(164446829) Molecular Genetic

8/13/2019 (164446829) Molecular Genetic

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MOLECULARGENETICPART IGene Regulation in Protein

Synthesis


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1 GeneRegulationSynthesisinProteinA.

B.

C.

D.

E.

Nucleic Acid Structure

Replication of DNA

Transcription of Bacterial DNA

Transcription of Eukaryote DNA

Translation


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A.Nucleic AcidStructure

Two types of nucleic acids- DNA & RNA

DNA (DeoxyriboNucleic Acid) & RNA

(RiboNucleic Acid)

Covalent link between atoms IN nucleic

acids building blocksprotect the

biological polymers

Weak bond BETWEEN different parts

allow functions


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DNA

Molecule in which the genetic materialstored

Nucleoside: purine base (Adenine,Guanine) pyrimidine base (Thymine,Cytosine)

1. is

2.

3.

4.

5.

F(x) as a

F(x) as a

DNA canA form,

template for its self-replication

template for RNA synthesis

adopt different conformationsB form, Z form


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DNA

DNA can exist in two conditionssupercoiled & relaxed

Naturally occurred in negatively supercoiledcondition

Enzyme alter DNA structure

6.

7.

8.Topoisomerase I (A & B) for single strand break

Topoisomerase II for double strand break,multimeric enzyme, require ATP hydrolysis to

complete two DNA strands cleavageProkaryotes has a special topoisomerase IIknown as DNA gyrase which introduce negativesupercoil


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DNA

Stabilize by hydrogen bonding, stackinginteractions and ionic interactions

9.

10.Undergo denaturation when heated and

anneal when cooled


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bond

PhosphodiesterHydrogen

bond


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RNA

1.

2.

3.

Chemically very similar to

Three differences:

DNA

deoxyribose vs ribose

thymine vs uracil

Typically found in

cell as singlepolynucleotide chain

backbone


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RNA

1.

Five functions:

Intermediate between the geneprotein synthesizing machinery

Adaptor between the codons inamino acids (tRNA)

and the(mRNA)mRNA and2.

3. Structural role as a components of theribosomes (rRNA)

Regulatory molecule which complement and

interferes with the translation of certainmRNAsEnzymes that catalyze essential reactions inthe cell (ribozymes)

4.

5.


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RNA

F(x) as a template for protein

Comes in various shapes,

sizes and some with catalytic

synthesis

property


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B. Replication of DNATHE CHEMISTRY OF DNA SYNTHESIS

Requires TWO key substratesdNTP(dATP, dGTP, dCTP, dTTP) and

primer:template junction


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DNA is synthesized by extending theend of the primer in antiparallelorientation

Matching dNTP form a base pair byhydrogen bonding

3

-

- hydroxyl group from 3end of primer attack

the P (-phosphoryl) from dNTP to formphosphodiester bond and release thepyrophosphate (- and -phosphoryl)


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Hydrolysis of pyrophosphate as adriving force for DNA synthesis

- Polymerization of nucleotides is promoted

by additional free energy provided by the

rapid hydrolysis of pyrophosphate


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B. Replication of DNAINITIATION OF DNA REPLICATION

Involves a replicator and initiatorcomponents

Replicator is a set of cis-acting DNA

sequence sufficient to direct an initiationreplication

Binding site for the initiator protein which

nucleates the assembly of replicationinitiation machinery

Consist of AT-rich region which unwindreadily

of

-

-


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B. Replication of DNA

Initiator is a sequence-specific DNAbinding protein

- Binds to a specific sequencereplicator

Unwind the DNA region

Attract other factors required

of replication

within the

-

-for initiation


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Unwinding of duplex DNA strandscatalyzed by DNA helicase

To stabilize the unwound single-strand

DNA , a single-strand DNA bindingprotein (SSBs) bind to ssDNA

Replication fork formation

Primase synthesis RNA primers onssDNA to initiate DNA synthesis by DNApolymerase


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B. Replication of DNAREPLICATION FORK

DNA is synthesized semidiscontinuously

Both DNA strands are synthesized

simultaneously

The junction between newly separated

strands and the unreplicated duplex DNA

is known as replication fork


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B. Replication of DNAREPLICATION FORK

Synthesis of DNA occur only from 5

direction. to 3

In lagging strand, DNA is synthesized

discontinuously and form a short new

fragments, known as Okazaki fragments


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B. Replication of DNADNA Polymerase

Key enzyme for DNA replication

Have single active site which catalyze

addition of any four dNTPs

the

Bound to primer:template junction on DNAstrand

Monitor the accuracy of base-pairing bycatalysis rate. High rate=matched, slow

rate=mismatched


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B. Replication of DNADNA Polymerase

When mismatched occur, primer:template

junction will slide out from catalytic site of

DNA polymerase into proofreading

exonuclease site

Exonuclease will remove the unmatched

base from 3-end

Primer:template junction will slide in into

the catalytic site of DNA polymerase


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RNA primers are removed by RNAse Hthrough degradation

The final ribonucleotide bond to DNA end is

removed by exonuclease which result in gaps

DNA ligase use ATP to create

phosphodiester bond between 5phosphate

and 3OH in a newly synthesized DNA

strands to close this gap


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REPLICATION TRANSCRIPTION

Synthesis of new Synthesis of new

deoxyribonucleotide strand ribonucleotide strand

Catalyze by DNA polymerase Catalyze by RNA polymerase

DNA polymerase require a RNA polymerase doesntprimer to initiate synthesis require any primer to initiate

synthesis The newly synthesized DNA The newly synthesized RNAform a base-pair with DNA doesnt remain base-pairedtemplate to the DNA template

Involve extensive Lack of extensive

proofreading proofreading Copy the entire genome Copy certain parts of the

once in every cell division genome


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C. Transcription of DNA

1.2.

3.

Key enzyme is RNA polymerase

Involve three phases:

InitiationElongation

Termination


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C. Transcription of Bacterial DNAINITIATION

Binding of polymerase to a promoter

sequence, form a closed complex

Upon binding, DNA strands separate

around the start site and form a

transcription bubble (open complex)

First two ribonucleotides are bought into

active site, aligned and joined

Polymerase move ahead opening the DNA

duplex


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C. Transcription of Bacterial DNAINITIATION

Formation of first 10 bp ribonucleotides is

a rather inefficient process, usually being

released and transcription start again-

abortive initiation

When transcribed sequence reach morethan 10 bp, a stable ternary complex is

established

Followed by elongation phase


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C. Transcription of Bacterial DNAELONGATION

DNA duplex enter polymerase

separated at the catalytic cleft

Ribonucleotides enter the site

and

and joinedthe growing RNA chain guided by the DNAtemplate

Only 8-9 growing RNA chain remain on theDNA template and the previously

generated are peeled off and directed out

from polymerase


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C. Transcription of Bacterial DNAELONGATION

During elongation, polymerase carries out twoproofreading functions:

Pyrophosphorolytic editing

1.

Polymerase uses its active site to catalyzeremoval of incorrect ribonucleotide by

incorporating the pyrophosphate (PPi)

2. Hydrolytic editingPolymerase is backtrack by one or more

the

mismatched nucleotides and remove the

containing sequence

error-


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C. Transcription of Bacterial DNATERMINATION

Termination is triggered by a specific sequence

known as terminators

Bacteria: rho-independent OR rho-dependent

- Rho-independent involve a stem-loop structureformed by self base-pairing

Rho-dependent require Rho protein, atranscription termination factor which require

ATP to remove RNA chain from template and

polymerase

-


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D. Transcription of Eukaryote DNAINITIATION

Polymerase II + general transcription factors +

promoter pre-initiation complex

Promoter consist of 4 elements: recognitionelement (BRE), TATA box, initiator (In r),

downstream promoter element (DPE)

Formationpromoter

hydrolysis

of pre-initiation complexmeltingof ATP)

DNA unwinding (require


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D. Transcription of Eukaryote DNAINITIATION

In vivo environment, mediator complex and

transcriptionally regulatory proteins are

needed

Different circumstances and promoters requires

nucleosome modifiers and chromatin

remodellers


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D. Transcription of Eukaryote DNAELONGATION

Pol II C-terminal domain (CTD)

phosphorylated at Ser residue

initiation factors with elongation

processing factors

exchange ofand RNA

Protein kinase p-TEFb phosphorylate CTD

and two other elongation factors

elongation

stimulate

TFIIS proofread RNA sequence byhydrolyticinherence RNAse activity, similar to

editing in DNA replication


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D. Transcription of Eukaryote DNAPOST ELONGATION

mRNA packaged and exported from nucleus

cytoplasm

Require active process (export protein +

GTPase)

Through a nuclear pore complex

to


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E. Translation of DNA

Generation of amino acid sequences frommRNA

Involve mRNA, tRNA, aminoacyl tRNA

synthetase and ribosome

Translation start at 5ORF (openreading

frame), end

Start codon:

Stop codon:

at 3ORF.

AUG, GUG, UUG

UAG, UAA, UGA

E T l ti f DNA


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Codons are degenerate. 4 nucleotide with

possibilities only code for 20 amino acids.

64

binding site

Exit

Peptidyl-tRNAAminoacyl-tRNA

binding site


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E T l i f DNA


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E. Translation of DNABACTERIA

Translation occur once mRNA emergeRNA polymerase

from

Ribosome binding to RBS (Shine-Dalgarno

sequence 5AGGAGG 3)Complementary sequence (5CCUCCU 3)

16S rRNA bind to this element in

Initiator tRNA (fMet-tRNA) bind to P site in smallribosomecodon

pairing of anticodon with start

Association of large rRNA subunit GO

E T l ti f DNA


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E. Translation of DNAEUKARYOTE

Ribosome binding is recruited by 5cap

Translation efficiency increased by Kozak

sequence (5G/ANNAUGG 3)

Initiator tRNA (Met-tRNA) bind to P site inribosome

small

Recognition mediator proteins bind to mRNA at

5cap, attract RNA helicase and unwind hairpinstructure binding to small ribosome

Start scanning until 5AUG 3is found

E T l ti f DNA


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E. Translation of DNAEUKARYOTE

Correct base-pairing of Met-tRNA and 5AUG 3result in release of recognition mediator factors

Association of large rRNA GO

f


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E. Translation of DNAELONGATION and TRANSLOCATION

E T l i f DNA


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E. Translation of DNATERMINATION

Release factor (RF) recognize stop codon

Two class RF:

Class I: recognize stop codon and trigger

hydrolysis of peptide chain

Class II: stimulate the dissociation of class

factor from ribosome

-

- I


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END OF PART I

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(164446829) Molecular Genetic