Download DNA structure and protein synthesis

The structure of DNA
• Deoxyribonucleic acid
• DNA is made of nucleotides
• Each nucleotide is composed of phosphate, sugar
(deoxyribose) and a nitrogen base
• 4 nitrogen bases – Adenine, Thymine, Guanine,
Cytosine (A,T,G,C)
• A-T, C-G
• Bases are linked by hydrogen bonds
DNA is a double helix
DNA strands
• Run in opposite directions
Base Pairing
DNA Replication
• The enzyme helicase breaks H bonds, unzips
DNA and another enzyme, DNA polymerase
adds new base pairs to make two new double
helix strands
Protein Synthesis – Ch. 17
1. Transcription
The transfer of genetic info. From DNA to messenger
RNA (mRNA)
2. Translation
The transfer of mRNA to protein
Genes are pieces of DNA that code for proteins
mRNA – Uracil instead of Thymine
Figure 17.4
DNA
template
strand
5
3
A C C
A A
A C
T
T
T
G G
T
C G A G
G G C
T
T
C A
3
5
DNA
molecule
Gene 1
TRANSCRIPTION
Gene 2
U G G
mRNA
U U
U G G C U
C A
5
3
Codon
TRANSLATION
Protein
Trp
Phe
Gly
Ser
Gene 3
Amino acid
Overview of Protein Synthesis
Transcription
• DNA codes for single strand of mRNA
• This happens in the nucleus
• RNA polymerase binds to the promoter region
on DNA template
• Sigma factor recognizes binding site on DNA
• mRNA detatches at the terminator region of
the DNA template
Important vocabulary in transcription
• The stretch of DNA that is transcribed is called a
transcription unit
• Transcription factors (sigma) – initiate the binding of
the RNA polymerase
• The completed assembly of transcription factors and
RNA polymerase II bound to a promoter is called a
transcription initiation complex
• A promoter called a TATA box is crucial in forming the
initiation complex in eukaryotes
Figure 17.8
1 A eukaryotic promoter
Promoter
Nontemplate strand
DNA
5
3
3
5
T A T A A AA
A T AT T T T
TATA box
Transcription
factors
Start point
Template strand
2 Several transcription
factors bind to DNA
5
3
3
5
3 Transcription initiation
complex forms
RNA polymerase II
Transcription factors
5
3
5
3
RNA transcript
Transcription initiation complex
3
5
Nontemplate
strand of DNA
RNA nucleotides
Transcription
RNA
polymerase
A
3
T
C
C
A A
5
3 end
C A
U
C
C A
T
A
G
G T
5
5
C
3
T
Direction of transcription
Template
strand of DNA
Newly made
RNA
Prokaryotes vs. Eukaryotes
• In prokaryotes, translation of mRNA can begin
before transcription has finished
• In a eukaryotic cell, the nuclear envelope
separates transcription from translation
• Eukaryotic RNA transcripts are modified
through RNA processing to yield the finished
mRNA
• A primary transcript is the initial RNA
transcript from any gene prior to processing
Transcription
RNA modification
• Enzymes in the eukaryotic nucleus modify premRNA (RNA processing) before the genetic
messages are dispatched to the cytoplasm
• During RNA processing, both ends of the primary
transcript are usually altered
• Also, usually some interior parts of the molecule
are cut out, and the other parts spliced together
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Alteration of mRNA Ends
• Each end of a pre-mRNA molecule is modified in a
particular way
– The 5 end receives a modified nucleotide 5 cap
– The 3 end gets a poly-A tail
– In eukaryotes, RNA polymerase II transcribes the
polyadenylation signal sequence; the RNA
transcript is released 10–35 nucleotides past this
polyadenylation sequence
• These modifications share several functions
1. They seem to facilitate the export of mRNA to the
cytoplasm
2. They protect mRNA from hydrolytic enzymes
3. They help ribosomes attach to the 5 end
© 2011 Pearson Education, Inc.
Figure 17.10
5
G
Protein-coding
segment
P P P
5 Cap 5 UTR
Polyadenylation
signal
AAUAAA
Start
codon
Stop
codon
3 UTR
3
AAA … AAA
Poly-A tail
Split Genes and RNA Splicing
• noncoding regions of eukaryotic DNA/RNA are
called introns
• exons are eventually expressed and translated
into amino acid sequences
• RNA splicing removes introns and joins exons,
creating an mRNA molecule with a continuous
coding sequence
• Spliceosomes consist of a variety of proteins and
several small nuclear ribonucleoproteins
(snRNPs) that recognize the splice sites
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Figure 17.11
5 Exon Intron Exon
Pre-mRNA 5 Cap
Codon
130
31104
numbers
Intron
Exon 3
Poly-A tail
105
146
Introns cut out and
exons spliced together
mRNA 5 Cap
Poly-A tail
1146
5 UTR
Coding
segment
3 UTR
Figure 17.12-3
RNA transcript (pre-mRNA)
5
Exon 1
Intron
Protein
snRNA
Exon 2
Other
proteins
snRNPs
Spliceosome
5
Spliceosome
components
5
mRNA
Exon 1
Exon 2
Cut-out
intron
Ribozymes
• Ribozymes are catalytic RNA molecules that
function as enzymes and can splice RNA
• Not all catalysts are proteins
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Figure 17.13
Gene
DNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1
Polypeptide
Animation: RNA Processing
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mRNA Degradation
• The life span of mRNA molecules in the cytoplasm
is a key to determining protein synthesis
• Eukaryotic mRNA is more long lived than
prokaryotic mRNA
• Nucleotide sequences that influence the lifespan
of mRNA in eukaryotes reside in the untranslated
region (UTR) at the 3 end of the molecule
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Figure 18.15
Hairpin
Hydrogen
bond
miRNA
Dicer
5 3
(a) Primary miRNA transcript
miRNA
miRNAprotein
complex
mRNA degraded Translation blocked
(b) Generation and function of miRNAs
Noncoding RNAs play multiple roles in
controlling gene expression
• Noncoding RNAs regulate gene expression at two
points: mRNA translation and chromatin
configuration
• MicroRNAs (miRNAs) are small single-stranded
RNA molecules that can bind to mRNA
• These can degrade mRNA or block its translation
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• The phenomenon of inhibition of gene expression
by RNA molecules is called RNA interference
(RNAi)
• RNAi is caused by small interfering RNAs
(siRNAs)
• siRNAs and miRNAs are similar but form from
different RNA precursors
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Animation: mRNA Degradation
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Figure 18.14
Ubiquitin
Proteasome
Protein to
be degraded
Ubiquitinated
protein
Proteasome
and ubiquitin
to be recycled
Protein entering
a proteasome
Protein
fragments
(peptides)
Translation
•
•
•
•
•
The transfer of mRNA into a protein
This happens at the ribosome
Every 3 base pairs of mRNA is called a codon
tRNA hold anti-codons and amino acids
tRNA bring amino acids down to the
ribosomes using the corresponding anticodon.
• Accurate translation requires two steps
1. a correct match between a tRNA and an amino
acid, done by the enzyme aminoacyl-tRNA
synthetase
2. a correct match between the tRNA anticodon and
an mRNA codon
Flexible pairing at the third base of a codon is called
wobble and allows some tRNAs to bind to more
than one codon
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• A ribosome has three binding sites for tRNA
– The P site holds the tRNA that carries the growing
polypeptide chain
– The A site holds the tRNA that carries the next
amino acid to be added to the chain
– The E site is the exit site, where discharged tRNAs
leave the ribosome
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Translation
Figure 17.14
Amino
acids
Polypeptide
Ribosome
tRNA with
amino acid
attached
tRNA
C
G
Anticodon
U G G U U U G G C
5
Codons
mRNA
3
Translation
Figure 17.19-4
Amino end of
polypeptide
E
3
mRNA
Ribosome ready for
next aminoacyl tRNA
P A
site site
5
GTP
GDP  P i
E
E
P A
P A
GDP  P i
GTP
E
P A
Figure 17.15
3
Amino acid
attachment
site
5
Amino acid
attachment
site
5
3
Hydrogen
bonds
Hydrogen
bonds
A A G
3
Anticodon
(a) Two-dimensional structure
Anticodon
(b) Three-dimensional structure
5
Anticodon
(c) Symbol used
in this book
The Genetic Code
Initiation of Translation
• The initiation of translation of selected
mRNAs can be blocked by regulatory proteins that
bind to sequences or structures of the mRNA
• Alternatively, translation of all mRNAs
in a cell may be regulated simultaneously
• For example, translation initiation factors are
simultaneously activated in an egg following
fertilization
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Animation: Blocking Translation
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Protein Processing and Degradation
• After translation, various types of protein
processing, including cleavage and the addition of
chemical groups, are subject to control
• Proteasomes are giant protein complexes that
bind protein molecules and degrade them
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Animation: Protein Processing
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Animation: Protein Degradation
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© 2011 Pearson Education, Inc.
Termination of Translation
• Termination occurs when a stop codon in the
mRNA reaches the A site of the ribosome
• The A site accepts a protein called a release
factor
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Figure 17.20-3
Release
factor
Free
polypeptide
5
3
3
5
5
Stop codon
(UAG, UAA, or UGA)
2
GTP
2 GDP  2 P i
3
Polyribosomes
• A number of ribosomes can translate a single
mRNA simultaneously, forming a polyribosome
(or polysome)
• Polyribosomes enable a cell to make many copies
of a polypeptide very quickly
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Figure 17.21
Growing
polypeptides
Completed
polypeptide
Incoming
ribosomal
subunits
Start of
mRNA
(5 end)
(a)
End of
mRNA
(3 end)
Ribosomes
mRNA
(b)
0.1 m
Targeting Polypeptides to Specific Locations
• Free ribosomes mostly synthesize proteins that
function in the cytosol
• Bound ribosomes make proteins of the
endomembrane system and proteins that are
secreted from the cell
• Ribosomes are identical and can switch from free
to bound
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• Polypeptide synthesis always begins in the
cytosol
• Synthesis finishes in the cytosol unless the
polypeptide signals the ribosome to attach to
the ER
• Polypeptides destined for the ER or for
secretion are marked by a signal peptide
• A signal-recognition particle (SRP) binds to
the signal peptide
• The SRP brings the signal peptide and its
ribosome to the ER
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Figure 17.22
1 Ribosome
5
4
mRNA
Signal
peptide
3
SRP
2
ER
LUMEN
SRP
receptor
protein
Translocation
complex
Signal
peptide
removed
ER
membrane
Protein
6
CYTOSOL
What Is a Gene? Revisiting the Question
– A discrete unit of inheritance
– A region of specific nucleotide sequence in a
chromosome
– A DNA sequence that codes for a specific
polypeptide chain
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Figure 17.26
DNA
TRANSCRIPTION
3
5
RNA
polymerase
RNA
transcript
Exon
RNA
PROCESSING
RNA transcript
(pre-mRNA)
AminoacyltRNA synthetase
Intron
NUCLEUS
Amino
acid
AMINO ACID
ACTIVATION
tRNA
CYTOPLASM
mRNA
Growing
polypeptide
3
A
Aminoacyl
(charged)
tRNA
P
E
Ribosomal
subunits
TRANSLATION
E
A
Anticodon
Codon
Ribosome
Evolution of the Genetic Code
• The genetic code is nearly universal, shared by
the simplest bacteria to the most complex
animals
• Genes can be transcribed and translated after
being transplanted from one species to another
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Figure 17.6
(a) Tobacco plant expressing
a firefly gene
(b) Pig expressing a jellyfish
gene
Mutations
• Mutations are changes in the genetic
material of a cell or virus
• Point mutations are chemical changes in
just one base pair of a gene
• Point mutations within a gene can be divided
into two general categories
– Nucleotide-pair substitutions
– One or more nucleotide-pair insertions or
deletions
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Figure 17.23
Wild-type hemoglobin
Sickle-cell hemoglobin
Wild-type hemoglobin DNA
C T T
3
5
G A A
5
3
Mutant hemoglobin DNA
C A T
3
G T A
5
mRNA
5
5
3
mRNA
G A A
Normal hemoglobin
Glu
3
5
G U A
Sickle-cell hemoglobin
Val
3
Substitutions
• A nucleotide-pair substitution replaces one
nucleotide and its partner with another pair of
nucleotides
• Silent mutations have no effect on the amino
acid produced by a codon because of
redundancy in the genetic code
• Missense mutations still code for an amino
acid, but not the correct amino acid
• Nonsense mutations change an amino acid
codon into a stop codon, nearly always leading
to a nonfunctional protein
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Insertions and Deletions
• Insertions and deletions are additions or losses
of nucleotide pairs in a gene
• These mutations have a disastrous effect on the
resulting protein more often than substitutions do
• Insertion or deletion of nucleotides may alter the
reading frame, producing a frameshift mutation
© 2011 Pearson Education, Inc.
Figure 17.24
Wild type
DNA template strand 3 T A C T T C A A A C C G A T T 5
5 A T G A A G T T T G G C T A A 3 
mRNA5 A U G A A G U U U G G C U A A 3
Protein
Met
Lys
Phe
Gly
Stop
Carboxyl end
Amino end
(b) Nucleotide-pair insertion or deletion
(a) Nucleotide-pair substitution
Extra A
A instead of G
3 T A C T T C A A A C C A A T T 5
5 A T G A A G T T T G G T T A A 3
3 T A C A T T C A A A C C G A T T 5
5 A T G T A A G T T T G G C T A A 3
Extra U
U instead of C
5 A U G A A G U U U G G U U A A 3
Met
Lys
Phe
Gly
Stop
Silent (no effect on amino acid sequence)
5 A U G U A A G U U U G G C U A A 3
Met
Stop
Frameshift causing immediate nonsense
(1 nucleotide-pair insertion)
T instead of C
A missing
3 T A C T T C A A A T C G A T T 5
5 A T G A A G T T T A G C T A A 3
3 T A C T T C A A C C G A T T 5T

5 A T G A A G T T G G C T A A 3A
A instead of G
U missing
5 A U G A A G U U U A G C U A A 3
Met
Lys
Phe
Ser
Stop
Missense
5 A U G A A G U U G G C U A A
Met
Lys
Leu
Ala
Frameshift causing extensive missense
(1 nucleotide-pair deletion)
A instead of T
3 T A C A T C A A A C C G A T T 5
5 A T G T A G T T T G G C T A A 3
U instead of A
5 A U G U A G U U U G G C U A A 3
Met
Nonsense
Stop
T T C missing
3 T A C A A A C C G A T T 5
5 A T G T T T G G C T A A 3
A A G missing
 A A
5 A U G U U U G G C U A A 3U
Met
Phe
Gly
Stop
No frameshift, but one amino acid missing
(3 nucleotide-pair deletion)
3
Gene Expression
• Gene expression is the act of going from
genotype to phenotype
• DNA to mRNA to Protein
• Genes are regulated by turning on and off
transcription
 The
lac operon in E.coli is the method by
which E. coli make enzymes that metabolize
lactose
 Regulatory gene – produces the repressor
 Repressor binds to the operator when lactose
is absent
 No Transcription – RNA polymerase cannot
bind to the promoter
 When
lactose is present, it binds to the
repressor pulling it off the operator
 RNA polymerase binds the promoter –
transcription begins
 Lactose-digesting enzymes are made
Regulatory
gene
DNA
Promoter
Operator
lacI
lacZ
No
RNA
made
3
mRNA
RNA
polymerase
5
Active
repressor
Protein
(a) Lactose absent, repressor active, operon off
lac operon
DNA
lacI
lacZ
lacY
lacA
RNA polymerase
3
mRNA
5
mRNA 5
-Galactosidase
Protein
Allolactose
(inducer)
Inactive
repressor
(b) Lactose present, repressor inactive, operon on
Permease
Transacetylase
 Trp
operon. E.coli will make tryptophan
from scratch, but if it is in the surroundings,
the E.coli will absorb it.
 Different from the lac operon
trp operon
Promoter
Promoter
Genes of operon
DNA
trpE
trpR
trpD
trpC
trpB
trpA
C
B
A
Operator
Regulatory
gene
3
RNA
polymerase
Start codon
Stop codon
mRNA 5
mRNA
5
E
Protein
Inactive
repressor
D
Polypeptide subunits that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on
DNA
No RNA
made
mRNA
Protein
Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off
Positive Gene Regulation
• Some operons are also subject to positive control
through a stimulatory protein, such as catabolite
activator protein (CAP), an activator of
transcription
• When glucose (a preferred food source of E. coli)
is scarce, CAP is activated by binding with cyclic
AMP (cAMP)
• Activated CAP attaches to the promoter of the lac
operon and increases the affinity of RNA
polymerase, thus accelerating transcription
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• When glucose levels increase, CAP detaches from
the lac operon, and transcription returns to a
normal rate
• CAP helps regulate other operons that encode
enzymes used in catabolic pathways
© 2011 Pearson Education, Inc.
Figure 18.5
Promoter
DNA
lacI
lacZ
CAP-binding site
cAMP
Operator
RNA
polymerase
Active binds and
transcribes
CAP
Inactive
CAP
Allolactose
Inactive lac
repressor
(a) Lactose present, glucose scarce (cAMP level high):
abundant lac mRNA synthesized
Promoter
DNA
lacI
CAP-binding site
lacZ
Operator
RNA
polymerase less
likely to bind
Inactive
CAP
Inactive lac
repressor
(b) Lactose present, glucose present (cAMP level low):
little lac mRNA synthesized
Proximal control elements are located close to
the promoter
 Distal control elements, groupings of which are
called enhancers, may be far away from a gene
or even located in an intron
 Some transcription factors function as
repressors, inhibiting expression of a particular
gene by a variety of methods
 A particular combination of control elements can
activate transcription only when the appropriate
activator proteins are present

Promoter
Activators
DNA
Enhancer
Distal control
element
Gene
TATA box
General
transcription
factors
DNAbending
protein
Group of mediator proteins
RNA
polymerase II
RNA
polymerase II
Transcription
initiation complex
RNA synthesis
Eukaryotic gene expression is regulated at
many stages
• All organisms must regulate which genes are
expressed at any given time
• In multicellular organisms regulation of gene
expression is essential for cell specialization
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Differential Gene Expression
• Almost all the cells in an organism are genetically
identical
• Differences between cell types result from
differential gene expression, the expression of
different genes by cells with the same genome
• Abnormalities in gene expression can lead to
diseases including cancer
• Gene expression is regulated at many stages
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Enhancer
Control
elements
Promoter
Albumin gene
Crystallin
gene
LENS CELL
NUCLEUS
LIVER CELL
NUCLEUS
Available
activators
Available
activators
Albumin gene
not expressed
Albumin gene
expressed
Crystallin gene
not expressed
(a) Liver cell
Crystallin gene
expressed
(b) Lens cell
Figure 18.6
Signal
NUCLEUS
Chromatin
DNA
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
Gene available
for transcription
Gene
Transcription
RNA
Exon
Primary transcript
Intron
RNA processing
Cap
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Degradation
of mRNA
Translation
Polypeptide
Protein processing, such
as cleavage and
chemical modification
Degradation
of protein
Active protein
Transport to cellular
destination
Cellular function (such
as enzymatic activity,
structural support)
Regulation of Chromatin Structure
• Genes within highly packed heterochromatin are
usually not expressed
• Chemical modifications to histones and DNA of
chromatin influence both chromatin structure and
gene expression
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Animation: DNA Packing
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Histone Modifications
• In histone acetylation, acetyl groups (COCH3)
are attached to positively charged lysines in
histone tails
• This loosens chromatin structure, thereby
promoting the initiation of transcription
• The addition of methyl groups (methylation) can
condense chromatin; the addition of phosphate
groups (phosphorylation) next to a methylated
amino acid can loosen chromatin
© 2011 Pearson Education, Inc.
Figure 18.7
Histone
tails
Amino acids
available
for chemical
modification
DNA
double
helix
Nucleosome
(end view)
(a) Histone tails protrude outward from a nucleosome
Acetylated histones
Unacetylated histones
(b) Acetylation of histone tails promotes loose chromatin
structure that permits transcription
DNA Methylation
• DNA methylation, the addition of methyl
groups(CH3) to certain bases in DNA, is
associated with reduced transcription in some
species
• DNA methylation can cause long-term inactivation
of genes
• The inheritance of traits transmitted by
mechanisms not directly involving the nucleotide
sequence is called epigenetic inheritance
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Animation: Initiation of Transcription
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