Pertemuan 5. Pengaturan Ekspresi Gen

Pengaturan Ekspresi Gen
(Regulation of Gene
Expression)
Central Dogma of Molecular
Biology
“The central dogma of molecular biology deals with
the detailed residue-by-residue transfer of
sequential information. It states that such
information cannot be transferred back from
protein to either protein or nucleic acid.”
Francis Crick, 1958

… in other words
 Protein information
cannot flow back to
nucleic acids
 Fundamental
framework to
understanding the
transfer of sequence
information between
biopolymers
The Basics: Cell Organization
Prokaryotes
Eukaryotes
Discovery of DNA
 Griffith’s Experiments (1928)

 Griffith’s experiments showed that hereditary
material can pass from one bacterial cell to
another.
 The transfer of genetic material to one cell from
another cell or from one organism to another
organism is called transformation.
Avery’s Experiments (1940’s)
 Avery’s work showed that DNA was the heredity

material that transfers information between
bacterial cells.
Hershery-Chase
Experiment
• Hershey and Chase
confirmed that DNA,
and not protein, is the
hereditary material
The Basics: Structure of DNA
The Basics: Additional Points

DNA => A T C G, RNA => A U C G

Almost always read in 5' and 3' direction

DNA and RNA are dynamic - 2° structure

Not all DNA is found in chromosomes
 Mitochondria
 Chloroplasts
 Plasmids
 BACs and YACs

Some extrachromosomal DNA can be useful in Synthetic
Biology
DNA codes for genes
 Gene - a segment of DNA that codes for a

protein, which in turn codes for a trait (skin
tone, eye color…etc.), a gene is a stretch of
DNA.
… an example of a plasmid vector
 Gene of interest
 Selective
markers
 Origin of
replication
 Restriction sites
The Basics: Gene Organization
… now to the main course

DNA Replication

The process of copying double-stranded DNA molecules

Semi-conservative replication
 Origin of replication
 Replication Fork

Proofreading mechanisms
DNA Replication: Prokaryotic
origin of replication
 1 origin of replication; 2
replication forks
DNA Replication: Enzymes
involved

Initiator proteins (DNApol clamp loader)

Helicases

SSBPs (single-stranded binding proteins)

Topoisomerase I & II

DNApol I – repair

DNApol II – cleans up Okazaki fragments

DNApol III – main polymerase

DNA primase

DNA ligase
DNA Replication:
DNA Replication: Proofreading
mechanisms
 DNA is synthesised from dNTPs. Hydrolysis of (two) phosphate
bonds in dNTP drives this reduction in entropy.
- Nucleotide binding error rate =>c.10−4, due to extremely short-lived imino and enol tautomery.
- Lesion rate in DNA => 10-9.
Due to the fact that DNApol has built-in 3’ →5’ exonuclease activity, can chew back
mismatched pairs to a clean 3’end.
Transcription
 Process of copying DNA to RNA

 Differs from DNA synthesis in that only one
strand of DNA, the template strand, is used to
make mRNA
 Does not need a primer to start
 Can involve multiple RNA polymerases
 Divided into 3 stages
 Initiation
 Elongation
 Termination
Types of RNA
1. Messenger RNA (mRNA):

 Carries genetic info from the nucleus to the
cytoplasm
2. Transfer RNA (tRNA):
 Carries specific amino acids to the ribosome to
build the protein
3. Ribosomal RNA (rRNA):
 Major component of the ribosome organelle
 Site of protein synthesis
 Most abundant type of RNA
Types of RNA
Steps of transcription
1. RNA polymerase binds to the promoter section of

DNA
2. DNA unwinds and separates
3. RNA polymerase adds nucleotides
complimentary to the DNA template strand
4. Process ends once RNA polymerase reaches the
termination signal on the DNA
Definition
 RNA polymerase: enzyme use to make an RNA

polymer from DNA
 Promoter: Starting point on DNA
 DNA template: Strand of DNA that RNA is
complementary to (create from)
 Termination signal: Ending point on DNA
Transcription: The final product
Transcription: Transcriptional
control
 Different promoters for different sigma factors

… Case study – Lac operon
 For control of lactose metabolism
 Consists of three structural genes, a promoter, a
terminator and an operator
 LacZ codes for a lactose cleavage enzyme
 LacY codes for ß-galactosidase permease
 LacA codes for thiogalactoside transcyclase
 When lactose is unavailable as a carbon source, the
lac operon is not transcribed
 The regulatory response requires the lactose repressor
 The lacI gene encoding repressor lies nearby the lac operon
and it is consitutively (i.e. always) expressed
 In the absence of lactose, the repressor binds very tightly to a
short DNA sequence just downstream of the promoter near the
beginning of lacZ called the lac operator
 Repressor bound to the operator interferes with binding of
RNAP to the promoter, and therefore mRNA encoding LacZ
and LacY is only made at very low levels
 In the presence of lactose, a lactose metabolite called
allolactose binds to the repressor, causing a change in its shape
 The repressor is unable to bind to the operator, allowing
RNAP to transcribe the lac genes and thereby leading to high
levels of the encoded proteins.
Products of Transcription:
 mRNA, tRNA, & rRNA
 All products move out of the nucleus and

go into the cytoplasm to be used in protein
synthesis
DNA  RNA mRNA

tRNA
rRNA
Protein synthesis
 The making of proteins at the ribosome

 The amount and kind of proteins produced in a
cell determine its structure & function
 Proteins carry out the genetic instruction in DNA
Protein review
 Monomer = amino acids

 20 different types
 Linked together by peptide bonds
 Sequence of amino acids determines the proteins

structure and function
The genetic code
 The correlation between nucleotide sequence

(DNA or RNA) and amino acid sequence (protein)
 Codons: combination of 3 mRNA nucleotides that

code for a specific amino acid
Types of codons
 64 codons code for 20 amino acids

 Thus more than one codon codes for an AA
 Start codon: (AUG) starts the process of translation
 Stop codons: (UAA, UAG, UGA) ends the process of

translation
Circular genetic code
Translation:
 The process of assembling

polypeptides (proteins) from
nucleotide sequence in mRNA
 “Translating” from one

language (nucleotides) into
another language (amino acids)
Steps of translation
 During translation, amino acids are assembled

from information encoded in mRNA
 As mRNA codons move through the ribosome,
tRNA’s add specific amino acids to the growing
polypeptide chain.
 The process continues until a stop codon is
reached and the newly made protein is released.
So what is the Central Dogma?
 The flow from DNA to RNA to Protein

Pertemuan 5. Pengaturan Ekspresi Gen

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Pengaturan Ekspresi Gen

Francis Crick, 1958

 Griffith’s Experiments (1928)

 Avery’s work showed that DNA was the heredity

 Gene - a segment of DNA that codes for a

… now to the main course

 Process of copying DNA to RNA

1. Messenger RNA (mRNA):

1. RNA polymerase binds to the promoter section of

 RNA polymerase: enzyme use to make an RNA

 Different promoters for different sigma factors

 mRNA, tRNA, & rRNA

 All products move out of the nucleus and

DNA  RNA mRNA

 The making of proteins at the ribosome

 Monomer = amino acids

 Linked together by peptide bonds

 Sequence of amino acids determines the proteins

 The correlation between nucleotide sequence

 Codons: combination of 3 mRNA nucleotides that

 64 codons code for 20 amino acids

 Start codon: (AUG) starts the process of translation

 Stop codons: (UAA, UAG, UGA) ends the process of

 The process of assembling

 “Translating” from one

 During translation, amino acids are assembled

 The flow from DNA to RNA to Protein

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