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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
TRANSCRIPTION
TRANSLATION
DNA
mRNA
Ribosome
Polypeptide
(a) Bacterial cell
Nuclearenvelope
TRANSCRIPTION
RNA PROCESSINGPre-mRNA
DNA
mRNA
TRANSLATION Ribosome
Polypeptide
(b) Eukaryotic cell
a) Bacterial cell. In a bacterial cell which
lacks a nucleus, mRNA produced by
transcription is immediately translated
without additional processing.
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.
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.
TRANSCRIPTIONDNA
mRNA
(a) Bacterial cell
TRANSCRIPTIONDNA
mRNA
TRANSLATIONRibosome
Polypeptide
(b) Eukaryotic cell
TRANSCRIPTION
Nuclearenvelope
DNA
Pre-mRNA
(b) Eukaryotic cell
TRANSCRIPTION
Nuclearenvelope
DNA
Pre-mRNARNA PROCESSING
mRNA
(b) Eukaryotic cell
TRANSCRIPTION
Nuclearenvelope
DNA
Pre-mRNARNA PROCESSING
mRNA
TRANSLATION Ribosome
Polypeptide
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
DNAmolecule
Gene 1
Gene 2
Gene 3
DNAtemplatestrand
TRANSCRIPTION
TRANSLATION
mRNA
Protein
Codon
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
Second mRNA base
Fir
st
mR
NA
ba
se (
5′′ ′′e
nd
of
co
do
n)
Th
ird
mR
NA
ba
se
(3
′′ ′′e
nd
of
co
do
n)
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
Start pointDNA
RNA polymerase
5′′′′5′′′′3′′′′3′′′′
Initiation1
2
3
5′′′′5′′′′3′′′′3′′′′
UnwoundDNA
RNAtranscript
Template strandof DNA
Elongation
RewoundDNA
5′′′′
5′′′′5′′′′
5′′′′
5′′′′
3′′′′3′′′′
3′′′′
3′′′′
RNAtranscript
Termination
5′′′′5′′′′3′′′′3′′′′
3′′′′5′′′′Completed RNA transcript
Newly madeRNA
Templatestrand of DNA
Direction oftranscription(“downstream”)
3′′′′ end
RNApolymerase
RNA nucleotides
Nontemplatestrand of DNA
Elongation
Promoter Transcription unit
DNAStart point
RNA polymerase
5′′′′5′′′′3′′′′
3′′′′
Initiation
3′′′′3′′′′
1
RNAtranscript
5′′′′5′′′′
UnwoundDNA
Template strandof DNA
2 Elongation
RewoundDNA
5′′′′
5′′′′5′′′′3′′′′
3′′′′3′′′′
RNAtranscript
3 Termination
5′′′′
5′′′′
5′′′′3′′′′3′′′′
3′′′′Completed RNA transcript
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.
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.
3) 3) Termination Eventually the RNA transcript is released and the
polymerase detaches from the DNA
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.
Elongation
RNA
polymerase
Nontemplatestrand of DNA
RNA nucleotides
3′′′′ end
Direction oftranscription(“downstream”) Template
strand of DNA
Newly madeRNA
3′′′′
5′′′′
5′′′′
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
3′′′′
1
2
3
Promoter
TATA box Start point
Template
TemplateDNA strand
5′′′′3′′′′5′′′′
Transcriptionfactors
5′′′′5′′′′3′′′′3′′′′
RNA polymerase II
Transcription factors
5′′′′5′′′′ 5′′′′3′′′′
3′′′′
RNA transcript
Transcription initiation complex
1) A eukaryotic promotercommonly 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
2) Several transcription factors one recognizing the TATA box must bind to the DNA before RNA polymerase
II can do so.
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
Protein-coding segment Polyadenylation signal
3′′′′
3′′′′ UTR5′′′′ UTR
5′′′′
5′′′′ Cap Start codon Stop codon Poly-A tail
G P PP AAUAAA AAA AAA…
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
Pre-mRNA
mRNA
Codingsegment
Introns cut out andexons spliced together
5′′′′ Cap
Exon Intron5′′′′
1 30 31 104
Exon Intron
105
Exon
146
3′′′′
Poly-A tail
Poly-A tail5′′′′ Cap
5′′′′ UTR 3′′′′ UTR1 146
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. βglobin 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
RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
Protein
snRNA
snRNPs
Otherproteins
5′′′′
5′′′′
Spliceosome
Spliceosomecomponents
Cut-outintron
mRNA
Exon 1 Exon 25′′′′
The roles of snRNPS and
spliceosomes in pre mRNA
splicing. The diagram shows only
a portion of the pre mRNA
transcript, additional introns and
exons lie downstream from the
pictured ones here. 1) Small
nuclear ribonucleoprotein
(snRNPs) and other proteins form
a molecular complex called a
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
mRNA releasing the intron and at
the same time splices the exons
together. The spliceosome then
comes apart releasing mRNA which now contains only exons.
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 Exon 2 Exon 3Intron Intron
Transcription
RNA processing
Translation
Domain 2
Domain 3
Domain 1
Polypeptide
Correspondence
between exons
and protein domains.
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
Polypeptide
Ribosome
Aminoacids
tRNA withamino acidattached
tRNA
Anticodon
Trp
Phe Gly
Codons 3′′′′5′′′′
mRNA
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.
The Structure and Function of Transfer RNA
ACC
� A tRNA molecule consists of a single RNA strand 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
Amino acidattachment site
3′′′′
5′′′′
Hydrogenbonds
Anticodon
(a) Two-dimensional structure
Amino acidattachment site
5′′′′
3′′′′
Hydrogenbonds
3′′′′ 5′′′′
AnticodonAnticodon
(c) Symbol usedin this book(b) Three-dimensional structure
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)
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’
� 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
Amino acid Aminoacyl-tRNAsynthetase (enzyme)
ATP
AdenosineP P P
AdenosineP
PP i
PPi
i
tRNA
tRNA
Aminoacyl-tRNAsynthetase
Computer model
AMPAdenosineP
Aminoacyl-tRNA(“charged tRNA”)
1) Active site binds to the amino acid and ATP
2) ATP loses two P groups and joins amino acids as AMP
3) Appropriate tRNA covalently bonds to amino acid displacing AMP
4) The tRNA charged with amino acid is released by the enzyme
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)
Growingpolypeptide Exit tunnel
Largesubunit
Smallsubunit
tRNAmolecules
E PA
mRNA5′′′′ 3′′′′
(a) Computer model of functioning ribosome
P site (Peptidyl-tRNAbinding site)
E site(Exit site)
A site (Aminoacyl-tRNA binding site)
E P A Largesubunit
mRNAbinding site
Smallsubunit
(b) Schematic model showing binding sites
Amino end Growing polypeptide
Next amino acidto be added topolypeptide chain
mRNAtRNAE
3′′′′
5′′′′ Codons
(c) Schematic model with mRNA and tRNA
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
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.
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.
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′′′′
3′′′′5′′′′5′′′′U
U
AA
C
GMet
GTP GDPInitiator
tRNA
mRNA
5′′′′ 3′′′′Start codon
mRNA binding site
Smallribosomalsubunit
5′′′′
P site
Translation initiation complex
3′′′′
E A
Met
Largeribosomalsubunit
The initiation of translation
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)
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
� 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 endof polypeptide
mRNA
5′′′′
3′′′′E
Psite
Asite
GTP
GDP
E
P A
E
P A
GDP
GTP
Ribosome ready fornext aminoacyl tRNA
E
P A
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) 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
The elongation cycle of translation.
The hydrolysis of GTP plays an
important role in the elongation process
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
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
3′′′′
The release factor hydrolyzes thebond between the tRNA in theP site and the last amino acid of thepolypeptide chain. The polypeptideis thus freed from the ribosome.
The two ribosomal subunitsand the other componentsof the assembly dissociate.
Releasefactor
Stop codon(UAG, UAA, or UGA)
5′′′′
3′′′′
5′′′′
3′′′′
5′′′′
Freepolypeptide
When a ribosome reaches a stopcodon on mRNA, the A site of the ribosome accepts a release factora protein shaped like a tRNA instead of an aminoacyl tRNA,
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
Ribosomes
mRNA
0.1 µµµµm
This micrograph shows a large polyribosome in a prokaryotic cell (TEM).
An mRNA molecule is generally translated simultaneouslyby several ribosomes in clusters called polyribosomes.
Incomingribosomalsubunits
Growingpolypeptides
End ofmRNA(3′′′′ end)
Start ofmRNA(5′′′′ end)
Polyribosome
Completedpolypeptides
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
Ribosome
mRNA
Signalpeptide
Signal-recognitionparticle (SRP)
CYTOSOLTranslocationcomplex
SRPreceptorprotein
ER LUMEN
Signalpeptideremoved
ERmembrane
Protein
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
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
polypetides synthesis
resumes with
simultaneous
translocation across the membrane (The signal
peptide stays attached to
the translocation complex)
5) The signal
cleaving
enzyme
cuts off the
signal peptide
6) The rest of
the completed
polypeptide
leaves the
ribosome and folds into its
final conformation
RNA plays multiple roles in the cell: a review
Type of RNA Functions
Messenger RNA
(mRNA)
Carries information specifying amino
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 Functions
Primary transcript
Serves as a precursor to mRNA, rRNA, or tRNA, before being
processed by splicing or cleavage
Small nuclear
RNA (snRNA)
Plays structural and catalytic
roles in spliceosomes
SRP RNA Is a component of the the signal-recognition particle (SRP)
Type of RNA Functions
Small nucleolar RNA (snoRNA)
Aids in processing pre-rRNA transcripts for ribosome subunit formation in the nucleolus
Small interfering RNA (siRNA) and microRNA (miRNA)
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
Polyribosome
mRNA
0.25 µmDirection oftranscription
DNA
RNApolymerase
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
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
A summary of transcription and
translation in a eukaryotic cell
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 isexpressed
� 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
DNA
Signal
Gene
NUCLEUS
Chromatin modification DNA unpacking involving histone acetylation and
DNA demethylation
Chromatin
Gene available
for transcription
Exon
Intron
Tail
RNA
Cap
RNA processing
Primary transcript
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
Transcription
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.
mRNA in cytoplasm
Translation
CYTOPLASM
Degradation
of mRNA
Polypeptide
Active protein
Cellular function (such as enzymatic activity,
structural support etc.)
Transport to cellulardestination
Degradation
of protein
Protein processing such as cleavage and chemical
modification
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
Histonetails
DNA
double helix
Aminoacidsavailablefor chemicalmodification
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.
(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
(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
Acetylated histonesUnacetylated histones
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)Proximal
control elements
Poly-A signalsequence
Terminationregion
DownstreamPromoter
UpstreamDNA
ExonExon ExonIntron Intron
Exon Exon ExonIntronIntronCleaved 3′′′′ endof primarytranscript
Primary RNATranscript(pre-mRNA)
Poly-Asignal
Transcription
5′′′′
Intron RNA
Coding segment
mRNA
5′′′′ CapStart
codonStop
codon 3′′′′ UTR Poly-A
tail
3′′′′
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.
RNA processingCap and tail added
introns excised and exons spliced
together
5’ UTR untranslated
region
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
� 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
Enhancers and Specific Transcription Factors
Enhancer TATAbox
PromoterActivators
DNAGene
Distal controlelement
Group ofmediator proteins
DNA-bendingprotein
Generaltranscriptionfactors
RNApolymerase II
RNApolymerase II
Transcriptioninitiation complex RNA synthesis
1) Activator proteins bind to distal control elements
grouped as an enhancer in the DNA. This enhancer has three binding sites.
2) A DNA bending protein brings the bound
activators closer to the promoter. General
transcription factors mediator proteins and RNA polymerase are nearby.
3) 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
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.
or
RNA splicing
mRNA
PrimaryRNAtranscript
Troponin T gene
Exons
DNA
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
Proteasomeand ubiquitinto be recycledProteasome
Proteinfragments(peptides)Protein entering a
proteasome
Ubiquitinatedprotein
Protein tobe degraded
Ubiquitin
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
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
3) Enzymatic components of the proteasome cut the protein into small peptides which can be further degraded by other enzymes in the cytosol.
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
miRNA-proteincomplex
(a) 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)
(b) Regulation of gene expression by miRNAs. RNA transcripts are processed into miRNAs which prevent expression of mRNAs containing complementary sequences.
Translation blocked
Hydrogenbond
(b) Generation and function of miRNAs
Hairpin miRNA
miRNA
Dicer
3′′′′
mRNA degraded
5′′′′
1) An enzyme cuts
each hairpin from
the primary miRNA transcript
2) A second enzyme
called Dicer, trims
the loop and the single stranded
ends from the
hairpin, cutting at the arrows.
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.
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)
� 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
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