From Genes to Proteins Chapter 17 AP Chapter 17. How was the fundamental relationship between genes and proteins discovered? In 1909, British physician

From Genes to ProteinsChapter 17

AP Chapter 17

How was the fundamental relationship between genes and proteins discovered?

In 1909, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions

He studied alkaptonuria.

• Alkaptonuria is best known for the darkening of urine from yellow to brown to black after it is exposed to the air.

• Garrod studied the patterns in several families, realized it followed an autosomal recessive pattern of inheritance, and postulated that it was caused by a mutation in a gene encoding an enzyme involved in the metabolism of a class of compounds called alkaptans.

http://en.wikipedia.org/wiki/Autosomal_recessive

http://upload.wikimedia.org/wikipedia/commons/4/46/Alkaptonuria.jpg

Nutritional Mutants in Neurospora: Scientific Inquiry

• George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal medium as a result of inability to synthesize certain molecules.

• They identified mutants that lacked certain enzymes necessary for synthesizing.

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

Fig. 17-2

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

Minimalmedium(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 OrnithineEnzyme B Enzyme B Enzyme B

Citrulline Citrulline Citrulline CitrullineEnzyme C Enzyme C Enzyme C Enzyme C

Arginine Arginine Arginine Arginine

The mutants couldnot produce theenzymes to movethrough the pathway.

They developed a one gene–one enzyme hypothesis, which states that each gene dictates production of a specific enzyme

Revision of the hypothesis…

• Some proteins aren’t enzymes, so researchers later revised the hypothesis: one gene–one protein

• Therefore, Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis

Note that it is common to refer to gene products as proteins rather than polypeptides.

Remember!

Proteins are made of amino acids.

RNA is a key-player in Protein Synthesis.

• RNA differs from DNA in three major ways.

– RNA has a ribose sugar.– RNA has uracil instead of thymine.– RNA is a single-stranded structure.

URACIL

Different sugars in DNA and RNA

ribose

deoxyribose

Has oneLess oxygen

atom

Fig. 17-4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNAtemplatestrand

TRANSCRIPTION

TRANSLATION

mRNA

Protein

Codon

Amino acid

Basic Principles of Transcription and Translation

• Transcription is the synthesis of mRNA (messenger RNA) under the direction of DNA

• Occurs in the nucleus of eukaryotes• Translation is the synthesis of a

polypeptide, which occurs under the direction of mRNA

• Ribosomes are the sites of translation


Fig. 17-3a-1

TRANSCRIPTIONDNA

mRNA

(a) Bacterial cell

Fig. 17-3a-2

(a) Bacterial cell

TRANSCRIPTIONDNA

mRNA

TRANSLATIONRibosome

Polypeptide

Differences in Euk and Prok 1. In a eukaryotic cell, the nuclear envelope

separates transcription from translation

2. Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA. A primary transcript is the initial RNA transcript from any gene.


Fig. 17-3

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

(a) Bacterial cell

Nuclearenvelope

TRANSCRIPTION

RNA PROCESSINGPre-mRNA

DNA

mRNA

TRANSLATION Ribosome

Polypeptide

(b) Eukaryotic cell

The central dogma is the concept that cells are governed by a cellular chain of command: DNA RNA protein.

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?

3

The Genetic Code is read in CODONS which are Triplets of Nucleotide Bases in mRNA that

code for amino acids.

• Example: AGT codes for the amino acid serine.


Cracking the Code

• All 64 codons were deciphered by the mid-1960s by Marshall Nirenberg

• Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation

• There is one “start” code AUG which also codes for the amino acid methionine.


Fig. 17-5Second 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)

What does this mean?

• The genetic code is redundant: an amino acid may have more than one code.

• but not ambiguous; no codon specifies more than one amino acid

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


Fig. 17-6

(a) Tobacco plant expressing a firefly gene

(b) Pig expressing a jellyfish gene

In a nutshell….

• During transcription, one of the two DNA strands called the template strand provides a template for ordering the sequence of nucleotides in a mRNA.

• Codons 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

Fig. 17-4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNAtemplatestrand

TRANSCRIPTION

TRANSLATION

mRNA

Protein

Codon

Amino acid

DNA:TAC AAA TGA GGA TCA GCT ACC CCA ACA ACT

mRNA:

AUG UUU ACU CCU AGU CGA UGG GGU UGU UGA

Amino acids

Met(start)–phe–thr–pro–ser-arg-trp-gly-cys-stop

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

• The three stages of transcription:– Initiation– Elongation– Termination


• 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


Fig. 17-7a-1Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

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

The DNA sequence where RNA polymerase attaches is called the promoter.

Fig. 17-7b

Elongation

RNApolymerase

Nontemplatestrand of DNA

RNA nucleotides

3 end

Direction oftranscription(“downstream”) Template

strand of DNA

Newly madeRNA

3

5

5

Promoters (TATA box area) signal the initiation of RNA synthesis.

Transcription factors mediate the binding of RNA polymerase and the initiation of transcription

Adds at 3’of growing end

Fig. 17-8A eukaryotic promoterincludes a TATA box

3

1

2

3

Promoter

TATA box Start point

Template

TemplateDNA strand

535

Transcriptionfactors

Several transcription factors mustbind to the DNA before RNApolymerase II can do so.

5533

Additional transcription factors bind tothe DNA along with RNA polymerase II,forming the transcription initiation complex.

RNA polymerase IITranscription factors

55 53

3

RNA transcript

Transcription initiation complex

Transcription factors


DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

5 5

UnwoundDNA

Template strandof DNA


DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

5 5

UnwoundDNA


2 Elongation

RewoundDNA

5

5 5 3 3 3

RNAtranscript


DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

5 5

UnwoundDNA


2 Elongation

RewoundDNA

5

5 5 3 3 3

RNAtranscript

3 Termination

5

5 5 33

3Completed RNA transcriptanimations

http://www.hhmi.org/biointeractive/dna/DNAi_transcription_vo1.html

http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html

http://www.hhmi.org/biointeractive/dna/DNAi_transcription_vo1.html

• A gene can be transcribed simultaneously by several RNA polymerases


Differences in transcription in Prokaryotes and Eukaryotes

1. Prok lack transcription factors2. No terminator sequence in Euk4. More types of RNA polymerases in

euk5. No RNA processing in Prok

Compare DNA polymerase and RNA polymerase

How they function:

• DNA polymerase used to bind DNA nucleotides to present DNA

• RNA polymerase used to bind RNA nucleotides to DNA

Requirement for a template and primer:

• RNA polymerase does not need a primer. Both use DNA as a template.

Direction of synthesis: 5’ to 3’

Type of nucleotides used:

DNA-A,T,C,G;

RNA – A,U,C,G (U instead of T)

What is a promoter?• It is a DNA sequence where

transcription will begin and where the RNA polymerase attaches.

• It is located upstream of a transcription unit.

What makes RNA polymerase start transcribing a gene at the right place on the DNA in a bacterial cell? In an eukaryotic cell?

• A promoter area

In an eukaryotic cell?

• A promoter area and transcription factors

Eukaryotic cells modify RNA after transcription

• 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

Fig. 17-9

Protein-coding segment Polyadenylation signal3

3 UTR5 UTR

5

5 Cap Start codon Stop codon Poly-A tail

G P PP AAUAAA AAA AAA…

G

(UTR – untranslated region)

These modifications share several functions:

–They facilitate the export of mRNA out of the nucleus

–They protect mRNA from hydrolytic enzymes

–They help ribosomes attach to the 5 end

Split Genes and RNA Splicing

• Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions called introns.

• Coding regions are exons

• RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

Fig. 17-10

Pre-mRNA

mRNA

Codingsegment

Introns cut out andexons spliced together

5 Cap

Exon Intron5

1 30 31 104

Exon Intron

105

Exon

146

3Poly-A tail

Poly-A tail5 Cap

5 UTR 3 UTR1 146

http://www.dnalc.org/resources/3d/

http://www.dnalc.org/resources/3d/

Remove the introns:

IJOILKJLOVEIUIOAPJKLBIOLOGYIOY

IJOILKJLOVEIUIOAPJKLBIOLOGYI

Remember the exons…..exit or go on

to make the proteins!

How are the pieces cut and spliced?

• Spliceosomes consist of a variety of proteins and small nuclear RNA (snRNA), “snRNP’s”, that recognize the splice sites

Did you call me? Oh, “snurps” not smurfs!

Fig. 17-11-1RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5


Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

5

Spliceosome


Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

5

Spliceosome

Spliceosomecomponents

Cut-outintronmRNA

Exon 1 Exon 25

• Ribozymes are catalytic RNA molecules that function as enzymes and can also splice RNA

The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins.

The Functional and Evolutionary Importance of Introns

• Alternative RNA splicing can enable some genes to encode more than one kind of protein depending on which segments are treated as exons.

• Introns allow more room for crossing-over which results in exon shuffling may result in the evolution of new proteins

A Closer Look at Translation

http://thednastore.com/dnastuff/bobbleheads.html

Fig. 17-4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNAtemplatestrand

TRANSCRIPTION

TRANSLATION

mRNA

Protein

Codon

Amino acid

Remember…

• Translation is using the mRNA code to order the amino acids in a polypeptide (protein).

How do the amino acids get to the ribosome?

t-RNA

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

Fig. 17-14

Amino acidattachment site

3

5

Hydrogenbonds

Anticodon

(a) Two-dimensional structure

Amino acidattachment site

5

3

Hydrogenbonds

3 5AnticodonAnticodon

(c) Symbol used in this book(b) Three-dimensional structure

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 a complementary codon on mRNA

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. 17-15-1

Amino acid Aminoacyl-tRNAsynthetase (enzyme)

ATP

AdenosineP P P

Fig. 17-15-2


ATP

AdenosineP P P

AdenosineP

PP i

PPi

i

Fig. 17-15-3


ATP

AdenosineP P P

AdenosineP

PP i

PPi

i

tRNA

tRNA

Aminoacyl-tRNAsynthetase

Computer model

AMPAdenosineP

Fig. 17-15-4


ATP

AdenosineP P P

AdenosineP

PP i

PPi

i

tRNA

tRNA

Aminoacyl-tRNAsynthetase

Computer model

AMPAdenosineP

Aminoacyl-tRNA(“charged tRNA”)

I’m really worried about my aminoacyl tRNA synthetases…

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)


• 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

Remember: P first, then A, then E!

Fig. 17-16a

Growingpolypeptide Exit tunnel

tRNAmolecules

Largesubunit

Smallsubunit

(a) Computer model of functioning ribosome

mRNA

E P A

5 3

Fig. 17-16b

P site (Peptidyl-tRNAbinding site) A site (Aminoacyl-

tRNA binding site)E site(Exit site)

mRNAbinding site

Largesubunit

Smallsubunit

(b) Schematic model showing binding sites

Next amino acidto be added topolypeptide chain

Amino end Growing polypeptide

mRNAtRNA

E P A

E

Codons

(c) Schematic model with mRNA and tRNA

5

3

Fig. 17-13

Polypeptide

Ribosome

Aminoacids

tRNA withamino acidattached

tRNA

Anticodon

TrpPhe Gly

Codons 35

mRNA

Codons along an mRNA molecule are read by translation machinery in the 5 to 3 direction

Building a Polypeptide

• The three stages of translation:– Initiation– Elongation– Termination

• All three stages require protein “factors” that aid in the translation process


Steps: Initiation1. First, a small ribosomal subunit binds

with mRNA and a special initiator tRNA

2. Then the small subunit moves along the mRNA until it reaches the start codon (AUG met) at the P site

3. Proteins called initiation factors bring in the large subunit that completes the translation initiation complex


Fig. 17-17

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

Elongation of the Polypeptide Chain

• tRNA bearing the next amino acid comes in at the A site by codon recognition

• Amino acids are joined by peptide bonds• tRNA that was in the P site moves over into

the E site. Everything shifts over.


More realistic images

Fig. 17-18-1

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

Fig. 17-18-2


mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

Fig. 17-18-3


mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

E

P A

Fig. 17-18-4


mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

E

P A

GDPGTP

Ribosome ready fornext aminoacyl tRNA

E

P A

http://www.dnalc.org/resources/3d/16-translation-advanced.html




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

Fig. 17-19-1

Releasefactor

3

5Stop codon(UAG, UAA, or UGA)

Fig. 17-19-2

Releasefactor

3


5

32

Freepolypeptide

2 GDP

GTP

Fig. 17-19-3

Releasefactor

3


5

32

Freepolypeptide

2 GDP

GTP

5

3H2O

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


Fig. 17-20

Growingpolypeptides

Completedpolypeptide

Incomingribosomalsubunits

Start ofmRNA(5 end)

Polyribosome

End ofmRNA(3 end)

(a)

Ribosomes

mRNA

(b) 0.1 µm

Protein Folding and Post-Translational Modifications

During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape

It may have sugarsand lipids bonded to it.

Targeting Polypeptides to Specific Locations

• Some will be free proteins and some will be bound 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

Let’s do this!

DNA: (in nucleus)

TAC AAA TGA GGA TCA GCT ACC CCA ACA ACT

RNA: (in cytoplasm, at ribosome)

Point mutations can affect protein structure and function

• Mutations are changes in the genetic material of a cell or virus

• Mutagens are physical or chemical agents that can cause mutations

• Point mutations are chemical changes in just one base pair of a gene

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


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 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

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. 17-23a

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)

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

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

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

NONPOLARPOLAR

http://upload.wikimedia.org/wikipedia/commons/2/23/Glutamic_Acid.png

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

Fig. 17-UN8

Documents

From Genes to Proteins Chapter 17 AP Chapter 17. How was the fundamental relationship between genes and proteins discovered? In 1909, British physician