CHAPTER 15 EXPRESSION OF BIOLOGICAL INFORMATION 6 hours Subtopics : 15.1 15.2 15.3 15.

4 DNA & genetic information DNA replication Protein synthesis Lactose Operon

15.1 DNA and genetic Information

a) Describe the structure of DNA based on the Watson & Crick model b) Explain DNA as the carrier of genetic information (F. Griffith 1931 & Avery et. al - 1944 ) c) Explain gene concept: one gene one polypeptide (1941, Beadle and Tatum)

15.1 DNA & genetic information

15.1(a) DNA Structure, based on the Watson & Crick Model.
5 end

3 end

3 end

3 end 5 end
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15.1(a) DNA Structure, Based on the Watson & Crick Model.

Long, thin, double strands of polynucleotides in helix shape. Both strands are antiparallel. Each strand has 3 end & 5 end, run in opposite direction. - 5 end: Free phosphate attach to 5th C of pentose sugar. - 3 end: Free - OH attach to 3rd C of pentose sugar.
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 The strands are held by Hydrogen bond to maintain the helix shape. Pentose sugar = Deoxyribose. Bases pairs : A-T, G-C. Same ratio * A - T : held by 2 H bonds. * G C : held by 3 H bonds. Phosphodiester bond joined the phosphate group to pentose sugar.
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15.1(b) DNA as the carrier of genetic information Experiments by F.Griffith (1931) and Avery et. al (1944). F. Griffith (1931): Studied 2 strains of Bacteria Streptococcus pneumoniae that cause pneumonia (disease). 1. S strain (smooth / wild) : - With capsule (protection against antibody) - pathogenic. - The capsule can be destroyed by heat treatment. 2. R strain (rough / mutant type) : - Without capsule, -non-pathogenic
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Frederick Griffith

Born: 1879 (1879-11-14T08:27:11) Died:1941 (1942) London, England Nationality: British Occupation: Physician, pathologist, bacteriologist

Known for discovery of Pneumococcal transformation

"could do more with a kerosene tin and a primus stove than most men could do with a palace".

1. 2. 3. 4.

Living R strain + mouse = mouse alive Living S strain + mouse = mouse die Heat-killed S strain + mouse = mouse alive Heat-killed S strain + living R strain + mouse = mouse die.
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F. Griffith (1931)
Conclusion: Living R strain was transformed into pathogenic S strain by unknown substance from dead S cells that allowed the R cells to make capsule.

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1944, Avery et. al Objective of Averys experiment : To determine the transforming substance observed in Griffiths experiments. Experiment : Avery extracted several substances from Heatkilled S strain bacteria, then mixed with living R strain, and injected into mouse.

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Result : Protein / RNA / carbohydrate of heat-killed S strain + living R strain + mouse = mouse alive = no transformation. DNA of Heat-killed S strain + living R strain + mouse = mouse die = there was transformation. Conclusion : - DNA is the transforming agent. - DNA is the carrier of genetic information.
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1931, Fred Griffith

1944, Avery et. al

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Avery DNA is the genetic carrier

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Identification of the transforming factor: Avery, et.al (1944)

(RNase)

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Gene concept : 1 gene 1 polypeptide

Beadle & Tatum : - 1941, proposed one gene one enzyme hypothesis.
Refer to One gene codes for the production of one protein / polypeptide / enzyme. Later, modified to "one gene one polypeptide" since many proteins (e.g hemoglobin) made of more than one polypeptide.
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Beadle & Tatum experiments (1941)

Use X rays to cause mutations in fungi (mold), Neurospora crassa. Mutation affects single genes that makes single enzymes in specific metabolic pathways. The mutant is crossed with the (wild) nonmutated to produce spores. The spores are grown on minimal medium (media with only one of the 20 essential amino acids).
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Beadle and Tatums evidence for the one gene-one enzyme hypothesis

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Any spore that cannot synthesize particular amino acid (affected by mutation), e.g Arginine, grows only if Arginine is supplied in the minimal medium. 1. Wild (non mutant) : can grow in a minimal medium, because can synthesize all amino acids & other chemicals need for growth. 2. Mutant : Cannot grow in minimal medium.
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Conclusion : - A single gene is mutated, causes a lack of a

single enzyme. - 1 gene synthesize 1 enzyme (polypeptide).

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15.2 DNA Replication

a) Describe 3 models of DNA replication: - Conservative model - Semi-Conservative model - Dispersive model b) Describe DNA replication during cell division c) List the enzymes involved and explain the function of each

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3 alternative models of DNA replication :

1. Conservative 2. Semi-conservative 3. Dispersive

26 1957, Meselson & Stahl experiment showed that : Semi conservative

Semi-conservative DNA replication : - Each strand of the original DNA remained intact as it served as the template for the synthesis of a complementary strand to make new DNA molecule. - One parental (original) DNA molecule replicates to make one new DNA molecule. - The new DNA molecule composed of two DNA identical strands that identical to the original DNA.
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Meselson & Stahl experiment :

2. (nitrogen-14)
1.
(nitrogen-15)

3.

1. Bacteria E. coli grown in a heavy medium (N15). The cells used N15 to synthesize DNA bases. 2. E. coli then transferred to light medium (with N14). 3. Cells DNA is extracted using density gradient centrifugation methods for few generation (P, F1, F2, F3).

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Result of Meselson & Stahl experiment

: Both of the DNA strands heavy. : Mix = 1 DNA strands heavy. another 1 DNA strands light. F2 (2nd generation) : 2 types of DNA = 1 heavy, 1 light = Both light F3 (3rd generation) : 1 heavy, 1 light, & more both light. Parental F1 (1st generation) Conclusion : DNA replication is semi conservative.
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DNA Replication During S phase of interphase in cell division.

1 parental DNA produce 1 new DNA Steps : 1. Initiation 2. Elongation 3. Termination.

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DNA Replication mechanism

Direction of new DNA strand formation is 5 to 3 .
Single-strand binding proteins stabilize the unwound parental strands Helicase unwind the parental strands

Topoisomerase breaks, swivels, and rejoins the parental DNA ahead of the replication fork, relieving the strain caused by unwinding.

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EXTRA NOTES
Eukaryotes have many origin of replication sites per chromosome.

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DNA Replication mechanism Steps : Initiation, Elongation, Termination.

1. Initiation - Helicases unwind the DNA parental strands at Origin of replication. - Forming replication bubble with replication fork. - The unwound parental DNA strands stabilized by Single-strand binding proteins.

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Cont..

DNA Replication mechanism

1. Initiation
- Each of the single-strand of parental DNA become a template strands for new complementary DNA strands. - Primase synthesizes RNA primers using the parental DNA as a template. - DNA polymerases add DNA nucleotides to the growing end (3 end) of the RNA primer, to the synthesize of new DNA strand.

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DNA Replication mechanism

Steps : Initiation, Elongation, Termination.

2. Elongation
DNA polymerases only add DNA nucleotides to the free 3 end of a growing DNA strand. New DNA strand elongate in the 5 to 3 direction. At one of the DNA template, DNA polymerase synthesize a complementary strand called leading strand. Leading strand elongates continuously in 5 to 3 direction towards the replication fork, using only one RNA primer. The complement of the leading strand, is called the lagging strand.

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Cont..

2. Elongation
Lagging strand elongates discontinuously, as a series of segments called Okazaki fragment, away from the replication fork, using several RNA primers. DNA ligase joined the okazaki fragments into a continuous DNA strand. The primers are converted to DNA before DNA ligase joins the fragments together.

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DNA Replication mechanism

Steps : Initiation, Elongation, Termination.

3. Termination - Occur when DNA Polymerase reach the end of the DNA parental strands.

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EXTRA NOTES
RNA primers = a short RNA chain. ( 5-10 nucleotides) = A starter to synthesize DNA chain.

DNA polymerases catalyze elongation of the new DNA strand, by continuously adding DNA nucleotides complementary to the parental DNA template strand, to the growing end (3end) of the new DNA strand.

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15.2(c) Enzymes involved in DNA replication

Enzyme 1. DNA helicase 2. Primase Function Untwist & unwind parental DNA strands at replication fork. Synthesize RNA primer, at 5 end of leading strand and of each Okazaki fragment of lagging strand by adding RNA nucleotides to the template.

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15.2(c) Enzymes involved in DNA replication

Enzyme 3. DNA polymerase **many types. Function Catalyze the synthesize of new DNA by adding DNA Nucleotides to a preexisting chain that complementary to the template strand.

DNA is synthesized in the 5' to 3' direction, because this enzyme can only add a nucleotide onto a preexisting 3'-OH group.
4. DNA Ligase Join DNA fragment (e.g Okazaki fragment) by formation of sugar-phosphate (phosphodiester) bond.

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15.3 PROTEIN SYNTHESIS

Importance : - to produce protein, e.g. hormones, enzymes. 2 Main Stages : 1. Transcription 2. Translation.

Eukaryotic cell has Pre-mRNA process RNA processing.

Prokaryotic has no RNA processing.

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2. Translation
Occurs in cytoplasm, 3 Stages : Initiation, Elongation, & Termination. Involves : RNA : mRNA, rRNA, & tRNA.

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Transcription : A process in which genetic information in DNA is transcribed (copied) into mRNA. Translation : A process of interpreting (translating) the genetic information (codon) in the mRNA.

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CODON & ANTICODON Genetic information - triplet bases (nucleotides), which refer to : a. Specific amino acid. e.g. UGG = trp. * 1 codon = 3 bases. b. Start / stop transcription or translation. e.g. Start codon = AUG (met) e.g. Stop codon = UAA, UGA, UAG

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CODON & ANTICODON

CODON = triplet bases in mRNA. ANTICODON = triplet bases in tRNA, that complementary to the CODON.
CODON & ANTICODON IS COMPLEMENTARY.

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Relations between genetic code (CODON) in mRNA and specific amino acids.
1 amino acid has more than 1 codon. Start codon : - AUG (methionine) -To initiate Protein synthesis. Stop codon : - UAA, UAG, UGA - To terminate Protein synthesis
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Protein synthesis : Transcription

Occur in nucleus. 3 stages : Initiation, elongation, termination. Basically involves : 1.DNA (only 1 strand) as a template 2.RNA polymerase ..add RNA nucleotides 3.Promoter (region in DNA with start signal, e.g AUG) 4.Terminator (region in DNA with stop signal, e.g UGA).

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Transcription stages

1. Initiation

2. elongation

3. Termination.

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Protein synthesis: Transcription stages

1. INITIATION

- Occur when RNA polymerase binds to Promoter, then unwind DNA strands (separate into 2 single strands and form bubble) - and initiates RNA synthesis by adding RNA nucleotides that complement to the template base (DNA). - * Only 1 DNA strand acts as template & be copied.
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Protein synthesis: Transcription stages

2. ELONGATION

- RNA polymerase moves downstream, unwinding the DNA and elongating RNA transcript from 5 3, by adding nucleotides to the 3end of growing polymer. - Behind the point of RNA synthesis, DNA double helix re-forms & mRNA is released. * To produce protein in large amounts, gene is transcribed by several RNA polymerase simultaneously to produce many mRNA.

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Protein synthesis: Transcription stages

3. TERMINATION - Transcription terminates (stop) when RNA polymerase reaches stop signal at termination site, mRNA (RNA transcript) released, and RNA polymerase detach from DNA.

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RNA processing (Pre-mRNA process) Gene has exon & intron :Exon = Coding sequence. = encodes a protein, Intron = Non-coding sequence. - Occur in eukaryotes only. - mRNA is modified before move out to the cytoplasm. - RNA splicing : - Removal of Introns, - Joining the Exons. - Addition of 5 cap at the 5end, and poly (A) tail at the 3end.

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Protein synthesis : Translation

Occurs in cytoplasm. 3 Stages : Initiation, Elongation, & Termination. Involves RNA : mRNA, rRNA, & tRNA.

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Amino acid attachment site Large subunit

small subunit

Anticodon

( mRNA )
- 1 strand. - has codons. - Made in nucleus - move to cytoplasm.

( tRNA )
- 1 strand. - cloverleaf shape. - has ANTICODON - has attachment site for amino acid.

( rRNA )
- Two subunits : 1. Large subunit - E site - P site - A site 2. Small subunits.

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rRNA (ribosomal RNA)

Ribosomes = rRNA + proteins

Consists of 2 subunits :

1.Large subunit has 3 binding site : P site (Peptidyl) : Holds tRNA carrying growing polypeptide chain. A site (Amino acyl) : Holds tRNA carrying the next amino acid. E site (Exit)
2. Small subunits. Function : To hold tRNA & mRNA close together, and catalyzes the addition of amino acid to the carboxyl end of the growing polypeptide chain. 59

tRNA (transfer RNA)

- Made in nucleus using DNA templates, move out to the cytoplasm. - Function : Transfer amino acid from cytosol, to ribosome. - Can be used repeatedly. Structure : - Single RNA strand, with 2-D cloverleaf shape, folded & twisted to form 3-D structure that roughly is L-shaped. - The loop protruding has ANTICODON (triplet base that binds to specific
mRNA codon).

- The 3end of the strand is the attachment site for amino acid.
tRNA = 45 types. Some tRNA anticodon recognize more than 1 codon. Activation of amino acids for attachment to tRNA - Amino acid is joined to the correct tRNA by aminoacyl-tRNA 60 synthetase (enzyme)..

Activation of amino acids for attachment to tRNA

Amino acid is joined to the correct tRNA by aminoacyltRNA synthetase (enzyme). The active site of each type of aminoacyl-tRNA synthetase fits only a specific combination of amino acid and tRNA.

There are 20 different synthetase for 20 different amino acid.

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PROTEIN SYNTHESIS : TRANSLATION

3 Stages of translation
1.Initiation 2.Elongation 3.Termination

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1. Initiation of translation

1. Small ribosomal subunit binds to mRNA, at binding site. 2. Initiator tRNA (with anticodon UAC, carries methionine) binds to the start codon, AUG . 3. Finally large ribosomal subunit bind to form Translation initiation complex. 4. Initiation factors (protein) bring all the translation 63 components together.

Initiation factor (protein) Large rRNA subunit

Met

Initiator tRNA

E site mRNA

P site
Start codon

A site

Small rRNA subunit

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2. Elongation of translation

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2. Elongation of translation
Elongation consists of a series of 3 step cycles as each amino acid is added to the proceeding one. 1. Codon recognition - The anticodon of incoming tRNA base-pairs with the complementary mRNA codon in A site. - elongation factor (protein) assists hydrogen bonding between the mRNA codon under the A site with the anticodon of tRNA carrying the correct amino acid. - Hydrolysis of GTP involved. 2. Peptide bond formation - rRNA catalyzes the formation of peptide bond between the polypeptide on tRNA in the P site with the new amino acid on tRNA in the A site. - This step separates the tRNA at the P site from the growing polypeptide chain and transfers the chain to the tRNA at the A site.
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3. Translocation
- The ribosome translocates (moves) the tRNA (with polypeptide chain) from A site to P site. - The empty tRNA in P site is moved to E site, then released. - Because the anticodon remains bonded to the mRNA codon, the mRNA moves along with it. The next codon is now available at the A site. - Translocation ensures that mRNA is read 5 to 3 codon by codon.

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Translation: 3. Termination
Elongation continues until a termination codon (stop codon) reaches A site. A release factor binds to stop codon & hydrolyzes the bond between polypeptide & tRNA in P site.

Stop codon : UAA, UAG, UGA .

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Polyribosome (or Polysomes) Polyribosomes : Several ribosome on a single mRNA simultenously . Importance (function) : to produce identical polypeptides in large amount in a short time. 1 mRNA can be used to make many copies of a polypeptide simultaneously.

The polypeptide will undergo modification before used as a particular protein.

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15.4 LACTOSE OPERON A system that show how gene control the production of a protein (enzymes) that induced by lactose in E. coli (bacteria) .

This concept was introduced by Jacob & Monod (1961) awarded the Nobel Prize.
Background : E.coli : lives in human colon, break down lactose (host drink). Lactose metabolism : Lactose -----------------> Glucose + Galactose

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15.4 LACTOSE OPERON

Proteins involved in lactose metabolism in the E. coli :a) -galactosidase hydrolyzes lactose to glucose & galactose b) -galactoside permease codes for permease to transports lactose into the cell c) -galactoside transacetylase - function still unclear.

Function in E.coli : - For the transport and metabolism of lactose. - To produce enzymes that break down lactose for energy or source for other use. The operon is activated when lactose is present.

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Components of lac (lactose) operon & its function

(R) (P) Lac Z Lac Y Lac A

(O)

Gene regulator (R) : contain gene to synthesize repressor (a protein). 1. Promoter (P) : Attachment site for RNA polymerase (to transcript gene).

2. Operator (O) : Attachment site for repressor. Operon switch

3. Structural genes : lac Z (S1) = gene to synthesize -galactosidase lac Y (S2) = gene to synthesize Galactoside permease 72 lac A (S3) = gene to synthesize Transacetylase

Mechanism of the lac (lactose) operon

Without Lactose : -R produce Repressor. -Repressor bind to Operator. This prevent RNA polymerase bind to Promoter =no transcription of structural genes =no enzyme produced =Operon not active (OFF).

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Mechanism of the lac (lactose) operon

With lactose (isomer - allolactose) : - R produce Repressor. - Allolactose bind to Repressor (change shape) cant bind to Operator). - Repressor cant bind to Operator - RNA polymerase bind to Promoter. = transcription occur, enzyme produced, Operon is active (ON). 74

Animation of lac operon.

Gene regulator

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The end

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