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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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B. Replication of DNATHE CHEMISTRY OF DNA SYNTHESIS
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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B. Replication of DNATHE CHEMISTRY OF DNA SYNTHESIS
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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B. Replication of DNA
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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B. Replication of DNA
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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E. Translation of DNA
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. Translation of DNA
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