Transcription and
Translation
From Gene to Protein
• The DNA inherited by an organism leads to specific
traits by dictating the synthesis of proteins and of
RNA molecules involved in protein synthesis.
• Gene expression is the process by which DNA directs
the synthesis of proteins.
• The process by which DNA directs protein synthesis,
gene expression includes two stages transcription
and translation
From Gene to Protein
• Proteins are the links between genotype and phenotype.
• A gene does not build a protein directly
• A gene dispatches instructions in the form of RNA, which
programs protein synthesis
The Human Genome
• Genome – An organisms genetic content.
• The human genome contains 3.2 billion chemical
nucleotide bases (A, C, T, and G).
• The average gene consists of 3000 bases, but
sizes vary greatly, with the largest known human
gene being dystrophin at 2.4 million bases.
• The total number of genes is estimated at 30,000
• Almost all (99.9%) nucleotide bases are exactly the
same in all people.
The
Human
Genome
Less than 2% of the genome codes for proteins.
•
• Repeated sequences that do not code for proteins
("junk DNA") make up at least 50% of the human
genome.
• Repetitive sequences are thought to have no direct
functions, but they shed light on chromosome
structure and dynamics.
• Over time, these repeats
– reshape the genome by rearranging it
– create entirely new genes, and
– modify and reshuffle existing genes.
• Transcription is the transfer of genetic information
from DNA to an RNA molecule
• Translation is the transfer of the information in the
RNA into a protein
Nuclear
envelope
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
mRNA
Ribosome
TRANSLATION
(b)
Polypeptide
Figure 17.3b
History
-
demonstrating the relationship between genes and enzymes
• In 1909, British physician Archibald Garrod
o Was the first to suggest that genes dictate phenotypes through
enzymes that catalyze specific chemical reactions in the cell
• The work of Beadle and Tatum
o Developed the “One gene-one enzyme hypothesis”
• Created mutants of bread mold that could not survive on
minimal medium
• Hypothesis states that the function of a gene is to
dictate the production of a specific enzyme
Basic Principles of Transcription and Translation
• Transcription
o Is the synthesis of RNA under the direction of DNA
o The message is transcribed onto RNA
o Produces messenger RNA (mRNA)
• Translation
o Is the actual synthesis of a polypeptide, which occurs
under the direction of mRNA
o There is a change in language as the sequence goes from
mRNA to the amino acid sequence of a polypeptide
o Occurs on ribosomes
• Flow of information
o DNA-------- RNA------- Protein
Some differences in the flow of information for
prokaryotes and eukaryotes
• Prokaryotes – no nucleus
o DNA not separated from ribosomes
o Translation of mRNA can begin while its transcription is still in progress
TRANSCRIPTION
DNA
mRNA
Ribosome
TRANSLATION
Polypeptide
Figure 17.3a
(a) Prokaryotic cell. In a cell lacking a nucleus, mRNA
produced by transcription is immediately translated
without additional processing.
Some differences in the flow of information for
prokaryotes and eukaryotes
• Eukaryotes – contain a nucleus
o Nuclear envelope separates transcription from translation
o Transcription occurs in nucleus
o Translation occurs in cytoplasm
o RNA will be modified before becoming functional mRNA
Figure 17.3a
The Genetic Code
• Think of nucleic acids and proteins as having languages
• We need to see how the chemical language of DNA is
translated into the different chemical language of proteins
• There are only 4 nucleotide bases to specify 20 amino acids
• How many bases correspond to an amino acid?
Codons: Triplets of Bases
• DNA and RNA are polymers made of nucleotide monomers
o DNA has 4 types of nucleotides (A,T,C and G)
o The same is true for RNA, although it has U instead of T
• If each arrangement of three consecutive bases specifies an
amino acid, there can be 64 (43) possible code words. Which is
enough.
• The flow of information is based on a triplet code.
o Three-nucleotide words (AGT for example will code for a particular amino acid)
• These triplet codes are called Codons
Overview: The Gene determines the sequence of bases
1. Gene 1 is a small
region of one of the
genes carried by a
DNA molecule
Gene 2
DNA
molecule
Gene 1
2. DNA’s language is
written as a linear
sequence of nucleotide
bases on a polynucleotide.
Gene 3
DNA strand 3
5
A C C A A A C C G A G T
(template)
TRANSCRIPTION
4. The red strand
represents the results
of transcription: an
RNA molecule.
U G G U U U G G C U C A
mRNA
5
3
Codon
6. The purple chain =
translation. The conversion
of the nucleic acid
language into the
polypeptide language.
3. These specific sequences of
bases, each with a beginning and
an end, make up the genes on a
DNA strand. A typical gene will
consist of hundreds or thousands
of nucleotides in a specific
sequence.
TRANSLATION
Protein
Trp
Amino acid
Phe
Gly
Ser
5. The DNA language
has been rewritten
(transcribed) as a
sequence of bases on
RNA. RNA is
complimentary to the
DNA strand.
• Remember, proteins consist of one or more polypeptides
o Polypeptides are polymers, but the monomers that make them up are the 20 amino
acids common to all organisms
Gene 2
DNA
molecule
Gene 1
Gene 3
DNA strand3
5
A C C A A A C C G A G T
(template)
TRANSCRIPTION
Triplets of bases are the smallest
“words” that can specify all the amino
acids.
mRNA
5
U G G U U U G G C U C A
Codon
TRANSLATION
Protein
Trp
Amino acid
Phe
Gly
Ser
3
The flow of information from gene to protein is based on a triplet code.
Codons and amino acids
Second mRNA base
U
C
UUU
Left to right in
groups of three.
C
A
UCC
UCA
UAC
Ser
Tyr
UGU
UGC
U
Cys
C
UCG
UAG Stop
UGG Trp
G
CUU
CCU
CAU
CGU
U
CUC
CCC
CAC
CUA
Leu
Leu
CCA
Pro
CAA
CUG
CCG
CAG
AUU
ACU
AAU
ACC
AAC
AUC
lle
AUA
ACA
Met or
start
Thr
AAG
GUU
GCU
GAU
GUC
GCC
GAC
GUG
Val
GCA
GCG
Ala
His
Gln
Asn
AAA
ACG
GUA
Figure 17.5
UAU
UGA Stop A
AUG
G
Phe
UCU
G
UAA Stop
UUG
First mRNA base (5 end)
Codons must be
read in the correct
Reading Frame.
UUC
U
UUA
A
Lys
CGA
Arg
Asp
CGG
G
AGU
U
AGC
Ser C
AGA
A
AGG Arg G
GGC
GGA
Glu
C
A
U
GGU
GAA
GAG
CGC
GGG
Gly
C
A
G
Third mRNA base (3 end)
•
Evolution of the Genetic
Code
• The genetic code is nearly universal
o Shared by organisms from the simplest bacteria to the most complex
animals
• CCG codes for proline in all organisms who's genetic code has
been studied
• Bacteria can translate human genetic messages and human cells
can translate bacterial RNA
• A language shared by all living things must have been
operating very early in the history of life. A shared genetic
vocabulary is a reminder of the kinship that bonds all life on
Earth.
Transcription a Closer Look
Transcription
Non-template
strand of DNA
Elongation
RNA nucleotides
–Transcription is catalyzed by RNA
polymerase, which pries the DNA strands apart
and joins the RNA nucleotides as they base
pair along the DNA.
RNA
polymerase
–Adds in the 5’to3’ direction
–No primer needed
A
T
C
C
A
A
3
3 end
Follows the same base-pairing
rules as DNA, except that in
RNA, uracil substitutes for
thymine
U
5
U
G
C
A
T
A
G
G
T
T
Direction of transcription
(“downstream”)
5
Only one strand of DNA
serves as a template. This
strand is called the
Template Strand
A
Newly made
RNA
Template
strand of DNA
Molecular Components of Transcription
• RNA polymerase
o The enzyme which pries the two strands of DNA apart
o Adds RNA nucleotides – base pair to DNA template strand
• Must add nucleotides to the 3’ end
o Transcription initiation
• Begins at a region of bases known as the Promoter
o This is a specific sequences of bases recognized by
RNA polymerase and where it attaches
• The end of transcription is a sequence called the
Terminator
• The promoter is said to be “upstream” of the
terminator and the direction of transcription is
“downstream”
Synthesis of an RNA Transcript
• The stages of transcription are
o 1. Initiation
Promoter
Transcription unit
5
3
3
5
Start point
DNA
RNA polymerase
o 2. Elongation
5
3
Unwound
o 3.Termination
DNA
The stretch of DNA
being transcribed is
called a
transcription unit
Bacteria have one type
of RNA polymerase
Eukaryotes have at least
Figure 17.7
3 types.
The portion of DNA that is being
transcribed is called the
transcription unit. RNA
polymerase carries out
transcription. It attaches to the
promoter region and pries apart
the double stranded DNA.
3
5
Template strand of
DNA
transcript
RNA
RNA polymerase moves along the
DNA, pairing up RNA nucleotides with
their DNA complements— adding
nucleotides to the end of the growing
RNA molecule.
Rewound
DNA
5
3
3
5
3
5
RNA
Only one strand of DNA serves
as a template and RNA
nucleotides are added to the 3’
end.
transcript
5
3
3
5
5
Completed RNA
transcript
3
Once transcription is
complete, RNA
polymerase releases the
completed RNA and
detaches from the DNA.
Promoters
• Prokaryotes
o Two short sequences of nucleotides which lie upstream
from the start point
o RNA polymerase binds directly to promoter
• Eukaryotes
o Very diverse, consist of a collection of proteins called
Transcription Factors
o Lie upstream from the start point
o Transcription factors must bind first, allowing RNA
polymerase II to then bind to DNA to begin transcription
o Promoter DNA sequence recognized is called a TATA box,
which is a sequence of nucleotides containing TATAAAA
Eukaryotic Promoter
• Initiation
• Elongation
o A single gene can be transcribed
Simultaneously by several molecules of
RNA polymerase. This increases the amount
of mRNA, which helps the cell make the protein
In large amounts.
Termination
Prokaryotes – proceeds through a terminator
Sequence and the polymerase detaches
Releasing mRNA
Eukaryotes – continues transcribing past
The point where the pre-mRNA is released.
pre-mRNA must go through processing
Modifying RNA after transcription
• Enzymes in nucleus modify pre-mRNA called RNA
processing
o
o
o
o
Both ends of the primary transcript are altered
Certain interior sections of the RNA molecule are cut out
Remaining parts are spliced together
mRNA molecule is now ready for translation
Modifying mRNA
• Each end is modified first
• RNA splicing occurs
o The sequence of DNA nucleotides that codes for a eukaryotic
polypeptide is usually not continuous; it is split into segments
o These noncoding segments are called introns
o Other regions, usually translated into amino acid sequences are
called exons.
o Introns are cut out and the exons are joined together, thus a
continuous coding sequence is formed
o This process is called RNA splicing
o snRNP’s or snurps join with other proteins and form spliceosomes
which release the introns and join the exons
o View campbell animation
video
http://www.youtube.com/watch?v=2zBBm2E0aZ4
Molecular Components of
Translation
• Translation – The RNA-directed synthesis of a polypeptide
• The interpreter of the protein coded on the mRNA strand is
called transfer RNA (tRNA)
• tRNA transfers amino acids from the cytoplasm to a
ribosome
• These amino acids are synthesized from other components or are taken up from
the surrounding cytoplasmic solution
• The ribosome adds each amino acid (from the tRNA) to the
growing end of a polypeptide chain
• Translation: the basic concept
TRANSCRIPTION
DNA
mRNA
Ribosome
TRANSLATION
Polypeptide
Amino
acids
Polypeptide
tRNA with
amino acid
Ribosome attached
Gly
tRNA
Anticodon
A A A
U G G U U U G G C
Codons
5
Figure 17.13
mRNA
3
tRNA
• Molecules of tRNA are not all identical
o Each carries a specific amino acid on one end
o Each has an anticodon, which is a nucleotide triplet on the
other end
o Anticodons base pair with complementary codons on
mRNA
• Example: mRNA codon-UUU, codes for amino acid
phenylalanine. The tRNA that would base pair has AAA
as its anticodon and carries the amino acid
phenylalanine on its other end
o The tRNA can read a nucleic acid word (the mRNA codon)
and interpret it as a protein word (the amino acid)
tRNA
• mRNA and tRNA are synthesized from DNA
templates
• In eukaryotes tRNA and mRNA are both
made in the nucleus and travel to the
cytoplasm
• tRNA molecules are used repeatedly
The Structure and Function of Transfer RNA
• A tRNA molecule
o Consists of a single RNA strand that is only about 80 nucleotides
A
long and is roughly L-shaped
C
5
3
Hydrogen
bonds
3
A
Amino acid
C
attachment site C
A AG
3
Anticodon
(b) Three-dimensional structure
C
Amino acid
attachment site
5
Anticodon
(c) Symbol used
in this book
A
C
G
C
U
U
A
A
U C
C A C AG
*
G
GU GU *
C
* *
U C
* GAG
G
U
Two-dimensional structure. The four base-paired regions and
three loops are characteristic of all tRNAs, as is the base
sequence of the amino acid attachment site at the 3 end. The
Figure 17.14a anticodon triplet is unique to each tRNA type. (The asterisks
mark bases that have been chemically modified, a characteristic
of tRNA.)
5
G
C
G
G
A
U
U
UA
* CUC
CGAG
*
AG *
*
G
A G G
C
Hydrogen
C
A
bonds
* G
A
A*
C
U
*
A
G
A
Anticodon
Joining amino acids to tRNA
• A specific enzyme called an aminoacyl-tRNA synthetases
o Joins each amino acid to the correct tRNA
Amino acid
P P
Aminoacyl-tRNA
synthetase (enzyme)
1 Active site binds the
amino acid and ATP.
P Adenosine
ATP
2 ATP loses two P groups
and joins amino acid as AMP.
P
Pyrophosphate
Pi
Phosphates
P
Adenosine
Pi
Pi
tRNA
3 Appropriate
tRNA covalently
Bonds to amino
Acid, displacing
AMP.
P Adenosine
AMP
4 Activated amino acid
is released by the enzyme.
Figure 17.15
Aminoacyl tRNA
(an “activated
amino acid”)
Ribosomes
• Ribosomes
o Facilitate the specific coupling of tRNA anticodons with mRNA codons
during protein synthesis
o Made of two subunits. The large and small
o Constructed of proteins and RNA molecules called ribosomal RNAs or
rRNA
o In eukaryotes units are made in the nucleolus
o The units will attach to form a functional ribosome only when
attached to a mRNA molecule
Large unit
Small unit
Ribosomes
• The ribosome has three binding sites for tRNA
o The P site
o The A site
o The E site
Growing polypeptide
Amino end
Next amino acid
to be added to
polypeptide chain
E
tRNA
E
P
A
A
3
mRNA
5
P
Codons
(c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its
anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the
growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added
Figure 17.16b
to the polypeptide chain. Discharged tRNA leaves via the E site.
Building a Polypeptide
• We can divide translation into three stages
o Initiation
o Elongation
o Termination
Ribosome Association and Initiation of Translation
• The initiation stage of translation
o Brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two
subunits of a ribosome
o The union of mRNA, initiator tRNA, and a small ribosomal subunit is followed by the
attachment of a large ribosomal subunit
• Initiator mRNA binds to P site
P site
3 U A C 5
5 A U G 3
Initiator tRNA
Large
ribosomal
subunit
GTP
GDP
E
A
mRNA
5
Start codon
mRNA binding site
Figure 17.17
3
Small
ribosomal
subunit
1 A small ribosomal subunit binds to a molecule of
mRNA. In a prokaryotic cell, the mRNA binding site
on this subunit recognizes a specific nucleotide
sequence on the mRNA just upstream of the start
codon. An initiator tRNA, with the anticodon UAC,
base-pairs with the start codon, AUG. This tRNA
carries the amino acid methionine (Met).
5
3
Translation initiation complex
2 The arrival of a large ribosomal subunit completes
the initiation complex. Proteins called initiation
factors (not shown) are required to bring all the
translation components together. GTP provides
the energy for the assembly. The initiator tRNA is
in the P site; the A site is available to the tRNA
bearing the next amino acid.
Elongation of the Polypeptide Chain
• In the elongation stage of translation
o Amino acids are added one by one to the preceding
amino acid.
TRANSCRIPTION
Amino end
of polypeptide
DNA
mRNA
Ribosome
TRANSLATION
Polypeptide
mRNA
Ribosome ready for
next aminoacyl tRNA
E
3
P A
site site
5
1 Codon recognition. The anticodon
of an incoming aminoacyl tRNA
base-pairs with the complementary
mRNA codon in the A site. Hydrolysis
of GTP increases the accuracy and
efficiency of this step.
2 GTP
2 GDP
E
E
P
P
A
GDP
Figure 17.18
3 Translocation. The ribosome
translocates the tRNA in the A
site to the P site. The empty tRNA
in the P site is moved to the E site,
where it is released. The mRNA
moves along with its bound tRNAs,
bringing the next codon to be
translated into the A site.
GTP
E
P
A
A
2 Peptide bond formation. An
rRNA molecule of the large
subunit catalyzes the formation
of a peptide bond between the
new amino acid in the A site and
the carboxyl end of the growing
polypeptide in the P site. This step
attaches the polypeptide to the
tRNA in the A site.
Termination of Translation
• The final stage of translation is termination
o Elongation will continue until a stop codon in the mRNA reaches the A site of the
ribosome
o
Release
factor
Free
polypeptide
5
3
3
5
5
3
Stop codon
(UAG, UAA, or UGA)
1 When a ribosome reaches a stop 2 The release factor hydrolyzes 3 The two ribosomal subunits
codon on mRNA, the A site of the
the bond between the tRNA in and the other components of
ribosome accepts a protein called
the P site and the last amino
the assembly dissociate.
a release factor instead of tRNA.
acid of the polypeptide chain.
The polypeptide is thus freed
from the ribosome.
Figure 17.19
polyribosomes
• Enable a cell to make many copies of a polypeptide very
quickly
• Polyribosomes are several ribosomes in clusters and can
translate an mRNA molecule simultaneously
In both
bacterial
and
eukaryotic
cells
Mutations
• Point mutations can affect protein structure and function
• Mutations
o Are changes in the genetic material of a cell that may involve large portions of a
chromosome or just one base pair of nucleotides, which are Point mutations
• Point mutations
o Are changes in just one base pair of a gene
Types of Gene Mutations
• Point Mutations
o Changes in nucleotides
• Include
oSubstitutions
oInsertions
oDeletions
Types of Point Mutations
Base-pair substitution
o Is the replacement of one nucleotide and its
complementary partner with another pair of nucleotides
Wild type
mRNA
Protein
A U G
5
Met
A A G U U U G G C U A A
Lys
Phe
Gly
3
Stop
Amino end
Because some codons code for the
same amino acid there are times this
has no effect on the protein. This is
called a silent mutation
Carboxyl end
Base-pair substitution
No effect on amino acid sequence
U instead of C
A U G A A G U U U G G U U A A
Met
If a substitution results in an
incorrectly coded amino acid (will
alter the character of the protein) its
called a missense mutation
If a substitution changes an amino acid
codon into a stop codon, prematurely –
creating a nonfunctional protein, its
called a nonsense mutation
Lys
Missense
Phe
Gly
Stop
A instead of G
A U G A A G U U U A G U U A A
Met
Lys
Phe
Ser
Stop
Nonsense
U instead of A
A U G U A G U U U G G C U A A
Met
Stop
Figure 17.24
Insertions and Deletions
• Base pair Insertions or deletions
o Are additions or losses of nucleotide pairs in a gene that are not in multiples
of three
• These change the reading frame and are called Frameshift mutations
• All nucleotides downstream from the mutation will be improperly
grouped. The codons are not read properly and create extensive missense
and nonsense.
Frameshift mutations
almost always produce
nonfunctional proteins
Figure 17.25
Other mutations
• Spontaneous mutations
o Include base-pair substitutions, insertions , deletions, and longer mutations.
o Can occur during DNA replication, recombination, or repair
Causes of “Other”
mutations:
• Mutagens
o Are physical or chemical agents that can cause mutations
o Include X-rays, UV light and various chemical agents.
What is a gene? revisiting
the question
• A gene
o Is a region of DNA whose final product is either a polypeptide or an RNA molecule
• A summary of transcription and translation in a
eukaryotic cell
DNA
TRANSCRIPTION
1 RNA is transcribed
from a DNA template.
3
5
RNA
transcript
RNA
polymerase
RNA PROCESSING
Exon
2 In eukaryotes, the
RNA transcript (premRNA) is spliced and
modified to produce
mRNA, which moves
from the nucleus to the
cytoplasm.
RNA transcript
(pre-mRNA)
Intron
Aminoacyl-tRNA
synthetase
NUCLEUS
Amino
acid
tRNA
FORMATION OF
INITIATION COMPLEX
CYTOPLASM 3 After leaving the
nucleus, mRNA attaches
to the ribosome.
mRNA
AMINO ACID ACTIVATION
4
Each amino acid
attaches to its proper tRNA
with the help of a specific
enzyme and ATP.
Growing
polypeptide
Activated
amino acid
Ribosomal
subunits
5
TRANSLATION
A succession of tRNAs
add their amino acids to
the polypeptide chain
Anticodon
as the mRNA is moved
through the ribosome
one codon at a time.
(When completed, the
polypeptide is released
from the ribosome.)
5
E
A
AAA
UG GU U U A U G
Codon
Figure 17.26
Ribosome