Download From DNA to Protein: Gene Expression

10
From DNA to Protein: Gene
Expression
Chapter 10 From DNA to Protein: Gene Expression
Key Concepts
10.1 Genetics Shows That Genes Code for
Proteins
10.2 DNA Expression Begins with Its
Transcription to RNA
10.3 The Genetic Code in RNA Is Translated
into the Amino Acid Sequences of
Proteins
Chapter 10 From DNA to Protein: Gene Expression
10.4 Translation of the Genetic Code Is
Mediated by tRNA and Ribosomes
10.5 Proteins Are Modified after Translation
Chapter 10 Opening Question
How do antibiotics target bacterial
protein synthesis?
Concept 10.1 Genetics Shows That Genes Code for Proteins
Identification of proteins as gene products
began in the early 20th century.
Children with a rare disease called
alkaptonuria had parents who were first
cousins.
Garrod realized they must be homozygous for
a recessive mutant gene.
He isolated homogentisic acid from the
children, which accumulated in the blood,
joints, and urine.
Concept 10.1 Genetics Shows That Genes Code for Proteins
Garrod speculated that homogentisic acid is
normally broken down to a harmless product
by an enzyme, but the dominant wild-type
allele for the enzyme was mutated in
alkaptonuria patients.
These and other studies led him to correlate
one gene to one enzyme.
Concept 10.1 Genetics Shows That Genes Code for Proteins
Homogentisic acid is part of a biochemical
pathway of protein breakdown.
Phenylketonuria is another genetic disease
that involves this pathway.
•  The enzyme that converts phenylalanine
to tyrosine is nonfunctional.
•  Untreated, it can lead to mental
retardation, but is easily detected in
newborns.
Figure 10.1 Metabolic Diseases and Enzymes
Concept 10.1 Genetics Shows That Genes Code for Proteins
Study of alkapatonuria and phenylketonuria
led to the “one gene–one enzyme”
hypothesis.
But studies of proteins made up of multiple
polypeptides showed that this hypothesis
was an oversimplification.
Concept 10.1 Genetics Shows That Genes Code for Proteins
Hemoglobin consists of 4 polypeptide chains.
•  Variations in human populations show
hundreds of single amino acid alterations
in the β-chains, such as the one that
results in sickle-cell disease.
The one gene–one enzyme relationship has
since been revised to the one gene–one
polypeptide relationship.
Figure 10.2 Gene Mutations and Amino Acid Changes
Concept 10.1 Genetics Shows That Genes Code for Proteins
Not all genes code for polypeptides.
•  Some are transcribed to RNA but not
translated to polypeptides; the RNAs have
other functions.
Molecular biology: the study of nucleic acids
and proteins; often focuses on gene
expression
Concept 10.1 Genetics Shows That Genes Code for Proteins
Gene are expressed in two steps:
•  Transcription: information from a DNA
sequence is copied to a complementary
RNA sequence
•  Translation: converts the RNA sequence
to an amino acid sequence
Concept 10.1 Genetics Shows That Genes Code for Proteins
Three types of RNA:
•  Messenger RNA (mRNA)—a DNA
sequence is copied to produce a
complementary mRNA strand, which
moves to a ribosome to be translated.
§ 
The nucleotide sequence of the
mRNA determines the sequence of
amino acids in the polypeptide chain.
Concept 10.1 Genetics Shows That Genes Code for Proteins
•  Ribosomal RNA (rRNA), along with
proteins, makes up ribosomes; one
catalyzes peptide bond formation.
•  Transfer RNA (tRNA) binds to specific
amino acids and to specific sequences on
mRNA by base pairing.
§ 
tRNA recognizes which amino acid
should be added next in a growing
polypeptide and carries it to the
ribosome.
Figure 10.3 From Gene to Protein
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
Transcription requires several components:
•  A DNA template for base pairings
•  The 4 nucleoside triphosphates (ATP,
GTP, CTP, UTP)
•  An RNA polymerase enzyme
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
RNA polymerases catalyze synthesis of RNA
from the DNA template.
RNA polymerases are processive—a single
enzyme–template binding results in
polymerization of hundreds of RNA bases.
Unlike DNA polymerases, RNA polymerases
do not need primers.
Figure 10.4 RNA Polymerase
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
Transcription occurs in three steps:
•  Initiation
•  Elongation
•  Termination
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
Initiation requires a promoter—a control
sequence of DNA that tells RNA polymerase
where to start transcription and which strand
to transcribe.
Each promoter has a transcription initiation
site.
Other proteins (sigma factors and transcription
factors) bind “upstream” from the initiation
site and help RNA polymerase bind and
determine which genes are expressed.
Figure 10.5 DNA Is Transcribed to Form RNA (Part 1)
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
Elongation: RNA polymerase unwinds DNA
and reads template in 3′-to-5′ direction.
RNA polymerase adds nucleotides to the 3′
end of the new strand; the RNA transcript is
antiparallel to the DNA template strand.
Uracil (not thymine) in the RNA molecule is
paired with adenine in the DNA molecule.
RNA polymerases can proofread but allow
more mistakes.
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
Termination is specified by a specific DNA
sequence.
Mechanisms of termination are complex and
vary among different genes and organisms.
In eukaryotes, multiple proteins are involved in
recognizing the termination sequence and
separating the newly formed RNA from the
DNA template and RNA polymerase.
Figure 10.5 DNA Is Transcribed to Form RNA (Part 2)
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
Coding regions are sequences of DNA that
are expressed as proteins.
In prokaryotes, most of the DNA is made up of
coding regions.
In prokaryotes and viruses, several adjacent
genes sometimes share one promoter.
Table 10.1
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
In eukaryotes each gene has its own promoter
and most have noncoding sequences called
introns.
The transcribed regions are exons.
Introns and exons both appear in the primary
mRNA transcript (pre-mRNA); introns are
removed from the final mRNA.
Figure 10.6 Transcription of a Eukaryotic Gene
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
Nucleic acid hybridization can be used to
locate introns.
Target DNA is denatured by heat to separate
the strands.
It is then incubated with a probe—a nucleic
acid strand from another source.
If the probe has a complementary sequence,
it forms a hybrid with the DNA by base
pairing.
Figure 10.7 Nucleic Acid Hybridization
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
Researchers used this technique to examine
the β-globin gene with previously isolated βglobin mRNA as the probe.
Viewing the hybridized molecules by electron
microscopy, they saw that the introns formed
loops—stretches of DNA that did not have
complementary base sequences on the
mature mRNA.
If pre-mRNA was used, the result was a linear
matchup—complete hybridization.
Figure 10.8 Demonstrating the Existence of Introns
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
Introns interrupt, but do not scramble, the DNA
sequence of a gene.
Most eukaryotic genes contain introns.
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
The pre-mRNA is modified:
RNA splicing removes introns and splices
exons together.
Consensus sequences are short sequences
between exons and introns; they are first
bound by snRNPs—small nuclear
ribonucleoprotein particles.
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
Then other proteins accumulate to form a
large RNA–protein complex called a
spliceosome.
The complex cuts the pre-mRNA, releases
the introns, and joins the exons together to
produce mature mRNA.
Figure 10.9 The Spliceosome: An RNA Splicing Machine
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
In the disease beta thalassemia, a mutation at
an intron consensus sequence in the βglobin gene prevents the pre-mRNA from
being spliced correctly.
Nonfunctional β-globin mRNA is produced,
resulting in severe anemia.
This is an example of how mutations are used
to elucidate cause-and-effect relationships.
Concept 10.2 DNA Expression Begins with Its Transcription to
RNA
The pre-mRNA is also modified at both ends:
A 5′ cap (G cap) is added to the 5′ end. It
facilitates binding to a ribosome and
prevents digestion by ribonucleases.
A poly A tail is added to the 3′ end. It assists
in export from the nucleus and contributes
to the stability of the mRNA.
IN-TEXT ART, p. 202
Concept 10.3 The Genetic Code in RNA Is Translated into the
Amino Acid Sequences of Proteins
Translation of the nucleotide sequence of an
mRNA into an amino acid sequence occurs
at ribosomes.
In prokaryotes, transcription and translation
are coupled: ribosomes often bind to an
mRNA as it is being transcribed.
In eukaryotes, the nuclear envelope separates
mRNA production and translation.
Concept 10.3 The Genetic Code in RNA Is Translated into the
Amino Acid Sequences of Proteins
The genetic information is a series of
sequential, nonoverlapping, three-letter
“words” (3 bases) called codons.
Each codon specifies an amino acid.
Codons were first identified by using short
artificial polynucleotides instead of complex
mRNAs.
Figure 10.10 Deciphering the Genetic Code (Part 1)
Figure 10.10 Deciphering the Genetic Code (Part 2)
Concept 10.3 The Genetic Code in RNA Is Translated into the
Amino Acid Sequences of Proteins
There are more codons than amino acids.
AUG codes for methionine and is also the
start codon.
UAA, UAG, and UGA are stop codons.
For most amino acids, there is more than one
codon; the genetic code is redundant.
But it is not ambiguous—each codon specifies
only one amino acid.
Figure 10.11 The Genetic Code
Concept 10.3 The Genetic Code in RNA Is Translated into the
Amino Acid Sequences of Proteins
The genetic code is nearly universal: the
codons that specify amino acids are the
same in all organisms.
A few exceptions occur in mitochondria,
chloroplasts, and some protists.
The genetic code is ancient; it unifies life and
indicates that all life came from a common
ancestor.
It is also the basis for genetic engineering.
Concept 10.3 The Genetic Code in RNA Is Translated into the
Amino Acid Sequences of Proteins
Point mutations also confirm the genetic code.
Point mutations in a coding region are
compared with the amino acid sequences in
the resulting polypeptides.
•  Silent mutations can occur because of
redundancy
§ 
Example: CCG and CCU are both
translated as proline (Pro).
Figure 10.12 Mutations (Part 1)
Figure 10.12 Mutations (Part 2)
Concept 10.3 The Genetic Code in RNA Is Translated into the
Amino Acid Sequences of Proteins
•  Missense mutations result in a change in
the amino acid sequence.
§  Example: GAU is translated as
aspartic acid (Asp), whereas a
mutation that results in GUU is
translated as valine (Val).
Figure 10.12 Mutations (Part 3)
Concept 10.3 The Genetic Code in RNA Is Translated into the
Amino Acid Sequences of Proteins
•  Nonsense mutations result in a
premature stop codon, resulting in a
shortened protein that is usually not
functional.
Figure 10.12 Mutations (Part 4)
Concept 10.3 The Genetic Code in RNA Is Translated into the
Amino Acid Sequences of Proteins
•  Frame-shift mutations are insertions or
deletions of one or more bases.
§  This causes new sets of triplets to be
read and an altered sequence of
amino acids in the resulting
polypeptide.
Figure 10.12 Mutations (Part 5)
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
tRNAs link information in mRNA codons with
specific amino acids.
Two events must occur:
•  tRNAs must read mRNA codons correctly
•  tRNAs must deliver the correct amino
acids that correspond to each codon
For each amino acid, there is at least one
specific type of tRNA.
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
The structure of tRNAs facilitates their three
functions:
•  Bind to a specific amino acid and become
“charged”
•  Bind to mRNA at the anticodon—a triplet
that is complementary to the mRNA
codon for the particular amino acid
•  Interact with ribosomes noncovalently
Figure 10.13 Transfer RNA
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
Wobble: specificity for the base at the 3′ end
of the codon is not always observed.
•  Example: Codons for alanine—GCA,
GCC, and GCU—are recognized by the
same tRNA.
Wobble allows cells to produce fewer tRNA
species but does not allow the genetic code
to be ambiguous.
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
tRNAs are charged by specific aminoacyltRNA synthetases.
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
An experiment demonstrated the importance
of the specificity between a tRNA and its
amino acid:
•  Cysteine already bound to tRNA was
chemically changed to alanine.
•  During protein synthesis, the tRNA
anticodon was recognized, not the amino
acid:
§  The cysteine-specific tRNA delivered
its cargo (alanine) to every mRNA
codon for cysteine.
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
Translation occurs at ribosomes.
All prokaryotic and eukaryotic ribosomes
consist of one large and one small subunit.
The subunits exist separately when not
translating.
Ribosomes are not specific; they can make any
type of protein.
Figure 10.14 Ribosome Structure
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
The large subunit has 3 tRNA binding sites:
•  A (amino acid) site binds with the
anticodon of a charged tRNA
•  P (polypeptide) site is where tRNA adds
its amino acid to the polypeptide chain
•  E (exit) site is where tRNA sits before
being released from the ribosome
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
Ribosomes have a fidelity function: when
proper binding occurs, hydrogen bonds
form between the base pairs.
Small subunit validates the match—if
hydrogen bonds have not formed between
all three base pairs, the tRNA is rejected.
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
Translation also occurs in three steps:
•  Initiation
•  Elongation
•  Termination
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
Initiation:
Initiation complex—the small subunit binds
to its recognition sequence on mRNA.
Methionine-charged tRNA then binds to the
AUG start codon.
The large subunit then joins the complex with
the charged tRNA in the P site.
The complex is put together by a group of
proteins called initiation factors.
Figure 10.15 The Initiation of Translation (Part 1)
Figure 10.15 The Initiation of Translation (Part 2)
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
Elongation:
Another charged tRNA enters the A site and
the large subunit catalyzes two reactions:
•  Breaks bond between tRNA in P site and
its amino acid
•  Forms peptide bond between that amino
acid and the amino acid on tRNA in the A
site
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
The large subunit is said to have peptidyl
transferase activity.
The component with this activity is an rRNA, so
the catalyst is an example of a ribozyme
(from ribonucleic acid and enzyme).
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
When the first tRNA has released its
methionine, the ribosome moves so that the
first tRNA is at the E site.
The first tRNA is released and can become
charged again.
Elongation occurs as the steps are repeated.
Figure 10.16 The Elongation of Translation (Part 1)
Figure 10.16 The Elongation of Translation (Part 2)
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
Termination:
Translation ends when a stop codon enters
the A site.
It binds a protein release factor, which allows
hydrolysis of the bond between the
polypeptide chain and the tRNA in the P
site.
The newly completed polypeptide then
separates from the ribosome.
Figure 10.17 The Termination of Translation (Part 1)
Figure 10.17 The Termination of Translation (Part 2)
Figure 10.17 The Termination of Translation (Part 3)
Table 10.2
Concept 10.4 Translation of the Genetic Code Is Mediated by
tRNA and Ribosomes
Several ribosomes can simultaneously
translate a single mRNA molecule,
producing multiple polypeptides at the same
time.
A strand of mRNA with associated ribosomes
is called a polyribosome, or polysome.
Figure 10.18 A Polysome
Concept 10.5 Proteins Are Modified after Translation
Proteins can undergo modifications both
during and after translation.
As a polypeptide emerges from the ribosome
it may fold into its 3-D shape and perform its
role in the cytosol.
Or, polypeptides may contain a signal
sequence that “targets” them to the
nucleus, mitochondria, plastids, or
peroxisomes.
Concept 10.5 Proteins Are Modified after Translation
The signal sequence binds to a receptor
protein on the organelle surface.
A channel forms in the membrane and the
protein moves into the organelle.
Figure 10.19 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell (Part 1)
Concept 10.5 Proteins Are Modified after Translation
A nuclear localization signal (NLS) targets the
protein to the nucleus.
Function of NLS was established by
experiments in which proteins with or without
it were put into cells and then located by
labeling the proteins with fluorescent dyes.
Only proteins with the NLS were found in the
nucleus.
Figure 10.20 Testing the Signal (Part 1)
Figure 10.20 Testing the Signal (Part 2)
Concept 10.5 Proteins Are Modified after Translation
If a polypeptide has a signal directing it to the
rough endoplasmic reticulum (RER),
translation will pause, and the ribosome will
bind to a receptor at the RER membrane.
Then translation resumes, and as elongation
continues, the polypeptide crosses into the
RER lumen.
It may be retained in the lumen, sent to other
regions in the endomembrane system, or be
secreted from the cell.
Figure 10.19 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell (Part 2)
Concept 10.5 Proteins Are Modified after Translation
Most polypeptides are modified after
translation:
Proteolysis—cutting off signal sequences; or
cutting a long chain (a polyprotein) into final
products by proteases
Glycosylation—addition of carbohydrates to
form glycoproteins
Concept 10.5 Proteins Are Modified after Translation
Phosphorylation—addition of phosphate
groups by protein kinases
The charged phosphate groups change the
conformation of the protein, often exposing
the active site or other binding site.
Figure 10.21 Posttranslational Modifications of Proteins
Figure 10.22 An Antibiotic at the Ribosome
Answer to Opening Question
Strains of MRSA with resistance to
tetracyclines are emerging.
Genes that confer resistance are carried on
plasmids, which can move between bacteria
during conjugation.
Some resistance genes encode proteins that
transfer tetracyclines out of the cell; others
encode proteins that prevent antibiotics from
binding to the ribosomes.
Answer to Opening Question
To overcome resistance, new antibiotics are
being developed and tried.
The evolutionary race between drug
resistance and development of antibiotics
continues.