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CAMPBELL BIOLOGY IN FOCUS

Urry Cain Wasserman Minorsky Jackson Reece

14
Gene Expression:
From Gene to Protein

Lecture Presentations by
Kathleen Fitzpatrick and Nicole Tunbridge
Edited by Rena Quinlan, Ph.D.
2014 Pearson Education, Inc.

Overview: The Flow of Genetic Information


The information content of genes is in the form of
specific sequences of nucleotides in DNA
The DNA inherited by an organism leads to specific
traits by dictating the synthesis of proteins
Proteins are the links between genotype and
phenotype
Gene expression, the process by which DNA
directs protein synthesis, includes two stages:
transcription and translation

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What led to the striking phenotype of the albino


deer shown below?
Inheritedtraitsaredeterminedbygenes,andthetraitforalbinismis
causedbyarecessivealleleofapigmentationgene

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Figure 14..1.

Basic Principles of Transcription and Translation


RNA is the bridge between DNA and protein synthesis
RNA is chemically similar to DNA, but RNA has a ribose sugar and the base uracil
(U) rather than thymine (T); RNA is single-stranded
Getting from DNA to protein requires two stages: transcription and translation
Transcription is the synthesis of RNA using information in DNA
Translation is the synthesis of a polypeptide, using information in the mRNA
(messenger RNA)
Ribosomes are the sites of translation

DNA

RNA

Protein

Figure 14.UN01. The central dogma of molecular biology. The central dogma is the
concept that cells are governed by a cellular chain of command.

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Transcription and Translation in prokaryotes and


eukaryotes

In prokaryotes, translation of mRNA can begin before transcription has finished

In eukaryotes, the nuclear envelope separates transcription from translation

Initial Eukaryotic RNA transcripts are modified via RNA processing to yield the finished mRNA

A primary transcript (pre-mRNA) is the initial RNA transcript from any gene prior to processing

Eukaryotic mRNA must be transported out of the nucleus into the cytoplasm to be translated
Nuclear
envelope

TRANSCRIPTION
RNA PROCESSING

DNA
Pre-mRNA

mRNA
DNA

TRANSCRIPTION
mRNA

Ribosome

TRANSLATION
Polypeptide

(a) Bacterial cell


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TRANSLATION

Ribosome
Polypeptide

(b) Eukaryotic cell


Figure 14.4

The Genetic Code


How are the instructions for assembling amino acids
into proteins encoded into DNA?
There are 20 amino acids, but there are only four
nucleotide bases in DNA
How many nucleotides correspond to an amino acid?

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Codons: Triplets of Nucleotides


The flow of
information from
gene to protein is
based on a triplet
code (codon): a
series of threenucleotide words

DNA
template
strand

5
A

The words of a
gene are
transcribed into
complementary
three-nucleotide
words of mRNA, a
codon
These words are
then translated
into a chain of
amino acids,
forming a
polypeptide
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TRANSCRIPTION
U

mRNA

A
3

Codon
TRANSLATION

Protein

Trp
Amino acid

Phe

Gly

Ser
Figure 14.5

During
transcription, one
of the two DNA
strands, called the
template strand,
provides a template
for ordering the
sequence of
complementary
nucleotides in an
RNA transcript
During translation,
the mRNA base
triplets, called
codons, are read in
the 5 to 3
direction
Each codon
specifies the
amino acid (one of
20) to be placed at
the corresponding
position along a
polypeptide
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DNA
template
strand

5
A

TRANSCRIPTION
U

mRNA

A
3

Codon
TRANSLATION

Protein

Trp
Amino acid

Phe

Gly

Ser
Figure 14.5

Cracking the Code

Second mRNA base


UUU
U

UUC
UUA
UUG

First mRNA base (5 end of codon)

64 codons
Of the 64 triplets, 61 code
for amino acids; 3 triplets
are stop signals to end
translation
The genetic code is
redundant: more than
one codon may specify a
particular amino acid
But it is not ambiguous:
no codon specifies more
than one amino acid
Codons must be read in
the correct reading frame
(correct groupings) in
order for the specified
polypeptide to be
produced

Phe

Leu

CUU
C

CUC
CUA

Leu

UCU
UCC

UAU
Ser

UAC

UAG

Stop

UGG

Trp

CCU

CAU

CCC
CCA

Pro

CAC
CAA

ACC
ACA

Thr

AAC
AAA

AUG Met or

ACG

AAG

GUU

GCU

GAU

GUA
GUG

GCC
GCA
GCG

Ala

GAC
GAA
GAG

His

Gln

Asn

Lys

CGU

CGC

Arg

CGA
CGG
AGU

Ser

AGC
AGA

Arg

AGG

Asp

Glu

GGU
GGC
GGA

C
A
G
U
C
A
G
U

Gly

GGG

Fig. 14.6. The codon table for mRNA.


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UCG

AAU

Val

ACU

GUC

UGC

Cys
Stop

AUU

start

UGU

UGA

CAG

IIe

Tyr
Stop

CCG

AUC

UAA

UCA

CUG

AUA

C
A
G

Third mRNA base (3 end of codon)

Evolution of the Genetic Code


The genetic code is nearly universal, shared by the simplest bacteria and
the most complex animals
Genes can be transcribed and translated after being transplanted from one
species to another

Fig. 14.7

(a) Tobacco plant expressing


a firefly gene

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(b) Pig expressing a jellyfish


gene

Concept 14.2: Transcription is the DNA-directed


synthesis of RNA: a closer look
Transcription is the first stage of gene expression

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Molecular Components of Transcription

RNA synthesis is catalyzed by


RNA polymerase, which pries the
DNA strands apart and joins
together the RNA nucleotides

Promoter

Transcription unit

5
3

3
5
Start point

RNA polymerases assemble


polynucleotides in the 5 to 3
direction and can start a chain
without a primer
The DNA sequence where RNA
polymerase attaches or binds to is
called the promoter when RNA
polymerase binds to the
promoter it initiates Transcription
The stretch of DNA that is
transcribed into RNA is called a
transcription unit (RNA
transcript)

The three stages of transcription:


Initiation: RNA polymerase
binds to Promoter
Elongation
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Termination

RNA polymerase

1 Initiation
5
3

3
5
Template strand of DNA

Unwound
DNA

Elongation
5
3

Termination

RNA
transcript
Rewound
DNA

3
5

3
5

RNA
transcript

Direction of
transcription
(downstream)

5
3

3
5
5

Completed RNA transcript

Fig. 14.8. The stages of Transcription: initiation,


elongation, and termination.

Concept 14.3: Eukaryotic cells modify RNA after


transcription
Enzymes in the eukaryotic
nucleus modify pre-mRNA
(RNA processing) before
the genetic messages enter
the cytoplasm
During RNA processing,
both ends of the premRNA (or primary
transcript ) are altered
Also, usually some interior
parts of the molecule are
cut out and the other parts
spliced together

TRANSCRIPTION

RNA PROCESSING

Pre-mRNA

mRNA

TRANSLATION

Ribosome

Polypeptide
Figure 14.UN03

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DNA

Alteration of mRNA Ends


Each end of a pre-mRNA molecule is modified in a particular way
The 5 end receives a modified Guanine nucleotide 5 cap
At 3 end, Poly (A) polymerase adds a chain of adenine nucleotides called a poly-A tail
These modifications share several functions:
Facilitating the export of mRNA to the cytoplasm
Protecting mRNA from hydrolytic enzymes in the cytoplasm
Helping ribosomes attach to the 5 end

50250 adenine
nucleotides added
to the 3 end

A modified guanine
nucleotide added to
the 5 end
5
G

Protein-coding segment

AAUAAA

5 Cap

Polyadenylation
signal

5 UTR

Start
codon

Stop
codon

3 UTR

Fig. 14.11. RNA processing: Addition of 5 cap and poly-A-tail.


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3
AAA AAA

Poly-A tail

Split Genes and RNA Splicing


Most eukaryotic mRNAs have long noncoding stretches of nucleotides called
introns that lie between coding regions called exons
Exons are translated into amino acid sequences
RNA splicing removes introns and joins exons, creating an mRNA molecule with a
continuous coding sequence

Pre-mRNA
5 Exon Intron

Exon

Intron

Exon 3

5 Cap

Poly-A tail
130

105146

31104

Introns cut out and


exons spliced together
mRNA
5 Cap

Poly-A tail
1146

5 UTR

Fig. 14.12. RNA processing: RNA splicing.


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Coding
segment

3 UTR

Introns may contribute to protein variability by


increasing the coding capacity of genes via
alternative splicing
Many genes can give rise to
two or more different
polypeptides, depending on
which segments are used
as exons

Spliceosome

Small RNAs

5
Pre-mRNA

Exons may be joined in


different combinations to
produce different mRNAs
from a single gene - this
process is called alternative
RNA splicing
RNA splicing is carried out
by spliceosomes

Exon 2

Exon 1
Intron

Spliceosome
components
mRNA
5
Exon 1

Spliceosomes consist of
proteins and small RNAs
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Exon 2

Cut-out
intron

Fig. 14.13. A spliceosome splicing a pre-mRNA.

Ribozymes
Ribozymes are RNA molecules that function as
enzymes capable of splicing out introns
RNA splicing can occur without proteins, or even
additional RNA molecules
Introns can act as ribozymes and catalyze their
own splicing

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Concept 14.4: Translation is the RNA-directed


synthesis of a polypeptide: a closer look
Genetic information flows from mRNA to protein
through the process of translation

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Molecular Components of Translation


A cell translates an mRNA message into protein with the help of transfer
RNA (tRNA)
tRNAs transfer amino acids to the growing polypeptide in a ribosome
Translation is a complex process in terms of its biochemistry and mechanics

TRANSCRIPTION

DNA

mRNA
Ribosome
TRANSLATION
Polypeptide
Figure 14.UN04
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The Structure and Function of Transfer RNA


Each tRNA can
translate a
particular mRNA
codon into a given
amino acid
The tRNA
contains an amino
acid at one end
and at the other
end has a
nucleotide triplet
that can base-pair
with the
complementary
codon on mRNA
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Amino acids

Polypeptide

Ribosome

tRNA with
amino acid
attached

Trp

Gly

Phe

tRNA
A

CC

CG

Anticodon

AAA
UGGUUUGGC

Codons

mRNA
Fig. 14.14. Translation: the basic concept.

Flattened into one plane, a tRNA molecule looks like a cloverleaf


In three dimensions, tRNA is roughly L-shaped, where one end of the L
contains the anticodon that base-pairs with an mRNA codon
3

Amino acid A
attachment C
C
site
A 5
C
G
C
U
U
A
A
U C
*
C A C AG
G
G U G U*
C
* *
C
U
* GA
G
G
U
*
*
A
*
A

G
C
G
G
A
U
U
A G *
U
A* C U C
*
G
C G A G
A G G
*
C
C
A
G
A

Anticodon
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5
3

Hydrogen
bonds

Hydrogen
bonds

C
U
G

(a) Two-dimensional structure

Amino acid
attachment site

A A G
3

Anticodon
(b) Three-dimensional
structure

Anticodon
(c) Symbol used
in this book
Figure 14.15

Ribosomes
Ribosomes facilitate
specific coupling of
tRNA anticodons with
mRNA codons during
protein synthesis

tRNA
molecules

The large and small


ribosomal subunits
are made of proteins
and ribosomal
RNAs (rRNAs)
In bacterial and
eukaryotic ribosomes
the large and small
subunits join to form
a ribosome only
when attached to an
mRNA molecule

Large
subunit

E P
A

Small
subunit
5

mRNA

(a) Computer model of functioning ribosome


Figure 14.17a

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Exit tunnel

Growing polypeptide

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
P site
(Peptidyl-tRNA
binding site)
E site
(Exit site)
mRNA
binding site

Growing polypeptide
Exit tunnel

Amino end
Next amino acid
to be added to
polypeptide
chain

A site (AminoacyltRNA binding site)


E

Large
subunit

mRNA

Small
subunit

(b) Schematic model showing binding sites

tRNA
3
Codons

(c) Schematic model with mRNA and tRNA

Figure 14.17b
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Building a Polypeptide
The three stages of translation:
Initiation: brings together
mRNA, a tRNA with the first
amino acid, and the two
ribosomal subunits. The small
ribosomal subunit binds to the
start codon (AUG) of the
mRNA first followed by the large
ribosomal subunit this
completes the formation of the
initiation complex
Elongation: amino acids are
added one by one to the
previous amino acid at the Cterminus of the growing chain
Termination: occurs when a
ribosome reaches a stop codon
on the mRNA. The ribosomes A
site then accepts a protein called
a release factor. The Release
factor promotes hydrolysis of the
bond between the tRNA in the P
site and the last amino acid of
polypeptide, thus freeing the
polypeptide from the ribosome

All three stages require protein


factors that aid in the translation
process
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Amino acids

Polypeptide

Ribosome

P site

tRNA with
amino acid
attached

Trp
Phe

Gly

tRNA

Anticodon

A A A
U G G U U U G G C

Codons

mRNA
Figure 14.14

Completing and Targeting the Functional Protein


Often translation is not sufficient to make a functional protein
Polypeptide chains are modified after translation or targeted
to specific sites in the cell

Protein Folding and Post-Translational


Modifications
During synthesis, a polypeptide chain spontaneously coils and folds into
its three-dimensional shape
Proteins may also require post-translational modifications before doing
their jobs for example, certain amino acids may be chemically modified
by attachment of sugars, lipids, phosphate groups etc., or the polypeptide
may be enzymatically cleaved into two or more pieces
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Targeting Polypeptides to Specific Locations


Two populations of ribosomes are evident in cells: free
ribosomes (in the cytosol) and bound ribosomes
(attached to the ER)
Free ribosomes mostly synthesize proteins that function
in the cytosol
Bound ribosomes make proteins of the endomembrane
system (ie., nuclear envelope, ER, Golgi, lysosomes,
vacuoles, and plasma membrane) and proteins that are
secreted from the cell (eg., insulin)

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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 protein-RNA
complex called a signal-recognition particle (SRP) binds to the signal peptide

The SRP brings the signal peptide and its ribosome to the ER
1

Polypeptide
synthesis
begins.

SRP
binds to
signal
peptide.

SRP
binds to
receptor
protein.

4
SRP
detaches
and
polypeptide
synthesis
resumes.

5
Signalcleaving
enzyme cuts
off signal
peptide.

6
Completed
polypeptide
folds into
final
conformation.

Ribosome
mRNA

Signal
peptide

ER
membrane

SRP
CYTOSOL

Signal
peptide
removed

Protein

SRP receptor
protein

ER LUMEM
Translocation complex
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Fig. 14.21

Concept 14.5: Mutations of one or a few nucleotides can


affect protein structure and function
Mutations are changes in
the genetic material of a cell
or virus
Sickle-cell hemoglobin

Wild-type hemoglobin

Point mutations are


chemical changes in just one
or a few nucleotide pairs 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

Wild-type hemoglobin DNA


C T C
3
5
G A G
3
5

mRNA
5

mRNA
G A G

Normal hemoglobin

The change of a single


nucleotide in a DNA template
strand can lead to the
production of an abnormal
protein
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Mutant hemoglobin DNA


C A C
5
3
G T G
3
5

Glu

G U G

Sickle-cell hemoglobin
Val

Fig. 14.25. The molecular basis of sickle-cell disease: a


point mutation.

Wild type

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. Substitution
mutations are usually
missense mutations
Nonsense mutations
change an amino acid
codon into a stop
codon, nearly always
leading to a
nonfunctional
protein
Insertion or deletion
of nucleotides may
alter the reading
frame of the genetic
message, producing a
frameshift mutation.
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DNA template strand 3


5
mRNA 5
Protein
Amino end

T A C T T C A A A C C G A T T
A T G A A G T T T G G C T A A
A U G A A G U U U G G C U A
Lys

Met

(a) Nucleotide-pair substitution


A instead of G
3
5

T A C T T C A A A C C A A T T
A T G A A G T T T G G T T A A

Gly

Phe

Stop
Carboxyl end
(b) Nucleotide-pair insertion or deletion
Extra A

5
3

3
5

T A C A T T C A A A C C G A T T
A T G T A A G T T T G G C T A A

A U G U A A G U U U G G U U A A

A U G A A G U U U G G U U A A
Met

Lys

Phe

Gly

Stop
Silent (no effect on amino acid sequence)

Met

Stop
Frameshift causing immediate nonsense
(1 nucleotide-pair insertion)

T instead of C
3
5

T A C T T C A A A T C G A T T
A T G A A G T T T A G C T A A

5
3

3
5

Met

Lys

Phe

Ser

Stop

Lys

Leu

T T C

missing

5
3

3
5

A U G U A G U U U G G U U A A 3

A U G U U U G G C U A A

Met

Nonsense

Ala

T A C A A A C C G A T T
A T G T T T G G C T A A

T A C A T C A A A C C G A T T
A T G T A G T T T G G C T A A

U instead of A
5

missing

Frameshift causing extensive missense


(1 nucleotide-pair deletion)

A instead of T
3
5

5
3

A U G A A G U U G G G U A A
Met

Missense

missing

T A C T T C A A C C G A T T
A T G A A G T T G G C T A A

A instead of G
A U G A A G U U U A G C U A A

Stop

5
3

Extra U

U instead of C
5

5
3
A 3

A A G
Met

5
3

missing

Phe

Gly

Stop
No frameshift, but one amino acid missing
(3 nucleotide-pair deletion)

Figure 14.26. Types of small-scale mutations that affect mRNA sequence

Mutagens
Spontaneous mutations can occur during DNA
replication, recombination, or repair
Mutagens are physical or chemical agents that can
cause mutations (eg., X-rays, UV light, pesticides)
Researchers have developed methods to test the
mutagenic activity of chemicals
Most cancer-causing chemicals (carcinogens) are
mutagenic

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