Gene Expression Overview
By
Salwa Hassan Teama
M.D. N.C.I. Cairo university
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Gene Expression
Eukaryotic Cell
The Gene Structure
Protein Synthesis
Prokaryotes Vs Eukaryotes
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Gene Expression
Gene expression is the
process by which a genes
information is converted into
the structures and functions of
a cell by a process of
producing a biologically
functional molecule of either
protein or RNA (gene product)
is made.
Gene expression is
assumed to be controlled at
various points in the sequence
leading to protein synthesis.
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Eukaryotic Cell
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Gene Structure
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Eukaryotic gene structure:
Most eukaryotic genes in
contrast to typical
bacterial genes, the
coding sequence (exons)
are interrupted by
noncoding DNA (introns).
The gene must have
( Exon; start signals; stop
signals; regulatory control
elements).
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Protein Synthesis
Protein Synthesis is the
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process in which cells
build proteins from
information in a DNA
gene in a two major
steps:
I-Transcription and
II-Translation
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Transcription :
synthesis of an RNA
(mRNA) that is
complementary to one of
the strands of DNA.
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Translation : ribosomes
read a messenger RNA
and make protein
according to its
instruction.
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Protein Synthesis
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Transcription
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http://biology.unm.edu/ccouncil/Biology_124/Images/transcription.gif
Transcription
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RNA polymerase copies both the exons and the introns.
The stretch of DNA that is transcribed into an RNA molecule
is called a transcription unit.
A transcription unit that is translated into protein contains
coding sequence that is translated into protein and
sequences that direct and regulate protein synthesis;
Transcription proceeds in the 5' → 3' direction.
Transcription is divided into 3 phases: Initiation, Elongation
and Termination..
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RNA polymerase;
eukaryotic nuclei contain
three RNA polymerases .
RNA polymerase I is
found in the nucleolus;
the other two
polymerases are located
in the nucleoplasm. The
three nuclear RNA
polymerase have different
roles in transcription.
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Polymerase I makes a large
precursor to the major rRNA
(5.8S,18S and 28S rRNA in
vertebrates).
Polymerase II synthesizes
hnRNAs, which are precursors
to mRNAs. It also make
most small nuclear RNAs
(snRNAs).
Polymerase III makes the
precursor to 5SrRNA, the
tRNAs and several other small
cellular and viral RNAs.
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Initiation
The general transcription
factors combine with
RNA polymerase to
form a preinitiation
complex that is
competent to initiate
transcription as soon as
nucleotide are available.
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Initiation
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The enzyme RNA polymerase recognizes a promoter,
which lies upstream of the gene. The polymerase
binding causes the unwinding of the DNA double helix.
This is followed by initiation of RNA synthesis at the
starting point.
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The RNA polymerase starts building the RNA chain, it
assembles ribonucleotides triphosphates: ATP; GTP;
CTP and UTP into a strand of RNA.
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After the first nucleotide is in place, the polymerase joins
a second nucleotide to the first, forming the initial
phosphodiester bond in the RNA chain.
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Elongation
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RNA polymerase directs the sequential binding of
riboncleotides to the growing RNA chain in the 5`-3`
direction.
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Each ribonucleotide is inserted into the growing RNA
strand following the rules of base pairing. This process is
repeated till the desired RNA length is
synthesized…………………….
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Termination
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Other regions at the end of genes; called terminators,
signal termination. These work in conjunction with RNA
polymerase to loosen the association between RNA
product and DNA template. The result is that the RNA
dissociate from RNA polymerase and DNA and so stop
transcription.
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The product is immature RNA or pre mRNA (Primary
transcript).
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RNA Processing
Pre-mRNA → mRNA
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Capping: Synthesis of the cap. The 5` cap is a 7- methylguanosine
(m7G) . The cap protects the mRNA from being degraded by enzymes;
enhancement of mRNA translatability.
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Splicing: Step-by-step removal of introns present in the pre-mRNA and
joining of the remaining exons. The removal of introns and joining of exons
takes place on a special structures called spliceosomes.
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Polyadenylation: Synthesis of the poly (A) tail involves cleavage of its 3'
end and then the addition of about 200 adenine residues to form a poly (A)
tail; This completes the mRNA molecule (mature mRNA), which is now
ready for export to the cytosol for protein synthesis.
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RNA Processing
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RNA Splicing
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Alternative Splicing
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Alternative splicing: is a very common phenomenon in higher
eukaryotes. It is a way to get more than one protein product
out of the same gene and a way to control gene expression
in cells.
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Translation
http://www.nature.com/embor/journal/v4/n9/images/embor923-f3.jpg
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Translation
Translation is the process by which ribosomes read the
genetic message in the mRNA and produce a protein
product according to the message's instruction.
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Translation
Requirement for translation
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Ribosomes
tRNA
mRNA template
Amino Acids
Initiation factors
Elongation factors
Termination factors
Aminoacyl tRNA synthetase enzymes
Energy source
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Ribosomes are the site
of protein biosynthesis
using the mRNA as a
template, the ribosome
traverses each codon of
the mRNA, pairing it with
the appropriate amino
acid. This is done using
molecules of transfer
RNA (tRNA) containing a
complementary anticodon
on one end and the
appropriate amino acid
on the other.
http://www.molecularexpressions.com/cells/ribosomes/images/ribosomesfigure1.jpg
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tRNA
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Act as adaptors that can bind an
amino acid at one end and interact
with the mRNA at the other.
mRNA
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Source of coding information for
the protein synthesis system.
Contains start and stop signals for
translation.
Eukaryotic mRNA is capped. This is
used as the recognition feature for
ribosome binding.
The site at which protein synthesis
begins on the mRNA is especially
crucial, since it sets the reading
frame for the whole length of the
message. An error of one
nucleotide either way at this stage
would cause every subsequent
codon in the message to be
misread, so that a nonfunctional
protein would result, the rate of
initiation thus determines the rate at
which the protein is synthesized.
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Amino acids are the monomers
which are polymerized to produce
proteins. The amino acids are
loaded onto tRNA molecules for
use in the process of translation.
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Initiation factors help the
ribosome, initiator tRNA, and other
components assemble the at the
correct location on the mRNA and
ensure that protein synthesis
starts in the correct reading frame
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Elongation factors are responsible
for moving the ribosome along the
mRNA and maintain the correct
reading frame. Facilitate removal
of "used" tRNAs and bringing in
"new" tRNAs.
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Termination factors recognize
the stop codons and release
proteins and ribosomes.
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Aminoacyl tRNA synthetase
enzymes: It catalyze the
covalent attachment of an
amino acids to the end of the
corresponding tRNA.
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Energy source: ATP or GTP
which are synthesized in the
mitochondria.
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Translation
Preparatory steps for
protein synthesis:
First, aminoacyl tRNA
synthetase join amino
acid to their specific
tRNA.
Second, ribosomes must
dissociate into subunits at
the end of each round of
translation.
The protein synthesis occur in 3
phases:
1- Accurate and efficient initiation
occurs, the ribosomes binds to
the mRNA, and the first amino
acid attached to its tRNA.
2- Chain elongation, the
ribosomes adds one amino
acid at a time to the growing
polypepyide chain.
3- Accurate and efficient
termination, the ribosomes
releases the mRNA and the
polypeptide.
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Translation: Initiation
The initiation phase of
protein synthesis
requires over 10
eukaryotic Initiation
Factors (eIFs): Factors
are needed to recognize
the cap at the 5` end of
an mRNA and binding to
the 40s ribosomal
subunit.
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Binding the initiator Met-tRNAiMet
(methionyl- tRNA) to the 40S small
subunit of the ribosome.
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Scanning to find the start codon by
binding to the 5` cap of the mRNA
and scanning downstream until
they find the first AUG (initiation
codon).
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The start codon must be located
and positioned correctly in the P
site of the ribosome and the
initiator tRNA must be positioned
correctly in the same site.
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Once the mRNA and initiator tRNA
are correctly bound, the 60S large
subunit binds to form 80 s initiation
complex with release of the eIF
factors.
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The large ribosomal
subunit contains three
tRNA binding sites,
designated A, P, and E.
The A site binds an
aminoacyl-tRNA (a tRNA
bound to an amino acid);
the P site binds a
peptidyl-tRNA (a tRNA
bound to the peptide
being synthesized); and
the E site binds a free
tRNA before it exits the
ribosome.
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Elongation
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Transfer of proper
aminoacyl-tRNA from
cytoplasm to A-site of
ribosome;
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Peptide bond formation;
Peptidyl transferase
forms a peptide bonds
between the amino acid
in the P site and the
newly arrived aminoacyl
tRNA in the A site. This
lengthens the peptide by
one amino acids.
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Elongation
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Translocation;
translocation of the new
peptidyl t-RNA with its
mRNA codon in the A site
into the free P site occurs
Now the A site is free for
another cycle of
aminoacyl t-RNA codon
recognition and
elongation. Each
translocation events
moves mRNA , one
codon length through the
ribosomes.
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Termination
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Translational termination requires specific protein factors
identified as releasing factors, RFs in E. coli and eRFs in
eukaryotes.
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The signals for termination are the same in both prokaryotes
and eukaryotes. These signals are termination codons
present in the mRNA. There are 3 termination codons, UAG,
UAA and UGA.
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Termination
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After multiple cycles of
elongation and
polymerization of specific
amino acids into protein
molecules, a nonsense
codon = termination
codon of mRNA appear in
the A site. The is
recognized as terminal
signal by eukaryotic
releasing factors (eRF)
which cause the release
of the newly synthesized
protein from the
ribosomal complex.
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Protein Synthesis
http://bioinfo.bact.wisc.edu/themicrobialworld/lysozyme.gif 
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Reading the instruction means
translating the code in the RNA
from bases
(building block of DNA and RNA)
to amino acids (building block of
Protein proteins).
Synthesis
www.bseinquiry.gov.uk/report/volume2/fig1_2.htm
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Prokaryotic vs. Eukaryotic
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Eukaryotic DNA is wound around histones to form
nucleosomes and packaged as chromatin. Chromatin
has a strong influence on the accessibility of the DNA to
transcription factors and the transcriptional machinery
including RNA polymerase.
Eukaryote genes are not grouped in operons. each
eukaryote gene is transcribed separately, with separate
transcriptional controls on each gene.
Protein synthesis takes place in the cytoplasm while
transcription and RNA processing take place in the
nucleus.
Essentially all humans' genes contain introns. A notable
exception is the histone genes which are intronless.
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Prokaryotic vs. Eukaryotic
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Eukaryotic mRNA is modified through RNA splicing.
Eukaryotic mRNA is generally monogenic
(monocistronic); code for only one polypeptide.
Eukaryotes have a separate RNA polymerase for each
type of RNA.
Eukaryotic mRNA contain no Shine-Dalgarno sequence
to show the ribosomes where to start translating.
Instead, most eukaryotic mRNA have caps at their 5`
end which directs initiation factors to bind and begin
searching for an initiation codon.
Eukaryotic protein synthesis initiation begins with
methionine not N formyl- methionine.
In eukaryotes, polysomes are found in the cytoplasm.
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Prokaryotic vs. Eukaryotic
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Bacterial genetics are different.
Prokaryote genes are grouped in operons.
Prokaryotes have one type of RNA polymerase for all
types of RNA,
mRNA is not modified
The existence of introns in prokaryotes is extremely rare.
To initiate transcription in bacteria, sigma factors bind to
RNA polymerases. RNA polymerases/ sigma factors
complex can then bind to promoter about 40
deoxyribonucleotide bases prior to the coding region of
the gene.
In prokaryotes, the newly synthesized mRNA is
polycistronic (polygenic) (code for more than one
polypeptide chain).
In prokaryotes, transcription of a gene and translation of
the resulting mRNA occur simultaneously. So many
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polysomes are found associated with an active gene.
References & Further Reading
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Robert F.Weaver. Molecular Biology. Fourth Edition. Page 600. McGraw-Hill International Edition.
ISBN 978-0-07-110216-2
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Innis,David H. Gelfand,John J. Sninsky PCR Applications: Protocols for Functional Genomics: ISBN:0123721865
Daniel H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. Washington, DC: AACC Press, 1996, ISBN 0915274-84-1.
William B. Coleman,Gregory J. Tsongalis: Molecular Diagnostics: For the Clinical Laboratorian: ISBN 1588293564...
Robert F. Mueller,Ian D. Young. Emery's Elements of Medical Genetics: ISBN. 044307125X
Daniel P. Stites,Abba T. Terr. Basic Human Immunology: ISBN. 0838505430
Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the
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cell. ISBN. 9780815341055
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http://www.pubmedcentral.nih.gov/
www.medscape.com
www.ebi.ac.uk/2can good introduction to bioinformatics and molecular biology
http://www.genomicglossaries.com
http://www.gene.ucl.ac.uk/nomenclature/guidelines.html defines the nomenclature for human genes
http://www.accessexcellence.org
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Codons.html
http://www.web-books.com/MoBio/
http://www.expasy.org
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPROTSYn.html
Cell & Molecular Biology online: http://www.cellbio.com/recommend.html
http://www.ornl.gov/sci/techresources/Human_Genome/glossary/glossary.shtml%20
http://www.genome.gov/10000715
http://www.ncbi.nlm.nih.gov/About/primer/mapping.html
http://www.lilly.com/research/discovering/targets.html
http://www.informatics.jax.org/expression.shtml
www.wikipdia.com
http://www.biology.arizona.edu/cell_bio/tutorials/pev/page2.html
http://www.genome.ou.edu/protocol_book/protocol_index.html
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