Chapter 17

From Gene to

Protein

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

Central Dogma of Biology

RNA

DNA

Protein

Transcription

Translation

Replication

Concept 17.1Genes specify proteins via

transcription and translation

• The process of producing an mRNA transcript from a DNA template is known as transcription• Facilitated by RNA polymerase

• The process of producing polypeptides from mRNA is known as translation• Facilitated by ribosomes

• To determine the sequence of amino acids based on the DNA sequence, the genetic code can be used to interpret the DNA sequence

Archibald Garrod

Studied inherited metabolic conditions.

First to suggest that genes dictate phenotypes

through enzymes that catalyze specific chemical

reactions in the cell.

E.g. alkaptonuria

Symptom of black urine caused by alkapton

Garrod suggested that lack of the enzyme which

metabolizes alkapton is the inherited trait

Links gene to protein to phenotype

George Beadle and Edward Tatum

Worked with the bread mold Neurospora and

created mutants with x-rays

Mutants could not survive on the minimal medium

that was sufficient to support growth of the wild-

type Neurospora

Beadle and Tatum then hypothesized that the

mutants lacked the ability to synthesize certain

necessary molecules

George Beadle and Edward Tatum

To determine the individual specific defects for

each mutant, they selected for mutants by

including one nutrient at a time

This allowed them to identify mutants based on the

nutrient it could not synthesize

They hypothesized that these mutants lacked a

necessary enzyme for the synthesis of that nutrient

One gene – one enzyme hypothesis

The function of a gene is to dictate the production of

a specific enzyme

RESULTS

EXPERIMENT

CONCLUSION

Growth:Wild-typecells growing

and dividing

No growth:

Mutant cellscannot grow and divide

Minimal medium

Classes of Neurospora crassa

Wild type Class I mutants Class II mutants Class III mutants

Minimal

medium

(MM)(control)

MM +

ornithine

MM +

citrulline

Co

nd

itio

n

MM +

arginine

(control)

Class I mutants

(mutation in

gene A)Wild type

Class II mutants

(mutation in

gene B)

Class III mutants

(mutation in

gene C)

Gene A

Gene B

Gene C

Precursor Precursor Precursor PrecursorEnzyme A Enzyme AEnzyme A Enzyme A

Enzyme B

Ornithine Ornithine Ornithine Ornithine

Enzyme B Enzyme B Enzyme B

Citrulline Citrulline Citrulline Citrulline

Enzyme C Enzyme C Enzyme C Enzyme C

Arginine Arginine Arginine Arginine

Products of Gene Expression

One gene – one enzyme was a valid hypothesis at

the time.

But, not all proteins are enzymes. The phrase was

then modified to one gene – one protein.

However, proteins can be composed of multiple

polypeptides. The phrase was then again restated

as one gene – one polypeptide.

The processes involved with going from gene to

protein are known as transcription and translation

Transcription

Genes provide the instructions for protein synthesis

in the form of a nucleotide sequence.

This sequence is used to generate a single stranded

RNA molecule through the process of transcription

RNA differs from DNA in the following ways:

Single-stranded instead of double-stranded

Ribose instead of deoxyribose

Uracil (U) instead of thymine (T)

Transcription

This RNA is known as messenger RNA (mRNA).

In eukaryotes, the mRNA that is produced contains

some information that is not included in the final

polypeptide.

This initial mRNA known as pre-mRNA must undergo

RNA processing to form a mature mRNA that

contains only regions that code for a polypeptide

sequence.

This pre-mRNA is also known as a primary transcript

Translation

After the production of the mRNA from the gene

through transcription, a polypetide can be

synthesized from the mRNA through the process of

translation.

The instructions held in the sequence of the mRNA

direct the formation of a polypeptide by ribosomes,

which work to link amino acids together in the

correct order to form a polypeptide chain.

These instructions are given in the sequence of the

nucleotide bases which can be deciphered using

the genetic code.

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide

(a) Bacterial cell

Nuclearenvelope

TRANSCRIPTION

RNA PROCESSINGPre-mRNA

DNA

mRNA

TRANSLATION Ribosome

Polypeptide

(b) Eukaryotic cell

The Genetic Code

There are 4 nucleotide bases that make up DNA

sequences.

However, there are 20 amino acids for which they

provide the code.

This is possible through a triplet code. Each possible

combination of three nucleotide bases codes for an

amino acid.

The instructions for the sequence of amino acids in

a polypeptide are written in non-overlapping three-

nucleotide words.

Codons

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

The template strand is read in the 3’ to 5’ direction

During translation, the mRNA base triplets, called codons, are read by translation machinery in the 5to 3 direction

Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide

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

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

Evolution of the Genetic Code

The genetic code is highly conserved. Most

organisms from the simplest to the most complex

use the same code

Gene sequences from one species can be

transcribed and translated in another species when

transferred.

Examples:

tobacco plant expressing a firefly gene

Insulin production by yeast or E. coli

Concept 17.2Transcription is the DNA-directed

synthesis of RNA: a closer look

• Transcription involves three phases: initiation,

elongation, and termination.

• The initial transcript that is produced is the pre-

mRNA which needs RNA processing to produce

the final mature mRNA which will be used for

translation

Synthesis of an RNA Transcript

As with DNA replication, there are many enzymes involved with the synthesis of mRNA during transcription.

RNA polymerase produces the new mRNA strand from the template strand of the DNA

However, since RNA is being formed, uracil is used instead of thymine

There are three stages of transcription:

Initiation

Elongation

Termination

Initiation of Transcription

The stretch of DNA that is transcribed is called a transcription unit

The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator

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

Promoter Transcription unit

Start pointDNA

RNA polymerase

5533

Initiation1

2

3

5533

Unwound

DNA

RNAtranscript

Template strand

of DNA

Elongation

Rewound

DNA

5

55

5

5

33

3

3

RNA

transcriptTermination

5533

35Completed RNA transcript

Newly made

RNA

Template

strand of DNA

Direction oftranscription(“downstream”)

3 end

RNA

polymerase

RNA nucleotides

Nontemplate

strand of DNAElongation

Elongation of the RNA transcript

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

Concept 17.3Eukaryotic cells modify RNA after

transcription

• RNA processing of pre-RNA involves RNA splicing

which is the removal of introns and joining of exons of the primary transcript by spliceosomes

• The ends of the pre-RNA are also modified with a

5’ cap and a 3’ poly-A tail

Ends of the mRNA strand

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 segmentPolyadenylation signal3

3 UTR5 UTR

5

5 Cap Start codon Stop codon Poly-A tail

G P PP AAUAAA AAA AAA…

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

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 splicing

RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

Protein

snRNA

snRNPs

Otherproteins

5

5

Spliceosome

Spliceosomecomponents

Cut-outintron

mRNA

Exon 1 Exon 25

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

Alternative RNA Splicing

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

Concept 17.4Translation is the RNA-directed

synthesis of a polypeptide: a closer

look

• Translation is facilitated by ribosomes and tRNAs

• tRNAs have an anticodon that recognizes the complementary codon of the mRNA sequence

and directs the addition of amino acids

Molecular Components of

Translation

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

Codons 35

mRNA

The Structure and Function of

Transfer RNA (tRNA)

A tRNA molecule consists of a single RNA strand that

is only about 80 nucleotides long

When flattened into one plane, a tRNA molecule

looks like a cloverleaf due to base pairing in regions

of the molecule

However, due to hydrogen bonds, tRNA actually

twists and folds into a three-dimensional molecule

that forms a shape that is more similar to an capital

letter L

The bottom loop is where the anticodon is located

Amino acidattachment site

(a) Two-dimensional structure

Hydrogenbonds

Anticodon

3

5

Amino acidattachment site

3

3

5

5

Hydrogenbonds

Anticodon Anticodon

(b) Three-dimensional structure(c) Symbol used

in this book

Translation

Accurate translation requires two steps:

1. a correct match between a tRNA and an amino

acid, done by the enzyme aminoacyl-tRNA

synthetase

2. 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”)

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)

Binding Sites

A ribosome has three binding sites for

tRNA: The E site is the exit site, where discharged tRNAs leave

the ribosome

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

Growingpolypeptide Exit tunnel

Largesubunit

Smallsubunit

tRNAmolecules

E P A

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

Building a Polypeptide

The three stages of translation:

Initiation

Elongation

Termination

All three stages require protein “factors” that aid in

the translation process

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

35

5U

U

AA

C

G

GTP GDPInitiator

tRNA

mRNA

53

Start codon

mRNA binding site

Smallribosomalsubunit

5

P site

Translation initiation complex

3

E A

Largeribosomalsubunit

Elongation of a 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

3E

Psite

Asite

GTP

GDP

E

P A

E

P A

GDP

GTP

Ribosome ready fornext aminoacyl tRNA

E

P A

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

Releasefactor

3

5

Stop codon(UAG, UAA, or UGA)

5

3

2

Freepolypeptide

2 GDP

GTP

5

3

Ribosomes

mRNA

(b) 0.1 µm

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

Growingpolypeptides

Completedpolypeptide

Incomingribosomalsubunits

Start ofmRNA(5 end)

End ofmRNA(3 end)(a)

Post-Translational Modifications

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

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

Ribosome Location

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

Targeting Polypeptides

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

Concept 17.5Point mutations can affect protein

structure and function

• Mutations change DNA sequences

• A mutation in the coding region of DNA can change the resulting protein through changes in in amino acids

• Different mutations can have differences in severity in terms of the effect on the resulting protein

Mutagens

Mutations result in changes in the original DNA

sequence

Spontaneous mutations can occur during DNA

replication, recombination, or repair

Mutagens are physical or chemical agents that can

cause mutations

Point mutation – Substitution

A base-pair substitution replaces one nucleotide and its partner with another pair of nucleotides

Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code

Missense mutations still code for an amino acid, but not necessarily the right amino acid

Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

Point Mutation – Insertions and

Deletions

Insertions and deletions are additions or losses of

nucleotide pairs in a gene

These mutations have a disastrous effect on the

resulting protein more often than substitutions do

Insertion or deletion of nucleotides may alter the

reading frame, producing a frameshift mutation

Wild-type

3DNA template strand

5

5

5

3

3

Stop

Carboxyl endAmino end

Protein

mRNA

3

3

3

5

5

5

A instead of G

U instead of C

Silent (no effect on amino acid sequence)

Stop

T instead of C

3

3

3

5

5

5

A instead of G

Stop

Missense

A instead of T

U instead of A

3

3

3

5

5

5

Stop

Nonsense No frameshift, but one amino acid missing (3 base-pair deletion)

Frameshift causing extensive missense (1 base-pair deletion)

Frameshift causing immediate nonsense (1 base-pair insertion)

5

5

53

3

3

Stop

missing

missing

3

3

3

5

5

5

missing

missing

Stop

5

5

53

3

3

Extra U

Extra A

(a) Base-pair substitution (b) Base-pair insertion or deletion

Concept 17.6While gene expression differs

among the domains of life, the

concept of a gene is universal

• Gene expression differs between the different

domains of life but the general concept remains consistent

• DNA carries genetic information in the form of

genes, which can be transcribed and translated

to produce a final product in the form of a

polypeptide or a protein

Gene expression in different

domains

Differences in mechanics of gene expression between bacteria and eukarya: Different RNA polymerases

Different methods for termination of transcription

Different ribosomes

Archaea tend to be more similar to eukarya for these categories

Bacteria can simultaneously transcribe and translate the same gene

In eukarya, transcription and translation are separated by the nuclear envelope

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)

Genes

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

TRANSCRIPTION

RNA PROCESSING

DNA

RNAtranscript

3

5RNApolymerase

RNA transcript(pre-mRNA)

Intron

Exon

NUCLEUS

Aminoacyl-tRNAsynthetase

AMINO ACID ACTIVATION

Aminoacid

tRNACYTOPLASM

Growingpolypeptide

3

Activatedamino acid

mRNA

TRANSLATION

Ribosomalsubunits

5

E

P

A

AAnticodon

Ribosome

Codon

E

Sense and Antisense

3’ TACATCGCCCATAACGAGAAT 5’

5’ 3’

Template Strand

(Antisense)

Coding Strand

(Sense)

mRNA5’ 3’

polypeptide