Chapter 17

From Gene to Protein

Overview: The Flow of Genetic Information

The information content of DNA Is in the form of specific sequences of

nucleotides along the DNA strandsThe DNA inherited by an organism

Leads to specific traits by dictating the synthesis of proteins

The process by which DNA directs protein synthesis, gene expression Includes two stages, called transcription and

translation

Concept 17.1: Genes specify proteins via transcription and translation

How does a single faulty gene result in the dramatic appearance of an albino deer?

Evidence from the study of metabolic defectsIn 1909, British physician Archibald Garrod

Was the first to suggest that genes dictate phenotypes through enzymes that catalyze specific chemical reactions in the cell

Suggested that symptoms of an inherited disease Reflect a person’s inability to synthesize a

particular enzymeResearch several decades later supported

his hypothesis

Nutritional mutants in Neurospora crassa: Scientific Inquiry

Beadle and Tatum were finally able to establish the link between genes and enzymes

They created mutant bread mold with X-rays Creating mutants that could not survive on

minimal medium (agar, inorganic salts, glucose & vitamin biotin)

But they could survive on complete growth medium (which contained all 20 amino acids)

Fig. 17-2b

RESULTSClasses of Neurospora crassa

Wild type Class I mutants Class II mutants Class III mutants

Minimalmedium(MM)(control)

MM +ornithine

MM +citrulline

MM +arginine(control)

Co

nd

itio

n

Fig. 17-2c

CONCLUSION Class I mutants(mutation in

gene A)

Class II mutants(mutation in

gene B)

Class III mutants(mutation in

gene C)Wild type

Precursor Precursor Precursor PrecursorEnzyme AEnzyme AEnzyme AEnzyme A

Ornithine Ornithine Ornithine OrnithineEnzyme BEnzyme B Enzyme BEnzyme B

Citrulline Citrulline Citrulline CitrullineEnzyme CEnzyme CEnzyme CEnzyme C

Arginine Arginine Arginine Arginine

Gene A

Gene B

Gene C

The Products of Gene Expression: A Developing Story

Beadle and Tatum developed the “one gene–one enzyme hypothesis” Which states that the function of a gene is to

dictate the production of a specific enzyme

But, not all proteins are enzymes like… Keratin, the structural protein of hair Insulin, a hormone (type of protein)

The Products of Gene Expression: A Developing Story

Later research demonstrated that many proteins are composed of several polypeptides each of which has its own gene

As researchers learned more about proteins The made minor revision to the one gene–one enzyme

hypothesis Genes code for polypeptide chains or for RNA

moleculesNote that it is common to refer to gene

products as proteins rather than polypeptides

Basic Principles of Transcription & TranslationTranscription

A DNA strand provides a template for the synthesis of a complementary RNA strand

Produces messenger RNA (mRNA)

Translation Change of language The synthesis of a polypeptide, which occurs

under the direction of mRNA Occurs on ribosomes

Why can’t proteins be translated directly to DNA?

Using an RNA intermediate Provides protection for the DNA Allows more copies of a protein to be made

simultaneously

Cells are governed by a cellular chain of command DNA RNA protein

In prokaryotes

Transcription and translation occur in the same place

And at the same time

Figure 17.3a

Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing.

(a)

TRANSLATION

TRANSCRIPTION DNA

mRNA

Ribosome

Polypeptide

In eukaryotes

RNA transcripts are modified before becoming true mRNA

Figure 17.3b

Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNAtranscript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA.

(b)

TRANSCRIPTION

RNA PROCESSING

TRANSLATION

mRNA

DNA

Pre-mRNA

Polypeptide

Ribosome

Nuclearenvelope

Codons: Triplets of Bases

Genetic information for a polypeptide chain Is encoded as a sequence of nonoverlapping

three-nucleotides words, or codons

During transcription, one DNA strand, the template strand Provides a template for ordering the sequence

of nucleotides in a RNA transcript

During transcription

The codons are read in the 5’ 3’ direction

Figure 17.4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNA strand(template)

TRANSCRIPTION

mRNA

Protein

TRANSLATION

Amino acid

A C C A A A C C G A G T

U G G U U U G G C U C A

Trp Phe Gly Ser

Codon

3 5

35

Cracking the CodeA codon in messenger RNA

Is either translated into an amino acid or serves as a translational stop signal

Figure 17.5

Second mRNA baseU C A G

U

C

A

G

UUUUUCUUAUUG

CUUCUCCUACUG

AUUAUCAUAAUG

GUUGUCGUAGUG

Met orstart

Phe

Leu

Leu

lle

Val

UCUUCCUCAUCG

CCUCCCCCACCG

ACUACCACAACG

GCUGCCGCAGCG

Ser

Pro

Thr

Ala

UAUUAC

UGUUGC

Tyr Cys

CAUCACCAACAG

CGUCGCCGACGG

AAUAACAAAAAG

AGUAGCAGAAGG

GAUGACGAAGAG

GGUGGCGGAGGG

UGGUAAUAG Stop

Stop UGA StopTrp

His

Gln

Asn

Lys

Asp

Arg

Ser

Arg

Gly

U

CA

GUCAG

UCAG

UCAG

Fir

st m

RN

A b

ase

(5

end

)

Th

ird

mR

NA

bas

e (3

en

d)

Glu

The genetic code

There is redundancy but no ambiguityCodons must be read in the correct

reading frame For the specified polypeptide to be produced

In summary, genetic information is encoded as a sequence of nonoverlapping base triplets, or codons, each of which is translated into a specific amino acid during protein synthesis.

Evolution of the Genetic Code

The genetic code is nearly universal Shared by organisms from the simplest bacteria

to the most complex animalsIn laboratory experiments

Genes can be transcribed and translated after being transplanted from one species to another

This has permitted bacteria to be programmed To synthesize certain human proteins after

insertion of appropriate human genes

Fig. 17-6

(a) Tobacco plant expressing a firefly gene

(b) Pig expressing a jellyfish gene

Concept 17.2: Transcription is the DNA-directed synthesis of RNA: a closer look

RNA synthesis Is catalyzed by RNA polymerase, which pries

the DNA strands apart & hooks together the RNA nucleotides

RNA polymerase starts the chain (doesn’t need a primer like DNA)

Follows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine

Molecular components of transcription

RNA polymerase attaches and initiates transcription at the promoter

In prokaryotes The sequence that signals the end of the

transcription is called the terminator

The direction of transcription is “downstream” and the other direction is “upstream”

Elongation

RNApolymerase

Non-templatestrand of DNA

RNA nucleotides

3 end

C A E G C AA

U

T A G G T TA

AC

G

U

AT

CA

T C C A AT

T

GG

3

5

5

Newly madeRNA

Direction of transcription(“downstream”) Template

strand of DNA

The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit

RNA polymerase

Bacteria have a single type of RNA polymerase that synthesizes all RNA molecules

Eukaryotes have 3 RNA molecules (I, II & III) in their nuclei RNA polymerase II is used for mRNA synthesis

The stages of transcription are:

1. Initiation

2. Elongation

3. Termination

RNA Polymerase Binding & Initiation of Transcription

In prokaryotes, RNA polymerase can recognize and bind

directly to the promoter region

In eukaryotes, proteins called transcription factors mediate the binding of RNA polymerase & the initiation of transcription Only after certain transcription factors are

attached to the promoter does RNA polymerase II bind to it

Transcription initiation complex

•The completed assembly of transcription factors & RNA pol II bound to a promoter is called a transcription initiation complex

•A crucial promoter DNA sequence is called a TATA box

Elongation of the RNA Strand

As RNA polymerase moves along the DNA It continues to untwist the double helix,

exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

The enzyme adds nucleotides to the 3’ end of the growing strand

Behind the point of RNA synthesis, the double helix re-forms & the RNA molecule peels away

Elongation of the RNA Strand

Transcription progresses at a rate of 40 nucleotides a second in eukaryotes

A single gene can be transcribed simultaneously By several RNA polymerases at a time

This helps the cell make the encoded protein in large amounts

Termination of Transcription

In prokaryotes, RNA polymerase stops transcription at the end of the terminator DNA & RNA are released

In eukaryotes, the pre-mRNA is cleaved from the growing RNA chain While the RNA pol II continues to transcribe

Termination of Transcription - Eukaryotes

Specifically, the polymerase transcribes a DNA sequence called the polyadenylation signal sequence that codes for AAUAA in the pre-mRNA

At a point about 10-35 nucleotides past this sequence, the pre-mRNA is cut from the enzyme

Transcription is terminated when the polymerase falls off the DNA

Concept 17.3: Eukaryotic cells modify RNA after transcription

Enzymes in the eukaryotic nucleus Modify pre-mRNA in specific ways before the

genetic messages are dispatched to the cytoplasm

Usually, both end of the primary transcript are altered

Certain interior parts are cut out, and the remaining is 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 cap The 3 end gets a poly-A tail

Figure 17.9

A modified guanine nucleotideadded to the 5 end

50 to 250 adenine nucleotidesadded to the 3 end

Protein-coding segment Polyadenylation signal

Poly-A tail3 UTRStop codonStart codon

5 Cap 5 UTR

AAUAAA AAA…AAA

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATION

Ribosome

Polypeptide

G P P P

53

UTR is untranslated regions

pre-mRNA end modifications

They seem to facilitate the export of mRNA from the nucleus

They help protect the mRNA from hydrolytic enzymes

They help the ribosomes attach to the 5’ end of the mRNA

Split Genes and RNA Splicing in Eukaryotes

RNA splicing Removes introns and joins exons

Figure 17.10

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATION

Ribosome

Polypeptide

5 CapExon Intron

1

5

30 31

Exon Intron

104 105 146

Exon 3Poly-A tail

Poly-A tail

Introns cut out andexons spliced together

Codingsegment

5 Cap1 146

3 UTR3 UTR

Pre-mRNA

mRNA

Exon – expressed regionsIntrons – intervening regions

RNA Splicing

Is carried out by spliceosomes in some cases

snRNA – small nuclear RNA molecule

snRNP – small nuclear ribonucleoproteins

Figure 17.11

Ribozymes

Are catalytic RNA molecules that function as enzymes & can splice RNA

The intron RNA functions as a ribozyme & catalyzes its own excision

The discovery of ribozymes rendered obsolete the statement that “All biological catalysts are proteins”

RNA as ribozymes

Three properties of RNA allow some RNA molecules to function as ribozymes.

1. Because RNA is single-stranded, a region of the RNA molecule may base-pair with a complementary region elsewhere in the same molecule, thus giving the RNA a specific 3D structure that is key to its ability to catalyze reactions

2. Some of the bases in RNA contain functional groups that may participate in catalysis.

3. The ability of RNA to hydrogen-bond with RNA or DNA adds specificity to its catalytic activity

The Functional and Evolutionary Importance of Introns

Some introns play a regulatory roleSplicing may regulate the passage of

mRNA from the nucleus to the cytoplasmOne obvious benefit is to enable a gene to

code for more than one polypeptide

The presence of introns Allows for alternative RNA splicing

Proteins often have a modular architecture

Consisting of discrete structural and functional regions called domains

In many cases Different exons code for the different domains in a

protein

Figure 17.12

GeneDNA

Exon 1 Intron Exon 2 Intron Exon 3

Transcription

RNA processing

Translation

Domain 3

Domain 1

Domain 2

Polypeptide

Exon Shuffling

The presence of introns increases the possibility of beneficial crossing over between genes Increases opportunity between for

recombination between two alleles May also be occasional mixing and matching

between completely different genes

Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look

A cell interprets a series of codons along an mRNA molecule and builds a

polypeptide

The interpreter is transfer RNA (tRNA) which transfers amino acids from the

cytoplasmic pool to a ribosome

Translation: the basic concept

Figure 17.13

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

Polypeptide

Aminoacids

tRNA withamino acidattachedRibosome

tRNA

Anticodon

mRNA

Trp

Phe Gly

A G C

A A A

CC

G

U G G U U U G G C

Codons5 3

tRNA

Molecules of tRNA are not all identical Each carries a specific amino acid on one end Each has an anticodon on the other end

Each tRNA is used repeatedly picking up its designated amino acid in the

cytosol, deposition the amino acid at the ribosome, and returning to the cytosol to pick up another amino acid

The Structure and Function of Transfer RNA

ACC

A tRNA molecule Consists of a single RNA strand that is only

about 80 nucleotides long Is roughly L-shaped

Figure 17.14a

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. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.)

(a)

3

CCACGCUUAA

GACACCU*

GC

* *G U G U *CU

* G AGGU**A

*A

A GUC

AGACC*

C G A GA G G

G*

*GA

CUC*AUUUAGGCG5

Amino acidattachment site

Hydrogenbonds

Anticodon

A

3D Structure

(b) Three-dimensional structure

Symbol used in this book

Amino acidattachment site

Hydrogen bonds

AnticodonAnticodon

A A G

53

3 5

(c)

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

Fig. 17-15-4

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”)

Wobble

If each anticodon had to be a perfect match, we would expect to find 61 types of tRNA But there are about 45

The rules for base pairing between the 3rd base of the codon & anticodon are relaxed (wobble)

This explains why the synonymous codons for a given amino acid can differ in their 3rd base (but not usually other bases)

Ribosomes

Facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis

In eukaryotes, the subunits are made in the nucleolus

Prokaryotes & eukaryotes have enough differences that some antibiotics (like tetracycline & streptomycin)

can paralyze prokaryote ribosomes without inhibiting eukaryotic ribosomes

Computer model of ribosome

Are constructed of proteins and RNA molecules named ribosomal RNA or rRNA

Figure 17.16a

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide Exit tunnelGrowingpolypeptide

tRNAmolecules

EP A

Largesubunit

Smallsubunit

mRNA

Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins.

(a)

53

The ribosome has three binding sites for tRNA

The P siteThe A siteThe E site

Figure 17.16b

E P A

P site (Peptidyl-tRNAbinding site)

E site (Exit site)

mRNAbinding site

A site (Aminoacyl-tRNA binding site)

Largesubunit

Smallsubunit

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. This schematic ribosome will appear in later diagrams.

(b)

Figure 17.16c

Amino end Growing polypeptide

Next amino acidto be added topolypeptide chain

tRNA

mRNA

Codons

3

5

Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site.

(c)

5’ end first, with the ribosome moving 5’ – 3’ on the mRNA

Building a Polypeptide

We can divide translation into three stages Initiation Elongation Termination

All 3 phases require protein “factors” that aid in translation process

Both initiation and chain elongation require energy provided by the hydrolysis of GTP

3355U

UA

ACGMet

GTP GDPInitiator

tRNA

mRNA

5 3Start codon

mRNA binding site

Smallribosomalsubunit

5

P site

Translation initiation complex

3

E A

Met

Largeribosomalsubunit

Ribosome Association and Initiation of Translation

The initiation stage of translation Brings together mRNA, tRNA bearing the first

amino acid of the polypeptide, and two subunits of a ribosome

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

Fig. 17-18-4

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

E

P A

GDPGTP

Ribosome ready fornext aminoacyl tRNA

E

P A

Releasefactor

3

5Stop codon(UAG, UAA, or UGA)

5

32

Freepolypeptide

2 GDP

GTP

5

3

Termination of TranslationThe final stage of translation is termination

When the ribosome reaches a stop codon in the mRNA

PolyribosomesA number of ribosomes can translate a single

mRNA molecule simultaneously Forming a polyribosome

Figure 17.20a, b

Growingpolypeptides

Completedpolypeptide

Incomingribosomalsubunits

Start of mRNA(5 end)

End of mRNA(3 end)

Polyribosome

An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes.

(a)

Ribosomes

mRNA

This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

0.1 µm(b)

Completing and Targeting the Functional Protein During and after synthesis, a polypeptide coils

and folds to its 3D shape Chaperone proteins may aid correct folding Proteins may require posttranslational

modifications Require additions such as sugars, lipids, or phosphate

groups to amino acids Enzymes may remove some amino acids or cleave

whole polypeptide chains Two or more polypeptides may join to form a protein

Targeting Polypeptides to Specific LocationsTwo populations of ribosomes are evident

in cells Free and bound

Free ribosomes in the cytosol Initiate the synthesis of all proteins Synthesize proteins that reside in the cytosol

Bound and free ribosomes are identical

Proteins destined for the endomembrane system or for secretion Must be transported into the ER Have signal peptide region to which a signal-

recognition particle (SRP) binds, enabling the translation ribosome to bind to the ER

SRP consists of a protein-RNA complex

Ribosome

mRNA

Signalpeptide

Signal-recognitionparticle (SRP)

CYTOSOL Translocationcomplex

SRPreceptorprotein

ER LUMEN

Signalpeptideremoved

ERmembrane

Protein

The signal mechanism for targeting proteins to the ER

Concept 17.5: Point mutations can affect protein structure and function

Mutations are changes in the genetic material of a cell (or virus).

Mutations are the ultimate source of new genes.

Mutations include large-scale mutations, in which long segments of DNA are affected like translocations, duplications, and inversions,

& point mutations, chemical changes in just one base pair of a gene.

Point Mutations

If a point mutation occurs in a gamete or in a cell that produces gametes, it may be transmitted to future generations.

If the mutation has an adverse effect on the phenotype of an organism, the mutant condition is referred to as a genetic disorder or hereditary disease.

A change in a single nucleotide in the DNA’s template strand leads to an abnormal protein.

Fig. 17-22

Wild-type hemoglobin DNA

mRNA

Mutant hemoglobin DNA

mRNA

33

3

3

3

3

55

5

55

5

C CT T TTG GA A AA

A A AGG U

Normal hemoglobin Sickle-cell hemoglobin

Glu Val

Types of Point Mutations

Point mutations within a gene can be divided into two general categories Base-pair substitutions Base-pair insertions or deletions

Fig. 17-23a

Wild type

3DNA templatestrand

3

355

5mRNA

Protein

Amino end

Stop

Carboxyl end

A instead of G

33

3

U instead of C

55

5

Stop

Silent (no effect on amino acid sequence)

Substitutions

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

Fig. 17-23b

Wild type

DNA templatestrand

35

mRNA

Protein

5

Amino end

Stop

Carboxyl end

53

3

T instead of C

A instead of G

33

3

5

5

5

Stop

Missense

Fig. 17-23cWild type

DNA templatestrand

35

mRNA

Protein

5

Amino end

Stop

Carboxyl end

53

3

A instead of T

U instead of A

33

3

5

5

5

Stop

Nonsense

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

Fig. 17-23d

Wild type

DNA templatestrand

35

mRNA

Protein

5

Amino end

Stop

Carboxyl end

53

3

Extra A

Extra U

33

3

5

5

5

Stop

Frameshift causing immediate nonsense (1 base-pair insertion)

Fig. 17-23e

Wild type

DNA templatestrand

35

mRNA

Protein

5

Amino end

Stop

Carboxyl end

53

3

missing

missing

33

3

5

5

5

Frameshift causing extensive missense (1 base-pair deletion)

Fig. 17-23fWild type

DNA templatestrand

35

mRNA

Protein

5

Amino end

Stop

Carboxyl end

53

3

missing

missing

33

3

5

5

5

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

Stop

Mutagens

Spontaneous mutations can occur during DNA replication, recombination, or repair

Mutagens are physical or chemical agents that can cause mutations

Most carcinogens are mutagenic and most mutagens are carcinogenic.

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Concept 17.6: 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, as well as a few with bacteria

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

What is a gene? revisiting the questionThe 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

Fig. 17-25

TRANSCRIPTION

RNA PROCESSING

DNA

RNAtranscript

3

5RNApolymerase

Poly-A

Poly-A

RNA transcript(pre-mRNA)

Intron

Exon

NUCLEUS

Aminoacyl-tRNAsynthetase

AMINO ACID ACTIVATIONAminoacid

tRNACYTOPLASM

Poly-A

Growingpolypeptide

3

Activatedamino acid

mRNA

TRANSLATION

Cap

Ribosomalsubunits

Cap

5

E

P

A

AAnticodon

Ribosome

Codon

E

You should now be able to:

1. Describe the contributions made by Garrod, Beadle, and Tatum to our understanding of the relationship between genes and enzymes

2. Briefly explain how information flows from gene to protein

3. Compare transcription and translation in bacteria and eukaryotes

4. Explain what it means to say that the genetic code is redundant and unambiguous

5. Include the following terms in a description of transcription: mRNA, RNA polymerase, the promoter, the terminator, the transcription unit, initiation, elongation, termination, and introns

6. Include the following terms in a description of translation: tRNA, wobble, ribosomes, initiation, elongation, and termination