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