Download Basic Principles of Transcription and Translation

The Flow of Genetic Information
The information content of DNA is in the form of specific sequences of
nucleotides
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
The ribosome is part of the cellular machinery for translation,
polypeptide synthesis
Basic Principles of Transcription and
Translation
RNA is the intermediate between genes and the proteins
for which they code
Transcription is the synthesis of RNA under the direction
of DNA
Transcription produces messenger RNA (mRNA)
Translation is the synthesis of a polypeptide, which occurs
under the direction of mRNA
Ribosomes are the sites of translation
• In prokaryotes, mRNA produced by transcription is
immediately translated without more processing
• In a eukaryotic cell, the nuclear envelope separates
transcription from translation
• Eukaryotic RNA transcripts are modified through RNA
processing to yield finished mRNA
• A primary transcript is the initial RNA transcript from any
gene
• The central dogma is the concept that cells are governed
by a cellular chain of command: DNA → RNA → protein
• Eukaryotic RNA transcripts are modified through RNA
processing to yield finished mRNA
DNA
TRANSCRIPTION
mRNA
Ribosome
a) Bacterial cell. In a bacterial cell which
lacks a nucleus, mRNA produced by
transcription is immediately translated
without additional processing.
TRANSLATION
Polypeptide
(a) Bacterial cell
Nuclear
envelope
DNA
TRANSCRIPTION
b) Eukaryotic cell. The nucleus provides a
separate compartment for transcription.
The original RNA transcript called pre
mRNA is processed in various ways
before leaving the nucleus as mRNA.
Pre-mRNA
RNA PROCESSING
mRNA
TRANSLATION
Ribosome
Polypeptide
(b) Eukaryotic cell
Overview: the roles of transcription and
translation in the flow of genetic
information. In a cell inherited information
flows from DNA to RNA to protein. The
two main stages of information flow are
transcription and translation.
DNA
TRANSCRIPTION
mRNA
TRANSCRIPTION
DNA
mRNA
Ribosome
(a) Bacterial cell
TRANSLATION
Polypeptide
Nuclear
envelope
TRANSCRIPTION
DNA
Pre-mRNA
(b) Eukaryotic cell
Nuclear
envelope
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
mRNA
(b) Eukaryotic cell
Nuclear
envelope
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
mRNA
TRANSLATION
Ribosome
Polypeptide
(b) Eukaryotic cell
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 bases correspond to an amino acid?
Codons: Triplets of Bases
The flow of information from gene to protein is based on a
triplet code: a series of nonoverlapping, three-nucleotide
words
These triplets are the smallest units of uniform length that
can code for all the amino acids
Example: AGT at a particular position on a DNA strand
results in the placement of the amino acid serine at the
corresponding position of the polypeptide to be produced
During transcription, one of the two DNA strands called the
template strand provides a template for ordering the
sequence of 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 to be placed at the
corresponding position along a polypeptide
Colons along an mRNA molecule are read by translation
machinery in the 5′ to 3′ direction
Each codon specifies the addition of one of 20 amino acids
DNA
molecule
Gene 2
Gene 1
Gene 3
DNA
template
strand
TRANSCRIPTION
mRNA
Codon
TRANSLATION
Protein
Amino acid
The triplet code. For each
gene, one strand of DNA
functions as a template for
transcription. The base
pairing rules for DNA
synthesis also guide
transcription, but uracil (U)
takes the place of thymine
(T) in RNA. During
translation the mRNA is read
as a sequence of base
triplets called codons. Each
codon specifies an amino
acid to be added to the
growing polypeptide chain.
The mRNA is read in the
5’⇒ 3’ direction.
Cracking the Code
All 64 codons were deciphered by the mid-1960s
Of the 64 triplets, 61 code for amino acids; 3 triplets are
“stop” signals to end translation
The genetic code is redundant but 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
Third mRNA base (3′′ end of codon)
First mRNA base (5′′ end of codon)
Second mRNA base
The dictionary of the genetic
code. The three bases of an
mRNA codon are designated
here as the first, second and
third bases reading in the 5’
⇒ 3’ direction along the
mRNA. The codon AUG not
only stands for the amino
acid methionine (Met) but
also functions as a start
signal for ribosomes to begin
translating the mRNA at that
point. Three of the 64
codons function as “stop”
signals marking the end of a
genetic message
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
Because diverse forms of life share a common genetic
code, one species can be programmed to produce proteins
characteristic of a second species by introducing DNA from
the second species into the first
Transcription is the DNA-directed synthesis of
RNA: a closer look
Transcription, the first stage of gene expression, can be
examined in more detail
The three stages of transcription:
Initiation
Elongation
Termination
Molecular Components of Transcription
RNA synthesis is catalyzed by RNA polymerase, which
pries the DNA strands apart and hooks together the RNA
nucleotides
RNA synthesis follows the same base-pairing rules as
DNA, except uracil substitutes for thymine
The DNA sequence where RNA polymerase attaches is
called the promoter; in bacteria, the sequence signaling
the end of transcription is called the terminator
The stretch of DNA that is transcribed is called a
transcription unit
Promoter
Transcription unit
5′′
3′′
Start point
RNA polymerase
3′′
5′′
DNA
1 Initiation
5′′
3′′
RNA
transcript
3′′
Rewound
DNA
5′′
3′′
5′′
3 Termination
3′′
5′′
5′′
3′′
5′′
3′′ end
3′′
5′′
5′′
RNA
transcript
RNA
polymerase
Template strand
of DNA
2 Elongation
5′′
3′′
RNA nucleotides
3′′
5′′
Unwound
DNA
Nontemplate
strand of DNA
Elongation
Completed RNA transcript
3′′
Direction of
transcription
(“downstream”)
Newly made
RNA
Template
strand of DNA
Promoter
3′′
5′′
1) Initiation. After RNA polymerase binds
to the promoter, the DNA strands
unwind and the polymerase initiates
RNA synthesis at the start point on the
template strand.
3′′
5′′
2) 2) Elongation The polymerase moves
downstream unwinding the DNA and
elongating the RNA transcript 5’ 3’ In
the wake of transcription the DNA
strands re-form a double helix.
Transcription unit
5′′
3′′
Start point
RNA polymerase
DNA
1 Initiation
5′′
3′′
Unwound
DNA
RNA
transcript
Template strand
of DNA
2 Elongation
3) 3) Termination Eventually the RNA
transcript is released and the
polymerase detaches from the DNA
Rewound
DNA
5′′
3′′
3′′
5′′
3′′
5′′
RNA
transcript
The stages of transcription: initiation
elongation and termination. Thus
general depiction of transcription
applies to both bacteria and
eukaryotes but the details of
termination differ, as described in the
text. Also in a bacterium the transcript
is immediately usable as mRNA in a
eukaryote the RNA transcript must first
undergo processing.
3 Termination
5′′
3′′
3′′
5′′
5′′
Completed RNA transcript
3′′
Nontemplate
strand of DNA
Elongation
RNA
polymerase
3′′
RNA nucleotides
3′′ end
5′′
5′′
Direction of
transcription
(“downstream”)
Newly made
RNA
Template
strand of DNA
RNA Polymerase Binding and Initiation of
Transcription
Promoters signal the initiation of RNA synthesis
Transcription factors mediate the binding of RNA
polymerase and the initiation of transcription
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
1
Promoter
Template
5′′
3′′
3′′
5′′
TATA box Start point Template
DNA strand
2
2) Several transcription factors one
recognizing the TATA box must bind
to the DNA before RNA polymerase
II can do so.
Transcription
factors
5′′
3′′
3′′
5′′
3
RNA polymerase II
Transcription factors
5′′
3′′
1) A eukaryotic promoter
commonly includes a TATA box a
nucleotide sequence containing
TATA about 25 nucleotides
upstream from the transcription
start point. By convention
nucleotide sequences are given as
they occur on the nontemplate
strand
3′′
5′′
5′′
RNA transcript
Transcription initiation complex
3) Additional transcription factors
(purple) bind to the DNA along with
RNA polymerase II forming the
transcription initiation complex. The
DNA double helix then unwinds and
RNA synthesis begins at the start
point on the template strand.
The initiation of transcription at a
eukaryotic promoter. In eukaryotic
cells proteins called transcription
factors mediate the initiation of
transcription by RNA polymerase II
Elongation of the RNA Strand
As RNA polymerase moves along the DNA, it untwists the
double helix, 10 to 20 bases at a time
Transcription progresses at a rate of 40 nucleotides per
second in eukaryotes
A gene can be transcribed simultaneously by several RNA
polymerases
Termination of Transcription
The mechanisms of termination are different in
bacteria and eukaryotes
In bacteria, the polymerase stops transcription
at the end of the terminator
In eukaryotes, the polymerase continues
transcription after the pre-mRNA is cleaved
from the growing RNA chain; the polymerase
eventually falls off the DNA
Eukaryotic cells modify RNA after
transcription
Enzymes in the eukaryotic nucleus modify pre-mRNA
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
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
These modifications share several functions:
They seem to facilitate the export of mRNA
They protect mRNA from hydrolytic enzymes
They help ribosomes attach to the 5′ end
5′′
G
Protein-coding segment Polyadenylation signal
3′′
P P P
5′′ Cap
AAUAAA
5′′ UTR Start codon
Stop codon
3′′ UTR
AAA…AAA
Poly-A tail
RNA processing addition of the 5’ cap and poly A tail. Enzymes modify the two
ends of a eukaryotic pre mRNA molecule. The modified ends may promote the
export of mRNA from the nucleus and they help protect the mRNA from
degradation. When the mRNA reaches the cytoplasm the modified ends in
conjunction with certain cytoplasmic proteins facilitate ribosome attachment.
The 5’ cap and poly A tail are not translated into protein, nor are the regions
called the 5’ untranslated regions (5’ UTR) and 3’ untranslated regions (3’ UTR)
Split Genes and RNA Splicing
• Most eukaryotic genes and their RNA transcripts have long
noncoding stretches of nucleotides that lie between coding
regions
• These noncoding regions are called intervening
sequences, or introns
• The other regions are called exons because they are
eventually expressed, usually translated into amino acid
sequences
• RNA splicing removes introns and joins exons, creating
an mRNA molecule with a continuous coding sequence
5′′ Exon Intron
Exon
Exon
Intron
3′′
Pre-mRNA 5′′ Cap
Poly-A tail
1
30
31
Coding
segment
104
105
Introns cut out and
exons spliced together
mRNA 5′′ Cap
1
5′′ UTR
146
Poly-A tail
146
3′′ UTR
RNA processing: mRNA splicing. The RNA molecule shown here codes for β globin one
of the polypeptides of hemoglobin. The numbers under the RNA refer to the codons. β
globin is 146 amino acids long. The β globin gene and its pre mRNA transcript have
three exons corresponding to sequences that will leave the nucleus as RNA. (The 5’
UTR and 3’ UTR are parts of exons because they are included in the mRNA however
they do not code for protein). During RNA processing the introns are cut out and the
exons are spliced together. In many genes the introns are much larger relative to the
exons than they are in the β globin gene. The mRNA is not drawn to scale.
In some cases, RNA splicing is carried out by
spliceosomes
Spliceosomes consist of a variety of proteins and several
small nuclear ribonucleoproteins (snRNPs) that recognize
the splice sites
5′′
RNA transcript (pre-mRNA)
Exon 1
Intron
Exon 2
The roles of snRNPS and
spliceosomes in pre mRNA
Protein
splicing. The diagram shows only
Other
snRNA
proteins
a portion of the pre mRNA
transcript,
additional introns and
snRNPs
exons lie downstream from the
pictured ones here. 1) Small
Spliceosome
nuclear ribonucleoprotein
(snRNPs) and other proteins form
a molecular complex called a
5′′
spliceosome on a pre mRNA
molecules containing exons and
introns. 2) Within the spliceosome
snRNA base pairs with nucleotides
at specific sites along the intron. 3)
The spliceosome cuts the pre
Spliceosome
mRNA releasing the intron and at
components
the same time splices the exons
Cut-out
together. The spliceosome then
intron
mRNA
comes apart releasing mRNA
5′′
which now contains only exons.
Exon 2
Exon 1
Ribozymes
Ribozymes are catalytic RNA molecules that function as
enzymes and can splice RNA
The discovery of ribozymes rendered obsolete the belief
that all biological catalysts were proteins
Three properties of RNA enable it to function as an
enzyme
It can form a three-dimensional structure because of its ability to
base pair with itself
Some bases in RNA contain functional groups
RNA may hydrogen-bond with other nucleic acid molecules
The Functional and Evolutionary Importance
of Introns
Some genes can encode more than one kind of
polypeptide, depending on which segments are treated as
exons during RNA splicing
Such variations are called alternative RNA splicing
Because of alternative splicing, the number of different
proteins an organism can produce is much greater than its
number of genes
Proteins often have a modular architecture consisting of
discrete regions called domains
In many cases, different exons code for the different
domains in a protein
Exon shuffling may result in the evolution of new proteins
Gene
DNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Correspondence
between exons
and protein
domains.
Translation
Domain 3
Domain 2
Domain 1
Polypeptide
Translation is the RNA-directed synthesis of a
polypeptide: a closer look
A cell translates an mRNA message into protein with the
help of transfer RNA (tRNA)
Molecules of tRNA are not identical:
Each carries a specific amino acid on one end
Each has an anticodon on the other end; the anticodon
base-pairs with a complementary codon on mRNA
Amino
acids
Polypeptide
Tr
p
Ribosome
tRNA with
amino acid
attached
P he
Gly
tRNA
Translation: the basic concept. As
a molecule of mRNA is moved
through a ribosome codons are
translated into amino acids one by
one. The interpreters are tRNA
molecules, each type with a
specific anticodon at one end and
a corresponding amino acid at the
other end. A tRNA adds its amino
acid cargo to a growing
polypeptide chain when the
anticodon hydrogen bonds to a
complementary codon on the
mRNA.
Anticodon
Codons
5′′
3′′
mRNA
The Structure and Function of Transfer RNA
A
A tRNA molecule consists of a single RNA CCstrand that is
only about 80 nucleotides long
Flattened into one plane to reveal its base pairing, a tRNA
molecule looks like a cloverleaf
Because of hydrogen bonds, tRNA actually twists and
folds into a three-dimensional molecule
tRNA is roughly L-shaped
3′′
Amino acid
attachment site
5′′
Hydrogen
bonds
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 anticodon triplet is
unique to each tRNA type as are some
sequences in the other two loops. (the
asterisks mark bases that have been
chemically modified a characteristic of
tRNA)
Anticodon
(a) Two-dimensional structure
5′′
3′′
Amino acid
attachment site
Hydrogen
bonds
3′′
Anticodon
(b) Three-dimensional structure
5′′
Anticodon
The structure of transfer RNA (tRNA).
Anticodons are conventionally written 3’∏
5’ to align properly with codons written 5’
∏3’. For base pairing RNA strands must
be antiparallel like DNA. For example
anticodon 3’ AAG 5’ pairs with mRNA
codon 5’ UUC 3’
(c) Symbol used
in this book
Accurate translation requires two steps:
First: a correct match between a tRNA and an amino
acid, done by the enzyme aminoacyl-tRNA synthetase
Second: 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
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P
1) Active site binds to the amino acid
and ATP
Adenosine
ATP
2) ATP loses two P groups and joins
amino acids as AMP
P
P Pi
Pi
Adenosine
tRNA
Aminoacyl-tRNA
synthetase
Pi
tRNA
P
3) Appropriate tRNA covalently bonds to
amino acid displacing AMP
4) The tRNA charged with amino acid is
released by the enzyme
Adenosine
AMP
Computer model
Aminoacyl-tRNA
(“charged tRNA”)
An aminoacyl tRNA synthethase joining
a specific amino acid to a tRNA.
Linkage of the tRNA and amino acid
is an endergonic process that occurs
at the expense of ATP. The ATP
loses two phosphate groups
becoming AMP (adenosine
monophosphate)
Ribosomes
Ribosomes facilitate specific coupling of tRNA anticodons
with mRNA codons in protein synthesis
The two ribosomal subunits (large and small) are made of
proteins and ribosomal RNA (rRNA)
Growing
polypeptide
Exit tunnel
tRNA
molecules
EP
Large
subunit
A
Small
subunit
5′′
mRNA 3′′
a) Computer model of functioning ribosome. This
is a model of a bacterial ribosome showing its
overall shape. The eukaryotic ribosome is roughly
similar. A ribosomal subunit is an aggregate of
ribosomal RNA molecules and proteins
(a) Computer model of functioning ribosome
P site (Peptidyl-tRNA
binding site)
E site
(Exit site)
A site (AminoacyltRNA binding site)
E P A
mRNA
binding site
Large
subunit
Small
subunit
(b) Schematic model showing binding sites
Growing polypeptide
Amino end
Next amino acid
to be added to
polypeptide chain
mRNA
5′′
E
b) Schematic model showing binding sites. A
ribosome has an mRNA binding site and three
tRNA binding sites known as the A, P and E sites.
tRNA
3′′
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 polypetide. The
A site holds the tRNA carrying the next amino acid
to be added to the polypeptide chain. Discharged
tRNAs leaves from the E site.
Codons
(c) Schematic model with mRNA and tRNA
The anatomy of a functioning ribosome.
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
Building a Polypeptide
The three stages of translation:
Initiation
Elongation
Termination
All three stages require protein “factors” that aid in the
translation process
Ribosome Association and Initiation of
Translation
The initiation stage of translation brings together mRNA, a
tRNA with the first amino acid, and the two ribosomal
subunits
First, a small ribosomal subunit binds with mRNA and a
special initiator tRNA
Then the small subunit moves along the mRNA until it
reaches the start codon (AUG)
Proteins called initiation factors bring in the large subunit
that completes the translation initiation complex
3′′ U A C 5′′
Met 5′′ A U G 3′′
Initiator
tRNA
P site
Met
Large
ribosomal
subunit
GTP GDP
E
mRNA
5′′
Start codon
mRNA binding site
3′′
Small
ribosomal
subunit
1) A small ribosomal subunit binds to a
molecule of mRNA. In a bacterial 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)
A
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.
The initiation of translation
Elongation of the Polypeptide Chain
During the elongation stage, amino acids are added one by
one to the preceding amino acid
Each addition involves proteins called elongation factors
and occurs in three steps: codon recognition, peptide bond
formation, and translocation
Amino end
of polypeptide
The elongation cycle of translation.
The hydrolysis of GTP plays an
important role in the elongation
process
Ribosome ready for
next aminoacyl tRNA
E
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
3′′
mRNA
P A
site site
5′′
GTP
GDP
E
E
P A
3) Translocation The ribosome
translocates the tRNA in the A 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
P A
GDP
GTP
E
P A
2) Peptide bond formation. An
rRNA molecule of the large
ribosomal subunit catalyses
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 removes the polypeptide
from the tRNA in the P site
and attaches it to the amino
acid on the tRNA in the A site
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
The release factor causes the addition of a water molecule
instead of an amino acid
This reaction releases the polypeptide, and the translation
assembly then comes apart
Release
factor
Free
polypeptide
5′′
3′′
5′′
3′′
3′′
5′′
Stop codon
(UAG, UAA, or UGA)
When a ribosome reaches a stop
codon on mRNA, the A site of the
ribosome accepts a release factor
a protein shaped like a tRNA instead
of an aminoacyl tRNA,
The release factor hydrolyzes the
bond between the tRNA in the
P site and the last amino acid of the
polypeptide chain. The polypeptide
is thus freed from the ribosome.
The two ribosomal subunits
and the other components
of the assembly dissociate.
The termination of translation. Like elongation, termination requires GTP
hydrolysis as well as additional factors which are not shown here
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
Completed
polypeptides
Growing
polypeptides
Incoming
ribosomal
subunits
Polyriboso
me
Start of
mRNA
(5′′ end)
End of
mRNA
(3′′ end)
An mRNA molecule is generally translated simultaneously
by several ribosomes in clusters called polyribosomes.
Ribosomes
mRNA
0.1 µm
This micrograph shows a large polyribosome in a prokaryotic cell (TEM).
Completing and Targeting the Functional
Protein
Often translation is not sufficient to make a functional
protein
Polypeptide chains are modified after translation
Completed proteins are targeted to specific sites in the cell
Protein Folding and Post-Translational
Modifications
During and after synthesis, a polypeptide chain
spontaneously coils and folds into its three-dimensional
shape
Proteins may also require post-translational modifications
before doing their job
Some polypeptides are activated by enzymes that cleave
them
Other polypeptides come together to form the subunits of a
protein
Targeting Polypeptides to Specific Locations
Two populations of ribosomes are evident in cells: free
ribsomes (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 and proteins that are secreted from the cell
Ribosomes are identical and can switch from free to bound
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
1)
Polypetide
synthesis
begins on a
free
ribosomo in
the cytosol.
2) An SRP binds
to the signal
peptide.
halting
synthesis
momentarily
3) The SRP binds to a receptor
protein in the ER
membrane. This receptor
is part of a protein
complex (a translocation
complex) that has a
membrane pore and a
signal cleaving enzyme
4) The SRP leaves and
5) The signal
polypetides synthesis
cleaving
resumes with
enzyme
simultaneous
cuts off the
translocation across the
signal
peptide
membrane (The signal
peptide stays attached to
the translocation
complex)
6) The rest of
the completed
polypeptide
leaves the
ribosome and
folds into its
final
conformation
Ribosome
mRNA
Signal
peptide
Signal
peptide
removed
Signalrecognition
particle (SRP)
CYTOSOL
ER LUMEN
SRP
receptor
protein
ER
membrane
Protein
Translocation
complex
The signal mechanism for targeting proteins to the ER. A polypeptide destined for the endomembrane
system or for secretion from the cell begins with a signal peptide a series of amino acids that targets it
for the ER. This figure shows the synthesis of a secretory protein and its simultaneous import into the
ER. In the ER and then in the Golgi, the protein will be processed further. Finally a transport vesicle will
convey it to the plasma membrane for release from the cell
RNA plays multiple roles in the cell: a review
Type of RNA
Functions
Messenger RNA Carries information specifying amino
(mRNA)
acid sequences of proteins from DNA
to ribosomes
Transfer RNA
(tRNA)
Serves as adapter molecule in protein
synthesis; translates mRNA codons
into amino acids
Ribosomal RNA
(rRNA)
Plays catalytic (ribozyme) roles and
structural roles in ribosomes
Type of RNA
Primary
transcript
Functions
Serves as a precursor to mRNA,
rRNA, or tRNA, before being
processed by splicing or
cleavage
Small nuclear Plays structural and catalytic
RNA (snRNA) roles in spliceosomes
SRP RNA
Is a component of the the signalrecognition particle (SRP)
Type of RNA
Small
nucleolar RNA
(snoRNA)
Small
interfering
RNA (siRNA)
and microRNA
(miRNA)
Functions
Aids in processing pre-rRNA
transcripts for ribosome subunit
formation in the nucleolus
Are involved in regulation of
gene expression
• RNA’s diverse functions range from structural to
informational to catalytic
Properties that enable RNA to perform many
different functions:
Can hydrogen-bond to other nucleic acids
Can assume a three-dimensional shape
Has functional groups that allow it to act as a catalyst
(ribozyme)
While gene expression differs among the
domains of life, the concept of a gene is
universal
Archaea are prokaryotes, but share many features of gene
expression with eukaryotes
Comparing Gene Expression in Bacteria,
Archaea, and Eukarya
Bacteria and eukarya differ in their RNA polymerases,
termination of transcription and ribosomes; archaea tend to
resemble eukarya in these respects
Bacteria can simultaneously transcribe and translate the same
gene
In eukarya, transcription and translation are separated by the
nuclear envelope. In addition extensive RNA processing
occurs in the nucleus
In archaea, transcription and translation are likely coupled
RNA polymerase
DNA
mRNA
Polyribosome
RNA
polymerase
Direction of
transcription
0.25 µm
DNA
Polyribosome
Polypeptide
(amino end)
Ribosome
mRNA (5′′ end)
Coupled transcription
and translation in
bacteria. In bacterial
cells, the translation
of mRNA can begin
as soon as the
leading (5’) end of the
mRNA molecule
peels away from the
DNA template. The
micrograph (TEM)
shows a strand of E
coli DNA being
transcribed by RNA
polymerase
molecules. Attached
to each RNA
polymerase molecule
is a growing strand of
mRNA which is
already being
translated by
ribosomes. The
newly synthesized
polypeptides are not
visible in the
micrograph but are
shown in the
diagram.
What Is a Gene? Revisiting the Question
The idea of the gene itself is a unifying concept of life
We have considered a gene as:
A discrete unit of inheritance
A region of specific nucleotide sequence in a
chromosome
A DNA sequence that codes for a specific polypeptide
chain
In summary, a gene can be defined as a region of DNA
that can be expressed to produce a final functional product,
either a polypeptide or an RNA molecule
A summary of transcription and
translation in a eukaryotic cell
1) Transcription- RNA is transcribed from a DNA template.
2) RNA processing- In eukaryotes the RNA transcript (pre mRNA) is
spliced and modified to produce mRNA, which moves from the
nucleus to the cytoplasm
3) The mRNA leaves the nucleus and attaches to a ribosome
4) Amino acid activation- Each amino acid attaches to its proper
tRNA with the help of a specific enzyme and ATP.
5) Translation- A succession of tRNAs add their amino acids to the
polypeptide chain as the mRNA is moved through the ribosome
one codon at a time (When completed the polypeptide is released
from the ribosome
Conducting the Genetic Orchestra
Prokaryotes and eukaryotes alter gene expression in
response to their changing environment
In multicellular eukaryotes, gene expression regulates
development and is responsible for differences in cell types
RNA molecules play many roles in regulating gene
expression in eukaryotes
Individual bacteria respond to environmental
change by regulating their gene expression
A bacterium can tune its metabolism to the changing
environment and food sources
This metabolic control occurs on two levels:
Adjusting activity of metabolic enzymes
Regulating genes that encode metabolic enzymes
Bacteria often respond to environmental
change by regulating transcription
Natural selection has favored bacteria that produce only
the products needed by that cell
A cell can regulate the production of enzymes by feedback
inhibition or by gene regulation
Gene expression in bacteria is controlled by the operon
model
Operons: The Basic Concept
An operon is the entire stretch of DNA that includes the
operator, the promoter, and the genes that they control
In bacteria, genes are often clustered into operons,
composed of
An operator, an “on-off” switch
A promoter
Genes for metabolic enzymes
A cluster of functionally related genes can be under
coordinated control by a single on-off “switch”
The regulatory “switch” is a segment of DNA called an
operator usually positioned within the promoter
The operon can be switched off by a protein repressor
The repressor prevents gene transcription by binding to
the operator and blocking RNA polymerase
The repressor is the product of a separate regulatory
gene
The repressor can be in an active or inactive form,
depending on the presence of other molecules
A corepressor is a molecule that cooperates with a
repressor protein to switch an operon off
Eukaryotic gene expression can be regulated
at any stage
All organisms must regulate which genes are expressed at
any given time
In multicellular organisms gene expression is essential for
cell specialization
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
In each type of differentiated cell, a unique subset of genes is
expressed
Many key stages of gene expression can be regulated in eukaryotic
cells
Errors in gene expression can lead to diseases including cancer
Gene expression is regulated at many stages
All our cells start off with the same set of genes
A small percentage of these genes are expressed in all our
cells- housekeeping genes like for example for glycolysis
Through development and our lives different cells selectively
express different genes
For example RBCs transcribe hemoglobin genes whereas the
eye does not transcribe this gene and instead it expresses
crystalline
Signal
NUCLEUS
Chromatin
Chromatin modification
DNA unpacking involving
histone acetylation and
DNA demethylation
DNA
Gene available
for transcription
Gene
Transcription
RNA
Exon
Primary transcript
Intron
RNA processing
Tail
Cap
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
Stages in gene expression that
can be regulated in eukaryotic
cells. In this diagram the colored
boxes indicate the processes
most often regulated; each color
indicates the type pf molecule
that is affected (blue=DNA,
orange= RNA, purple= protein).
The nuclear envelope
separating transcription from
translation in eukaryotic cells
offers an opportunity for post
transcriptional control in the
form of RNA processing that is
absent in prokaryotes. In
addition eukaryotes have a
greater variety of control
mechanisms operating before
transcription and after
translation. The expression of
any given gene, however does
not necessarily involve every
stage shown; for example not
every polypeptide is cleaved.
CYTOPLASM
mRNA in cytoplasm
Degradation
of mRNA
Translation
Polypeptide
Protein processing such
as cleavage and chemical
modification
Active protein
Degradation
of protein
Transport to cellular
destination
Cellular function (such as
enzymatic activity,
structural support etc.)
Controls before transcription
Promoters
Enhancers
Methylation and acetylation
Rearrangement- multiplication- for example polytene
chromosomes contain several copies of genes allowing
more RNA and subsequently more proteins to get
produced
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
Histone Modifications
In histone acetylation, acetyl groups are attached to positively
charged lysines in histone tails
This process 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
The histone code hypothesis proposes that specific
combinations of modifications help determine chromatin
configuration and influence transcription
Histone
tails
Amino
acids
available
for chemical
modification
DNA
double helix
(a) Histone tails protrude outward from a nucleosome.
This is an end view of a nucleosome. The amino acids
in the N termincal tails are accessible for chemical
modification
Unacetylated histones
Acetylated histones
A simple model of histone
tails and the effect of
histone acetylation. In
addition to acetylation
histones can undergo
several other types of
modifications that also
help determine the
chromatin configuration in
a region.
(b) Acetylation of histone tails promotes loose chromatin
structure that permits transcription. A region of
chromatin in which nucleosomes are unacetylated forms
a compact structure (left) in which the DNA is not
transcribed. When nucleosomes are highly acetylated
(right) the chromatin becomes less compact, and the
DNA is accessible for transcription
DNA Methylation
DNA methylation, the addition of methyl groups to certain
bases in DNA, is associated with reduced transcription in
some species
DNA methylation can cause long-term inactivation of genes
in cellular differentiation
In genomic imprinting, methylation regulates expression
of either the maternal or paternal alleles of certain genes at
the start of development
Chemical Modifications
Methylation of DNA can
inactivate genes
Acetylation of histones
allows DNA unpacking
and transcription
Methylation of histone or of DNA usually turns a gene off.
Acetylation of histone usually turns a gene on.
Epigenetic Inheritance
Although the chromatin modifications just discussed do not
alter DNA sequence, they may be passed to future
generations of cells
The inheritance of traits transmitted by mechanisms not
directly involving the nucleotide sequence is called
epigenetic inheritance
Regulation of Transcription Initiation
Chromatin-modifying enzymes provide initial control of
gene expression by making a region of DNA either
more or less able to bind the transcription machinery
Organization of a Typical Eukaryotic Gene
Associated with most eukaryotic genes are control
elements, segments of noncoding DNA that help regulate
transcription by binding certain proteins
Control elements and the proteins they bind are critical to
the precise regulation of gene expression in different cell
types
Enhancer
(distal control elements)
Poly-A signal
sequence
Termination
region
Proximal
control elements
Exon
Intron
Exon
Intron Exon
DNA
Upstream
Downstream
Promoter
Transcription
Exon
Primary RNA
5′′
Transcript
(pre-mRNA)
Intron
Exon
Intron Exon
Cleaved 3′′ end
of primary
transcript
RNA processing
Cap and tail added
introns excised Poly-A
and exons spliced signal
together
Intron RNA
Coding segment
mRNA
3′′
5′′ Cap
Start
5’ UTR
codon
untranslated
region
Stop
codon
3′′ UTR Poly-A
tail
A eukaryotic gene and its transcript. Each eukaryotic gene has a promoter a DNA sequence where
RNA polymerase binds and starts transcription proceeding downstream. A number of control
elements (gold) are involved in regulating the initiation of transcription; these are DNA sequences
located near (proximal to) or far from (distal to) the promoter. Distal control elements can be
grouped together as enhancers one of which is shown for this gene. A polyadenylation (poly-A)
signal sequence in the last exon of the gene is transcribed into an RNA sequence that signals
where the transcript is cleaved and the poly A tail added. Transcription may continue for hundreds
of nucleotides beyond the poly A signal before terminating. RNA processing of the primary
transcript into a functional mRNA involves three steps: addition of the 5’ cap addition of the poly A
tail and splicing. In the cell the 5’ cap is added soon after transcription is initiated splicing and poly
A tail addition may also occur while transcription is till under way.
The Roles of Transcription Factors
To initiate transcription, eukaryotic RNA polymerase
requires the assistance of proteins called transcription
factors
General transcription factors are essential for the
transcription of all protein-coding genes
In eukaryotes, high levels of transcription of particular
genes depend on control elements interacting with specific
transcription factors
Enhancers and Specific Transcription Factors
Proximal control elements are located close to the
promoter
Distal control elements, groups of which are called
enhancers, may be far away from a gene or even located
in an intron
An activator is a protein that binds to an enhancer and
stimulates transcription of a gene
Bound activators cause mediator proteins to interact with
proteins at the promoter
Promoter
Activators
DNA
Enhancer
1)
2)
3)
Distal control
element
Activator proteins bind to distal control elements
grouped as an enhancer in the DNA. This
enhancer has three binding sites.
A DNA bending protein brings the bound
activators closer to the promoter. General
transcription factors mediator proteins and RNA
polymerase are nearby.
TATA
box
General
transcription
factors
DNA-bending
protein
The activators bind to certain mediator proteins
and general transcription factors, helping them
form an active transcription initiation complex on
the promoter.
A model for the action of enhancers and
transcription activators: Bending of the DNA by a
proteins enables enhancers the influence a
promoter hundreds or even thousands of
nucleotides away. Specific transcription factors
called activators bind to the enhancer DNA
sequences and then to a group of mediator
proteins, which in turn bind to general
transcription factors assembling the transcription
initiation complex. These protein protein
interactions facilitate the correct positioning of
the complex on the promoter and the initiation of
RNA synthesis. Only one enhancer (with three
orange control elements) is shown here, but a
gene may have several enhancers that act at
different times or on different cell types
Gene
Group of
mediator proteins
RNA
polymerase II
RNA
polymerase II
Transcription
initiation complex
RNA synthesis
Coordinately Controlled Genes in Eukaryotes
Unlike the genes of a prokaryotic operon, each of the
coordinately controlled eukaryotic genes has a promoter
and control elements
These genes can be scattered over different
chromosomes, but each has the same combination of
control elements
Copies of the activators recognize specific control
elements and promote simultaneous transcription of the
genes
Mechanisms of Post-Transcriptional
Regulation
Transcription alone does not account for gene expression
Regulatory mechanisms can operate at various stages
after transcription
Such mechanisms allow a cell to fine-tune gene expression
rapidly in response to environmental changes
Control of RNA processing
In alternative RNA splicing, different mRNA molecules
are produced from the same primary transcript,
depending on which RNA segments are treated as
exons and which as introns
Alternative splicing- For example different muscle cells
express slightly different forms of the troponin gene by
utilizing alternative splicing. This generates proteins with
somewhat unique functions
Nuclear envelope- UTRs contain zipcodes which allow
the RNA to exit the nucleus with the aid of proteins which
recognize it and selectively bind to it. In addition these
unique zipcodes specify to which cytoplasmic location
these RNA must move. This is crucial during embryonic
development.
Exons
DNA
Troponin T gene
Primary
RNA
transcript
RNA splicing
mRNA
or
Alternative RNA splicing of the troponin T gene. The primary transcript of this gene can be
spliced in more than one way, generating different mRNA molecules. Notice that one mRNA
molecule has ended up with exon 3 (green) and the other with exon 4 (purple). These two
mRNAs are translated into different but related muscle proteins.
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
The mRNA life span is determined in part by sequences in
the leader and trailer regions
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
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
1) Multiple ubiquitin
molecules are attached to a
protein by enzymes in the
cytosol
2) The ubiquitin tagged protein is
recognized by a proteasome which
unfolds the protein and sequesters
it within a central cavity
Ubiquitin
Proteasome
Protein to
be degraded
Ubiquitinated
protein
3) Enzymatic components
of the proteasome cut the
protein into small peptides
which can be further
degraded by other
enzymes in the cytosol.
Proteasome
and ubiquitin
to be recycled
Protein entering a
proteasome
Protein
fragments
(peptides)
Degradation of a protein by a proteasome. A proteasome, an enormous protein
complex shaped like a trash can, chops up unneeded proteins in the cell. In
most cases the proteins attacked by a proteasome have been tagged with short
chains of ubquitin a small protein. Steps 1 and 3 require ATP. Eukaryotic
proteasomes are as massive as ribosomal subunits and are distributed
throughout the cell. Their shape somewhat resembles that of chaperone
proteins, which protect protein structure rather than destroy it
Control after translation (post-translational)
Example: phosphorylation and other protein
modifications which occur after the protein has been
synthesized can change their activity
Noncoding RNAs play multiple roles in
controlling gene expression
Only a small fraction of DNA codes for proteins, rRNA,
and tRNA
A significant amount of the genome may be transcribed
into noncoding RNAs
Noncoding RNAs regulate gene expression at two points:
mRNA translation and chromatin configuration
Effects on mRNAs by MicroRNAs and Small
Interfering RNAs
MicroRNAs (miRNAs) are small single-stranded RNA
molecules that can bind to mRNA
These can degrade mRNA or block its translation
1)
Hairpin
miRNA
Hydrogen
bond
2)
Dicer
5′′ 3′′
One strand of the
double stranded
RNA is degraded;
the other strand
(miRNA) then forms
a complex with one
or more proteins
4)
The miRNA in the
complex can bind to
any target mRNA
that contains at
least 6 bases of
complementary
sequence.
(b)
5)
If miRNA and
mRNA are
complementary all
along their length,
the mRNA is
degraded (left); if
the match is less
complete translation
is blocked (right)
miRNAprotein
complex
Primary miRNA transcript.
This RNA molecule is
transcribed from a gene in a
nematode worm. Each
double stranded region that
ends in a loop is called a
hairpin and generates one
miRNA (shown in orange)
Regulation of gene
expression by miRNAs. RNA
transcripts are processed
into miRNAs which prevent
expression of mRNAs
containing complementary
sequences.
mRNA degraded
Translation blocked
(b) Generation and function of miRNAs
A second enzyme
called Dicer, trims
the loop and the
single stranded
ends from the
hairpin, cutting at
the arrows.
3)
miRNA
(a)
An enzyme cuts
each hairpin from
the primary miRNA
transcript
The phenomenon of inhibition of gene expression by RNA
molecules is called RNA interference (RNAi).
This is caused by single-stranded small interfering RNAs
(siRNAs) and can lead to degradation of an mRNA or block
its translation
siRNAs and miRNAs are similar but form from different
RNA precursors
Chromatin Remodeling and Silencing of
Transcription by Small RNAs
siRNAs play a role in heterochromatin formation and can
block large regions of the chromosome
Small RNAs may also block transcription of specific
genes