the same.

mRNA translation to protein

• Initiation

- begin when the small ribosomal subunit

attaches to a special region near the 5’ end of the

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.

• Elongation

- begin when next tRNA bind to the A site of the

ribosome. The methionine is removed from the

first tRNA & attached to the amino acid on the

newly arrived tRNA.

- the first tRNA, which no longer carries an amino

acid, is released. After its released, the tRNA can

again bind with its specific amino acid, allowing

repeated deliveries to the mRNA during

translation.

- The remaining tRNA moves from the A side to

the P site. Now the A side is unoccupied and a

new codon is exposed. This is anologous to the

ribsome moving over one codon.

- A new tRNA carrying a new amino acid enter

the A side. The 2 amino acid on the tRNA in the P

site are transferred to the new amino acid,

forming a chain of three amino acid.

- the tRNA in a P site is released, the process is

repeated, polypeptide chain still growing.

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• Termination

- occurs when the ribosome encounters one of

the three “stop” codon.

- At termination, the complete polypeptide, the

last tRNA, and the two ribosomal subunits are

released.

- The ribosomal subunits can now attach to the

same or another mRNA and repeat the process.

• Once the polyeptide is completeed, interaction

among the amino acids give its secondary &

tertiary structure.

• Subsequent processing by the endoplasmic

reticulum or a Golgi apparatus may make final

modifications before the protein functions as

structural element or as an enzyme.

Summary of protein synthesis!!!

Applied Biotechnology

• Polymerase Chain Reaction!!