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GENE EXPRESSION: TRANSCRIPTION & TRANSLATIONIndwiani
AstutiDept of Pharmacology & TherapyFac of MedicineUniversitas
Gadjah Mada*[email protected]
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DNA and Gene Expression*[email protected]
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DNADeoxyribonucleic AcidDouble helixCarries genetic
informationLocated in the nucleusThe monomer is a nucleotideA
phosphateA ribose sugarA nitrogenous base*[email protected]
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What are the bases in DNAA adenineT thymineC cytosineG
guanineBase pair rules*[email protected]
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Where is DNA located in the Cell?*[email protected]
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Where are the genes located?
Genes are located on the chromosomes.Every species has a
different number of chromosomes.There are two types of chromosomes:
autosomes and sex chromosomes*[email protected]
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Genes are located on the chromosomes which are found in the
nucleus of a cell. When a cell is undergoing cell reproduction, the
chromosomes are visible. Chromosomes appear when the chromatin
condenses and become visible.Most of the time (90%) the genetic
material in the form of chromatin.A genome is the complete genetic
information contained in an individual.(gene + chromosome)
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What is gene expression?Gene expression is the activation of a
gene that results in a protein.Biological processes, such as
transcription, and in case of proteins, also translation, that
yield a gene product.A gene is expressed when its biological
product is present and active.Gene expression is regulated at
multiple levels.*[email protected]
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*[email protected]
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DNA is embedded in chromatin*[email protected]
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Regulation of gene expression at the level of
chromatinSequence-independentlinker histones: control DNA
compaction and accessibility to trans-acting factors
post-translational modifications of histone tails: control
compaction of DNA and serve as docking sites for trans-acting
factors
Range: Can act at the level of a single gene, often acts over
groups of genes and over larger domains (20-200kb), and can affect
gene expression over an entire chromosome*[email protected]
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*Eukaryotic Chromosome StructureEukaryotic DNA is packaged into
chromatin.Chromatin structure is directly related to the control of
gene expression.Chromatin structure begins with the organization of
the DNA into nucleosomes.Nucleosomes may block RNA polymerase II
from gaining access to [email protected]
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*Eukaryotic Chromosome StructureMethylation (the addition of
CH3) of DNA or histone proteins is associated with the control of
gene expression.Clusters of methylated cytosine nucleotides bind to
a protein that prevents activators from binding to DNA.Methylated
histone proteins are associated with inactive regions of
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Regulation of gene expressionPlasmidGene (red) with an intron
(green)Promoter2. TranscriptionPrimary transcript1. DNA
replication3. Posttranscriptional processing4. TranslationmRNA
degradationMature mRNA5. Posttranslational processingProtein
degradationinactiveproteinactiveproteinsingle copy vs. multicopy
plasmids*[email protected]
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*Control of Gene ExpressionControlling gene expression is often
accomplished by controlling transcription initiation.
Regulatory proteins bind to DNA to either block or stimulate
transcription, depending on how they interact with RNA
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Gene expression regulation at the level of
DNASequence-dependentcis-acting factors: promoters/regulatory
sequences of genestrans-acting factors: proteins and RNAs that bind
cis-elements and promote or repress gene expressionDNA methylation:
methylation of CpG islands promotes silencing
Range: Usually functions at level of single gene, or at most a
local group of genes*[email protected]
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*Control of Gene ExpressionProkaryotic organisms regulate gene
expression in response to their environment.
Eukaryotic cells regulate gene expression to maintain
homeostasis in the [email protected]
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Gene expression takes place differently in prokaryotes and
eukaryotes. What is a prokaryotes? Eukaryotes? ProkaryotesNo
membrane bound organelles (nucleus)More primitive organismsOnly one
circular chromosomeBacteria are the only organisms that are
prokaryotes.EukaryotesMembrane bound organelles ( specialize in
function nucleus, mitochondria, chloroplast)Chromosomes are in
pairs and not circularAll organisms that are not bacteria: protist,
fungi, plants and animals*[email protected]
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*Expression of Genetic InformationProduction of proteins
requires two steps:Transcription involves an enzyme (RNA
polymerase) making an RNA copy of part of one DNA strand. There are
four main classes of RNA:i. Messenger RNAs (mRNA), which specify
the amino acid sequence of a protein by using codons of the genetic
code.ii. Transfer RNAs (tRNA).iii. Ribosomal RNAs (rRNA).iv. Small
nuclear RNAs (snRNA), found only in eukaryotes.
Translation converts the information in mRNA into the amino acid
sequence of a protein using ribosomes, large complexes of rRNAs and
proteins.
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*Proteins Regulate Gene [email protected]
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*Gene structure relevant to metabolic
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In Eukaryotes, following mitosis or meiosis, DNA recoils but
certain regions remain relaxed for transcription. The areas of
relaxed DNA are called euchromatin.
Transcription is theReading of the DNA andChanging the code
tomRNA.Translation is changingThe mRNA into a trait byUsing tRNA to
interpret themRNA (The synthesis of a polypeptide chain using the
genetic code on the mRNA molecule as its
guide).*[email protected]
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DNA in eukaryotes has regions of coding and noncoding DNA. The
regions of DNA that code for proteins or traits are called EXONS,
while the regions that do not code for proteins are called
INTRONS.cytoplasm cytoplasm*[email protected]
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*Eukaryotic TranscriptionGeneral transcription factors bind to
the promoter region of the gene.RNA polymerase II then binds to the
promoter to begin transcription at the start site (+1).Enhancers
are DNA sequences to which specific transcription factors
(activators) bind to increase the rate of
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*Intron Splicing in EukaryotesExons: coding regionsIntrons:
noncoding regions Introns are removed by splicingGUat 5 endof
intronAGat 3 endof [email protected]
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*Splicesomes Roles in Spicing out IntronRNA splicing occurs in
small nuclear ribonucleoprotein particles (snRNPS) in
spliceosomesSpliceosomes: protein + small RNAs (U1-8) complementary
to the splice [email protected]
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*Eukaryotic TranscriptionCoactivators and mediators are also
required for the function of transcription factors.coactivators and
mediators bind to transcription factors and bind to other parts of
the transcription [email protected]
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Enhancer ControlEukaryote genes on a DNA strand also have
noncoding control sequences that facilitate transcription.These are
called enhancers.Transcription factors are additional proteins that
bind to RNA polymerase and enhancers to help with transcription.
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In prokayotes, transcription and translation occur in the
cytoplasm.In eukaryotes, transcription occurs inside the nucleus in
a two step sequence of events.Pre-mRNA includes both introns and
exons for the gene.mRNA is only the coding portion
(exons).Translation occurs in the cytoplasm at the
ribosomes.Reminder: The are three (3) types of RNAMessenager
(mRNA)Transfer (tRNA)Ribsomal (rRNA)*[email protected]
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RIBONUCLEIC ACID (RNA)Found all over the cell (nucleus,
mitochondria, chloroplasts, ribosomes and the soluble part of the
cytoplasm) 2007 Paul Billiet ODWS*[email protected]
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Structural characteristics of RNA moleculesSingle polynucleotide
strand which may be looped or coiled (not a double helix).Sugar
Ribose (not deoxyribose).Bases used: Adenine, Guanine, Cytosine and
Uracil (not Thymine). 2007 Paul Billiet ODWS*[email protected]
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mRNAA long molecule 1 million DaltonsEphemeralDifficult to
isolatemRNA provides the plan for the polypeptide chain 2007 Paul
Billiet ODWS*[email protected]
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rRNACoiledTwo subunits: a long molecule 1 million Daltons a
short molecule 42 000 DaltonsFairly stableFound in ribosomesMade as
subunits in the nucleolusrRNA provides the platform from protein
synthesis 2007 Paul Billiet ODWS*[email protected]
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tRNAShort molecule about 25 000 DaltonsSolubleAt least 61
different forms each has a specific anticodon as part of its
structure.tRNA translates the message on the mRNA into a
polypeptide chain 2007 Paul Billiet ODWS*[email protected]
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Transcription: The synthesis of a strand of mRNA (and other
RNAs)Uses an enzyme RNA polymeraseProceeds in the same direction as
replication (5 to 3)Forms a complementary strand of mRNAIt begins
at a promotor site which signals the beginning of gene is not much
further down the molecule (about 20 to 30 nucleotides)After the end
of the gene is reached there is a terminator sequence that tells
RNA polymerase to stop transcribing NB Terminator sequence
terminator codon 2007 Paul Billiet ODWS*[email protected]
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Editing the mRNAIn prokaryotes the transcribed mRNA goes
straight to the ribosomes in the cytoplasmIn eukaryotes the freshly
transcribed mRNA in the nucleus is about 5000 nucleotides longWhen
the same mRNA is used for translation at the ribosome it is only
1000 nucleotides longThe mRNA has been edited The parts which are
kept for gene expression are called EXONS (exons = expressed)The
parts which are edited out (by snRNP molecules) are called INTRONS
2007 Paul Billiet ODWS*[email protected]
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*Posttranscriptional RegulationControl of gene expression
usually involves the control of transcription initiation.But gene
expression can be controlled after transcription, with mechanisms
such as:RNA interferencealternative splicingRNA editingmRNA
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*Posttranscriptional RegulationRNA interference involves the use
of small RNA moleculesThe enzyme Dicer chops double stranded RNA
into small pieces of RNAmicro-RNAs bind to complementary RNA to
prevent translationsmall interfering RNAs degrade particular mRNAs
before [email protected]
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*Posttranscriptional RegulationIntrons are spliced out of
pre-mRNAs to produce the mature mRNA that is translated.Alternative
splicing recognizes different splice sites in different tissue
types.The mature mRNAs in each tissue possess different exons,
resulting in different polypeptide products from the same
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*Posttranscriptional RegulationRNA editing creates mature mRNA
that are not truly encoded by the genome.For example apolipoprotein
B exists in 2 isoformsone isoform is produced by editing the mRNA
to create a stop codonthis RNA editing is
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*Posttranscriptional RegulationMature mRNA molecules have
various half-lives depending on the gene and the location (tissue)
of expression.The amount of polypeptide produced from a particular
gene can be influenced by the half-life of the mRNA
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TranslationRNASingle strandedDoes not contain thymine but has
uracil instead.tRNA carries 3 base pair code for specific amino
acid.Amino acids compose polypeptid chains.One or more polypeptide
chains compose a protein proteins provide the blueprints for our
characteristics and functions.*[email protected]
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Transcription planTranscriptionDNAmessenger RNAGeneNucleus 2007
Paul Billiet ODWS*[email protected]
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Translation planTRANSLATIONRibosomesStop codonStart codon 2007
Paul Billiet ODWS*[email protected]
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SKEMA TRANSLASI*[email protected]
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TranslationLocation: The ribosomes in the cytoplasm that provide
the environment for translationThe genetic code is brought by the
mRNA molecule 2007 Paul Billiet ODWS*[email protected]
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What is the genetic code?The genetic code consists of the
sequence of bases found along the mRNA moleculeThere are only four
letters to this code (A, G, C and U)The code needs to be complex
enough to represent 20 different amino acids used to build proteins
2007 Paul Billiet ODWS*[email protected]
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How many combinations?If one base represented one amino acid
this would only be able to produce 4 different combinations. (A, C,
G and U)If pairs of bases represented each amino acid this would
only be able to produce 4 x 4 = 16 combinations. (AA, AC, AG, AU,
CA, CC, CG, CU etc)If triplets of bases represented each amino
acid, this would be able to produce 4 x 4 x 4 = 64 combinations
This is enough combinations to code for the 20 amino acids but is
the code actually made of triplets? 2007 Paul Billiet
ODWS*[email protected]
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Nature is logical!Over 10 years biochemists synthesised bits of
mRNA with different combinationsThen they used them to synthesise
polypeptidesThe results proved the logical answer was correctThe
genetic code is made of triplets of bases called codons 2007 Paul
Billiet ODWS*[email protected]
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07_33_mRNA.encode.jpgRibosomes initiate translation at
ribosome-binding sites in polycistronic procaryotic mRNAs, which
can encode more than one protein*[email protected]
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07_35_polyribosome.jpgA polyribosome from a eucaryotic
cell*[email protected]
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Initiation of Translation in
EukaryotesAAAAAAAAACapAUG----------------Stop5UTR3UTRImportant
points:No direct binding between mRNA and rRNASmall ribosome
subunit binds directly to cap requires specific initiation factor
eIF4eOther initiation factors can unwind double stranded regions in
the mRNA eIF4 groupSmall subunit scans mRNA till it finds correct
AUGCorrect AUG is embedded in preferred sequence
GccAccAUGG G
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ProteinsEssential for all biological eventsDNA carries the
informationProtein does the business
Enormous diversityfunctionalstructuralSimple building-blocks -
L-amino acids*[email protected]
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Kuliah Selanjutnya ProteinSelamat [email protected]*
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*this ability to selectively activate/repress genes underlies
much of the differences in gene expression in different tissues and
at different times. additionally it is important to note that such
a mechanism allows for concomitant activation of specific cohorts
of genes by sharing common sequence elements. it also lends itself
to formation of cascades or networks that move a program forward in
time (say a developmental program) and also allow homeostasis to
occur. given the program, I might need to say more about sequence
specific regulation through regulatory motifs, etc. transcription
factors are composite proteins with multiple domains, one of which
will bind in a sequence-specific manner to DNA, and other domains
that bind to other transcriptional regulatory proteins or the core
transcriptional machinery.
sequence elements can be close by or several hundred kb
away.
cooperativity multiple copies of the same protein tandemly
repeated, also binding between
proteins***************07_33_mRNA.encode.jpg*07_35_polyribosome.jpg
