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Gene Expression

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0% found this document useful (0 votes)
10 views49 pages

Gene Expression

Uploaded by

bryanrale21
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Benjamin B.

Jularbal
DNA Structure
DEOXYRIBUNUCLEIC ACID

• Polymer of DEOXYRIBONUCLEOSIDE
MONOPHOSPHATES covalently linked by
3'→5'–PHOSPHODIESTER BONDS.
• DNA exists as a double- stranded molecule,
two strands forms a DOUBLE HELIX.
• DNA is found associated with nucleoprotein
present in the nucleus.
CHEMICAL COMPONENTS

• The polynucleotide chain of DNA contains


NUCLEOTIDES, which consist of a
NITROGENOUS BASE (A, G, C, OR
T), DEOXYRIBOSE, and PHOSPHATE
• The bases are the PURINES:
ADENINE(A) and GUANINE(G), and
PYRIMIDINES: CYTOSINE(C) and
thymine (T).
• PHOSPHODIESTER BONDS join the
3′-carbon of one sugar to the 5′-carbon of
the next sugar
PHOSPHODIESTER BONDS

• Join the 3’-OH of the DEOXYPENTOSE


of one nucleotide to the 5’-OH of the
DEOXYPENTOSE of an adjacent
nucleotide through a phosphate group.
• The long, unbranched chain has polarity, 5'-
end- the end with the free Pi and a 3'-end-
the end with the free OH, that are not
attached to other nucleotides.
• Bases along the resulting deoxyribose
phosphate backbone are written in sequence
from the 5'-end of the chain to the 3'-end.
• Can be cleaved hydrolytically by chemicals, or
hydrolyzed enzymatically by
DEOXYRIBONUCLEASES for DNA and
RIBONUCLEASES for RNA
BASE PAIRING

• Bases of one strand of DNA are paired with the


bases of the second strand
• ADENINE is always paired with a THYMINE
• CYTOSINE is always paired with a GUANINE.
• POLYNUCLEOTIDE CHAIN of the DNA
double helix is always the complement of the
other.
• Base pairing in DNA leads to the CHARGAFF
RULE: the amount of adenine equals the amount
of thymine, the amount of guanine equals the
amount of cytosine, and the total amount of
purines equals the total amount of pyrimidines.
• The base pairs are held together by
HYDROGEN BONDS: 2 H-bonds between A
and T and 3 bonds G and C.
• H BONDS, and HYDROPHOBIC
INTERACTIONS between the stacked bases,
stabilize the structure of the double helix.
DNA Replication
DNA REPLICATION

• DNA synthesis, occurs during the S phase of the


cell cycle in eukaryotes, and is catalyzed by a
complex of proteins that includes the enzyme
DNA POLYMERASE.
• Each strand of the parent DNA acts as a
template for the synthesis of its
COMPLEMENTARY STRAND.
• DEOXYRIBONUCLEOSIDE
TRIPHOSPHATES (ATP and GTP) are the
precursors for DNA synthesis.

DNA POLYMERASE

• Copies the template strand in the 3′ to 5′


direction and synthesizes the new strand in the
5′ to 3′ direction.
• DNA polymerase cannot initiate the synthesis of
a new strand.
• A short stretch of RNA serves as a PRIMER.
• Other proteins and enzymes are required to
unwind the parental strands and allow both
strands to be copied simultaneously.
BIDIRECTIONAL

• Replication begins at a site of origin


and simultaneously moves out in both
directions from this point.
• Prokaryotes have one site of origin on
each chromosome.
• Eukaryotes have multiple sites of
origin on each chromosome.

SEMICONSERVATIVE

• After replication, daughter molecule


of DNA contains one intact
parental strand and one newly
synthesized strand.
Unzipping of the double helix

Priming of the template strand,

Assembly of the new DNA segment.


UNZIPPING
REPLICATION FORKS
• Sites at which DNA synthesis is occurring
• Parental strands of DNA separate and the helix
unwinds ahead of a replication fork.

PRIMASE
• Synthesize RNA primers

HELICASES
• Unwind the helix.

SSBP
• Hold the single-stranded conformation.

TOPOISOMERASES
• Prevents the extreme supercoiling of the parental
helix that would result because of unwinding at a
replication fork.
• Topoisomerases break and rejoin DNA chains.
• DNA GYRASE, a topoisomerase inhibited by the
quinolone family of antibiotics, is found only in
prokaryotes.
PRIMING
DNA POLYMERASES

• Catalyze the synthesis of DNA.


• DNA polymerases only copy in the 3′ to 5′ direction
and produce in the 5′ to 3′ direction.

PROKARYOTES

• Have three DNA polymerases: pol I, pol II, and pol III.

EUKARYOTES

• Have many DNA polymerases, and the major ones are α,


β, δ, ε, and γ
• DNA polymerase α- involved in generating primers
for DNA replication.
• DNA polymerase β- used for repair.
• DNA polymerase δ- acts as the polymerase on the
lagging strand during replication.
• DNA Polymerase ε- polymerase on the leading strand
of DNA,
• DNA Polymerase γ- functions in the mitochondria.
DNA polymerase requires a PRIMER.

DNA polymerases cannot initiate the synthesis of


new strands.

RNA serves as the primer for DNA


polymerase.

RNA PRIMER

• Contains about 10 nucleotides.


• Formed by copying of the parental strand in a reaction by
DNA polymerase α then adds a few
deoxyribonucleotides to the RNA primer.
DNA polymerase adds deoxyribonucleotides to the
3′-hydroxyls of the RNA primers and subsequently to
the ends of the growing DNA strands.
ASSEMBLY
DNA TEMPLATE STRANDS
• Copied simultaneously at replication forks,
although they run in opposite directions.

LEADING STRAND
• Formed by continuous copying of the parental
strand that runs 3′ to 5′ toward the replication
fork.
LAGGING STRAND
• Formed by discontinuous copying of the
parental strand that runs 3′ to 5′ away from the
replication fork.
• As more of the helix is unwound, the synthesis of
the lagging strand begins from another primer
forming short fragments known as OKAZAKI
FRAGMENTS.
• The RNA primers are removed by
NUCLEASES then the resulting gaps are filled
with the appropriate deoxyribonucleotides by
another DNA polymerase.
• Okazaki fragments are joined by DNA
LIGASE, an enzyme that catalyzes the formation
of phosphodiester bonds between two
polynucleotide chains.
RNA Structure
RNA
• Similar to DNA except that RNA
contains RIBOSE and URACIL.
• Single-stranded and can act as
CATALYSTS of reactions
• RIBOZYMES- usually precursors of
rRNA, remove internal segments of
themselves, splicing the ends together.
• RIBONUCLEASES- cleaving other
RNA molecules
• PEPTIDYLTRANSFERASE- an
enzyme in protein synthesis.
MESSENGER RNA (mRNA)
• Provide the blueprint for an amino acid
sequence.
• mRNA is about 5% of total RNA.

TRANSFER RNA (tRNA)


• Match genetic information to an amino
acid sequence.
• tRNA is about 15% of total RNA.
• Have a cloverleaf shape with an acceptor
arm, which receives the amino acid, and a
THREE-BASE ANTICODON, which
can form base pairs with the
COMPLEMENTARY CODON in
mRNA.

RIBOSOMAL RNA (rRNA)


• Self-assemble with basic proteins to form
ribosomes.
• rRNA is about 80% of total RNA.
RNA Transcription
RNA TRANSCRIPTION

• Eukaryotic mRNA is synthesized as a


PRIMARY TRANSCRIPT that
must be processed to remove
INTRONS.
• Prokaryotic and eukaryotic genes have
PROMOTER REGIONS

PROMOTER REGIONS

• Point RNA polymerase in the right


direction and position it to start
transcription
RNA POLYMERASE

• Unwinds the DNA helix and transcribes the DNA template strand into RNA,
beginning at the transcription start site.
• The DNA strand complementary to the template strand is the CODING, or
SENSE STRAND.
• The RNA chain is elongated in the 5’ to 3’ direction.
• RIBONUCLEOSIDE TRIPHOSPHATES are added to the free 3’-OH
end of the RNA, producing a new 3’-OH terminal.

Eukaryotes contain three nuclear RNA polymerases that


produce different types of RNA.
• RNA POLYMERASE I produces rRNA.
• RNA POLYMERASE II produces mRNA.
• RNA POLYMERASE III produces tRNA.

Prokaryotes contain a single RNA polymerase that produces


all the types of RNA.
• Rifampin, an antibiotic used in the treatment of tuberculosis, inhibits RNA
polymerase in bacteria but not in eukaryotes.
Eukaryotic Transcription of mRNA
INITIATION

ELONGATION

TERMINATION

PROCESSING
INITIATION
INITIATION
• DNA unwinds and separates to form a
small open complex.
• RNA polymerase binds to the DNA of the
gene at a region called the PROMOTER
• INITIATION COMPLEX, containing
RNA polymerase II and transcription factors
is assembled near the Promoter region.

TATA BOX
• A conserved promoter sequence about 25
bases upstream of the start site.
• Helps orient the RNA polymerase.
CAAT BOX
• Conserved sequence within the promoter
region about 70 bases upstream of the start
site.
CG BOX
• Binds a specific TRANSCRIPTION
FACTOR (SP-1)
ELONGATION
ELONGATION

• RNA polymerase "walks" along one strand


of DNA, template strand, in the 3' to 5'
direction.
• ELONGATION continues until it
reaches a TERMINATION SIGNAL.
• The produced PRIMARY mRNA
TRANSCRIPT contains CODING and
NONCODING SEQUENCES.

EXONS

• Coding sequences within a gene.

INTRONS

• Intervening (noncoding) sequences within


a gene.
TERMINATION
TERMINATION

• Happens once the polymerase


transcribes a sequence of DNA
known as a TERMINATOR.

RHO-DEPENDENT
TERMINATION

• RNA contains a binding site for a


protein called RHO FACTOR.
• Rho factor binds to this sequence and
starts "climbing" up the transcript
towards RNA polymerase.
• When it catches up with the
polymerase, Rho pulls the RNA
transcript and the template DNA
strand apart, releasing the RNA
molecule and ending transcription.
PROCESSING
METHYLATION
• 7-methylguanosine cap is added to the 5’ end of the growing
mRNA before transcription is completed.

POLYADENYLATION
• Polyadenylation at the POLY(A) SITE leads to formation
of the POLY(A) TAIL consisting of ADENYLATE
RESIDUES at the 3’ end of the transcript.
SPLICING

• Removes introns from capped, tailed


transcripts and rejoins exons in a continuous
coding sequence.

SPLICE SITES

• The junctions between exons and introns,


• Marked by consensus sequences: GU at the 5’
end of an intron and AG at the 3’ end.
• SMALL NUCLEAR
RIBONUCLEOPROTEINS (snRNPs)
assemble at splice sites in mRNA, forming a
SPLICEOSOME.
• TRANSESTERIFICATION
REACTIONS catalyzed by snRNPs result in
excision of the intron and joining of exons.
• Systemic lupus erythematosus the
patient produces antibodies to snRNPs that
have been released into the blood due to
tissue damage.
Control of Gene
Expression
LAC OPERON

• POSITIVE CONTROL-factors
required to initiate RNA synthesis.
• NEGATIVE CONTROL - factors
required to prevent initiation of RNA
synthesis.

EUKARYOTIC GENE
EXPRESSION
• Regulated by controlling physical access
to the DNA by the RNA polymerase
and by controlling the rate of its
binding.
LAC OPERON SYSTEM

NEGATIVE CONTROL
• Occurs when only glucose is available as an
energy source and expression of the lac operon
is not needed.
• REPRESSOR PROTEIN is needed to inhibit
transcription
• LAC REPRESSOR PROTEIN is produced
constitutively and binds to the operon to
prevent transcription.

POSITIVE CONTROL
• Occurs when lactose is present, and expression
of the lac operon is needed.
• ACTIVATOR PROTEIN is needed to start
transcription
LACTOSE IS PRESENT

• Inducer, ALLOLACTOSE, is formed and


binds to the repressor inactivating it.

NO GLUCOSE IS PRESENT

• cAMP is elevated and combines with the


CATABOLITE ACTIVATOR
PROTEIN (CAP)
• CAP binds to the promoter stimulating
binding of RNA polymerase.

GLUCOSE IS PRESENT

• cAMP is not present, and the CAP-cAMP


complex cannot stimulate transcription;
this allows glucose to be used until it is
depleted.

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