Biology 201 Chapter 15

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1

Chapter 15

Lecture and Animation Outline

Chapter 15Genes and How They Work

2

The Nature of Genes

• Early ideas to explain how genes work came from studying human diseases

• Archibald Garrod – 1902 – Recognized that alkaptonuria is inherited via a

recessive allele– Proposed that patients with the disease lacked a

particular enzyme• These ideas connected genes to enzymes

3

4

Beadle and Tatum – 1941

• Deliberately set out to create mutations in chromosomes and verify that they behaved in a Mendelian fashion in crosses

• Studied Neurospora crassa– Used X-rays to damage DNA– Looked for nutritional mutations

• Had to have minimal media supplemented to grow

5

• Beadle and Tatum looked for fungal cells lacking specific enzymes– The enzymes were required for the biochemical

pathway producing the amino acid arginine– They identified mutants deficient in each enzyme of

the pathway• One-gene/one-enzyme hypothesis has been

modified to one-gene/one-polypeptide hypothesis

6


Experimental Procedure

Growth onminimalmediumplus arginine

Wild-typeNeurospora

crassaMutagenizewith X-rays

Grow onrich medium arg mutants

No growthon minimalmedium

Results

Mutationin Enzyme

PlusOrnithine

PlusCitruline

PlusArginosuccinate

PlusArginine

E

F

G

HE F G H

Glutamate Ornithine Citruline Arginosuccinate Arginine

Enzymesencodedby arggenes

arggenes

arg E arg F arg G arg H

Conclusion

Central Dogma

• First described by Francis Crick• Information only flows from

DNA → RNA → protein• Transcription = DNA → RNA • Translation = RNA → protein• Retroviruses violate this order using reverse

transcriptase to convert their RNA genome into DNA

7

8


3′3′

5′

5′

G

C

C

AT

T

C

G

G

UA

A

Transcription

Protein

Eukaryotes

Prokaryotes

Translation

DNA template strand

mRNA

9

• Transcription– DNA-directed synthesis of RNA– Only template strand of DNA used– U (uracil) in DNA replaced by T (thymine) in RNA– mRNA used to direct synthesis of polypeptides

• Translation– Synthesis of polypeptides– Takes place at ribosome– Requires several kinds of RNA

RNA

• All synthesized from DNA template by transcription

• Messenger RNA (mRNA)• Ribosomal RNA (rRNA)• Transfer RNA (tRNA)• Small nuclear RNA (snRNA)• Signal recognition particle RNA (SRP RNA)• Micro-RNA (miRNA)

10

Genetic Code

• Francis Crick and Sydney Brenner determined how the order of nucleotides in DNA encoded amino acid order

• Codon – block of 3 DNA nucleotides corresponding to an amino acid

• Introduced single nulcleotide insertions or deletions and looked for mutations– Frameshift mutations

• Indicates importance of reading frame11

12

One Bases Deleted

Met Pro Thr His Arg Asp Ala Ser

Met Pro HisAla

Met Pro Thr His Arg Asp Ala Ser

Met Pro His Arg Asp Ala Ser

Ser Thr Thr

Delete one bases

AUGCCUACGCACCGCGACGCAUCA

AUGCCUAGCACCGCGACGCAUCA

AUGCCUACGCACCGCGACGCAUCA

AUGCCUCACCGCGACGCAUCA

Amino acids do notchange after third deletion

All amino acids changedafter deletion

Amino acids

Amino acids

Delete three bases

Three Bases Deleted


Spaced or unspaced codons

• Spaced– Codon sequence in a gene punctuated

• Unspaced– codons adjacent to each other

13

14

SCIENTIFIC THINKING

Delete one letter

Only one word changed

WHY DID THE RED BAT EAT THE FAT RAT

WHY DID HE RED BAT EAT THE FAT RAT

Delete one letter

All words after deletion changed

WHYDIDTHEREDBATEATTHEFATRAT

WHYDIDHEREDBATEATTHEFATRAT

Sentence with Spaces

Sentence with No Spaces


15

• Marshall Nirenberg identified the codons that specify each amino acid

• Stop codons– 3 codons (UUA, UGA, UAG) used to terminate

translation• Start codon

– Codon (AUG) used to signify the start of translation• Code is degenerate, meaning that some amino

acids are specified by more than one codon

16

Code practically universal

• Strongest evidence that all living things share common ancestry

• Advances in genetic engineering

• Mitochondria and chloroplasts have some differences in “stop” signals

17

18

Prokaryotic transcription

• Single RNA polymerase• Initiation of mRNA synthesis does not

require a primer• Requires

– Promoter – Start site Transcription unit– Termination site

19

• Promoter– Forms a recognition and binding site for the

RNA polymerase– Found upstream of the start site– Not transcribed– Asymmetrical – indicate site of initiation and

direction of transcription

20


Holoenzyme

׳

a.

Prokaryotic RNA polymerase

Coreenzyme

21


Upstream

Downstream

5′b.

Codingstrand

Templatestrand

Start site (+1)

TATAAT– Promoter (–10 sequence)

TTGACA–Promoter (–35 sequence)

Holoenzyme

׳

a.


Coreenzyme

3′

5′3′

TATAAT

TTGACA

22


Upstream

Downstream

5′

binds to DNA

b.

Helix opens at–10 sequence

Codingstrand

Templatestrand

Start site (+1)



Holoenzyme

׳

a.


Coreenzyme

3′

5′3′

5′ 3′

5′3′

TATAAT

TTGACA

23


binds to DNA

dissociates

Start site RNAsynthesis begins

Transcriptionbubble

RNA polymerase boundto unwound DNA

Helix opens at–10 sequence

ATP

5′ 3′

5′3′


Upstream

Downstream

5′

b.

Codingstrand

Templatestrand

Start site (+1)



Holoenzyme

׳

a.


Coreenzyme

3′

5′3′

TATAAT

TTGACA

• Elongation– Grows in the 5′-to-3′ direction as

ribonucleotides are added– Transcription bubble – contains RNA

polymerase, DNA template, and growing RNA transcript

– After the transcription bubble passes, the now-transcribed DNA is rewound as it leaves the bubble

24

25


RNApolymerase

Start site

3'

5′

Codingstrand Unwinding

Template strand

Transcription bubble

Upstream

mRNA

Rewinding

5′

3'

3'

5′

DNA

Downstream

26

• Termination– Marked by sequence that signals “stop” to

polymerase• Causes the formation of phosphodiester bonds to

cease• RNA–DNA hybrid within the transcription bubble

dissociates• RNA polymerase releases the DNA• DNA rewinds

– Hairpin


DNA and RNAPolymerase dissociates

mRNAdissociatesfrom DNA

CytosineGuanineAdenineUracil

mRNA hairpincauses RNApolymerase to pause

RNA polymerase

DNA

3'

5′

3'

5′

Four or more Uribonucleotides

5′27

• Prokaryotic transcription is coupled to translation– mRNA begins to be translated before

transcription is finished– Operon

• Grouping of functionally related genes• Multiple enzymes for a pathway• Can be regulated together

28

29


0.25 µm

RNA polymerase DNA

mRNA

Ribosomes

Polyribosome

Polypeptidechains

© Dr. Oscar Miller

30

Eukaryotic Transcription

• 3 different RNA polymerases– RNA polymerase I transcribes rRNA– RNA polymerase II transcribes mRNA and

some snRNA– RNA polymerase III transcribes tRNA and

some other small RNAs• Each RNA polymerase recognizes its own

promoter

31

• Initiation of transcription– Requires a series of transcription factors

• Necessary to get the RNA polymerase II enzyme to a promoter and to initiate gene expression

• Interact with RNA polymerase to form initiation complex at promoter

• Termination– Termination sites not as well defined

32


TATA box

Transcriptionfactor

EukaryoticDNA

Other transcription factors

1. A transcription factor recognizes and binds to the TATA box sequence, which is part of the core promoter.

33


Other transcription factors RNA polymerase II

TATA box

Transcriptionfactor

EukaryoticDN A


2. Other transcription factors are recruited, and the initiation complex begins to build.

34


Other transcription factors RNA polymerase II

TATA box

Transcriptionfactor

EukaryoticDNA

Initiationcomplex


2. Other transcription factors are recruited, and the initiation complex begins to build.

3. Ultimately, RNA polymerase II associates with the transcription factors and the DNA, forming the initiation complex, and transcription begins.

35

• In eukaryotes, the primary transcript must be modified to become mature mRNA– Addition of a 5′ cap

• Protects from degradation; involved in translation initiation

– Addition of a 3′ poly-A tail• Created by poly-A polymerase; protection from

degradation– Removal of non-coding sequences (introns)

• Pre-mRNA splicing done by spliceosome

mRNA modifications

36


A A A A A A A

Methyl group

3′poly-A tail

5′cap

CH2

HO OH

P P P

G

mRNA

PP

P

+N+

CH35′

3′

CH3

Eukaryotic pre-mRNA splicing

• Introns – non-coding sequences • Exons – sequences that will be translated• Small ribonucleoprotein particles

(snRNPs) recognize the intron–exon boundaries

• snRNPs cluster with other proteins to form spliceosome– Responsible for removing introns

37

38


E1 I1 E2 I2 E3 I3 E4 I4

Transcription

Introns are removed

a.

ExonsIntrons

Mature mRNA

cap ׳poly-A tail5 ׳33

cap ׳5 poly-A tail ׳3

Primary RNA transcript

DNA template

39


b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine

E1 I1 E2 I2 E3 I3 E4 I4

Intron

Exon

DNA

1

2 34

5 67

a.

b. c.

ExonsIntrons

mRNA

Mature mRNA

3' poly-A tail5' cap

5' cap 3' poly-A tail


DNA template

Introns are removed

Transcription

40

snRNPs

Exon 2Exon 1 Intron

Branch point A

snRNA

5′ 3′

1. snRNA forms base-pairs with 5′end of intron, and at branch site.

A


41


snRNPs

Exon 2Exon 1 Intron

Branch point A

snRNA

5′

5′

3′

3′

2. snRNPs associate with other factors to form spliceosome.


SpliceosomeA

A

42


snRNPs

Exon 2Exon 1 Intron

Branch point A

snRNA

Lariat

5′

5′

3′

3′

5′ 3′



3. 5′end of intron is removed and forms bond at branch site, forming a lariat. The 3′end of the intron is then cut.

SpliceosomeA

A

A

43


snRNPs

Exon 2Exon 1 Intron

Branch point A

snRNA

Exon 1 Exon 2

Lariat

5′

5′

3′

3′

5′

5′

3′

3′


4. Exons are joined; spliceosome disassembles.


3. 5′end of intron is removed and forms bond at branch site, forming a lariat. The 3′end of the intron is then cut.

Mature mRNA

Excisedintron

SpliceosomeA

A

A

Alternative splicing

• Single primary transcript can be spliced into different mRNAs by the inclusion of different sets of exons

• 15% of known human genetic disorders are due to altered splicing

• 35 to 59% of human genes exhibit some form of alternative splicing

• Explains how 25,000 genes of the human genome can encode the more than 80,000 different mRNAs

44

tRNA and Ribosomes

• tRNA molecules carry amino acids to the ribosome for incorporation into a polypeptide– Aminoacyl-tRNA synthetases add amino acids

to the acceptor stem of tRNA– Anticodon loop contains 3 nucleotides

complementary to mRNA codons

45

4646


c: Created by John Beaver using ProteinWorkshop, a product of the RCSB PDB, and built using the Molecular Biology Toolkit developed by John Moreland and Apostol Gramada (mbt.sdsc.edu). The MBT is financed by grant GM63208

2D “Cloverleaf” ModelAcceptor end

Anticodonloop

׳3׳5

3D Ribbon-like Model Acceptor end

Anticodon loop

3D Space-filled Model

Anticodon loop

Acceptor end Icon

Anticodon end

Acceptor end

tRNA charging reaction

• Each aminoacyl-tRNA synthetase recognizes only 1 amino acid but several tRNAs

• Charged tRNA – has an amino acid added using the energy from ATP– Can undergo peptide bond formation without

additional energy• Ribosomes do not verify amino acid

attached to tRNA47

48

tRNAPiPi

NH3+

O–

C OC O

OH

OAMP

Aminogroup

Carboxylgroup

TrpNH

3+

Trp

ATP

Aminoacid site

Acceptingsite

Anticodonspecific to tryptophan

Aminoacyl-tRNAsynthetase

tRNAsite

1. In the first step of the reaction, the amino acid is activated. The amino acid reacts with ATP to produce an intermediate with the carboxyl end of the amino acid attached to AMP. The two terminal phosphates (pyrophosphates) are cleaved from ATP in this reaction.


49


PiPi

Aminogroup

Carboxylgroup

Trp

ATPAminoacid site


tRNAsite

NH3+ C

O–

O

50


tRNAPiPi

NH3+

O–

C OC O C

O

OH

OAMP O

OH

AMP

Aminogroup

Carboxylgroup

TrpNH

3+ NH

3 +Trp Trp

ATPAminoacid site

Acceptingsite



tRNAsite

51


tRNAPiPi

NH3+

O–

C OC OC

O CO

COOH

OAMP O

OH

AMPAMP

O

O

Charged tRNA travels to ribosomeAminogroup

Carboxylgroup

TrpNH

3+ NH

3 +

NH3 +

NH3+Trp Trp

TrpATPAminoacid site

Acceptingsite



tRNAsite

ChargedtRNA

dissociates

Trp

1. In the first step of the reaction, the amino acid is activated. The amino acid reacts with ATP to produce an intermediate with the carboxyl end of the amino acid attached to AMP. The two terminal phosphates (pyrophosphates) are cleaved from ATP in this reaction.

2. The amino acid-AMP complex remains bound to the enzyme. The tRNA next binds to the enzyme.

3. The second step of there action transfers the amino acid from AMP to the tRNA, Producing a charged tRNA and AMP. The charged tRNA consists of a specific amino acid attached to the 3′ accept or stem of it sRNA.

52

• The ribosome has multiple tRNA binding sites– P site – binds the tRNA attached to the

growing peptide chain– A site – binds the tRNA carrying the

next amino acid– E site – binds the tRNA that carried the

last amino acid

53


mRNA

90°

0°

3′

5′

Largesubunit

Smallsubunit

Largesubunit

Smallsubunit

LargesubunitSmallsubunit

54

• The ribosome has two primary functions– Decode the mRNA– Form peptide bonds

• Peptidyl transferase– Enzymatic component of the ribosome– Forms peptide bonds between amino acids

55

Translation

• In prokaryotes, initiation complex includes– Initiator tRNA charged with N-formylmethionine– Small ribosomal subunit– mRNA strand

• Ribosome binding sequence (RBS) of mRNA positions small subunit correctly

• Large subunit now added• Initiator tRNA bound to P site with A site

empty

56


3′

5′

AUG

U CA

GTP GDP+Pi

Smallsubunit

Initiationfactor

Initiationfactor

fMet 3′

5′

mRNA


U CAA GU

GTP GDP+

E site

Initiation complex Complete ribosomePi

tRNA inP site

A site

Largesubunit

5′ 5´

3′3′

• Initiations in eukaryotes similar except– Initiating amino acid is methionine– More complicated initiation complex– Lack of an RBS – small subunit binds to 5′

cap of mRNA

57

58

• Elongation adds amino acids– 2nd charged tRNA can bind to empty A

site– Requires elongation factor called EF-Tu

to bind to tRNA and GTP– Peptide bond can then form– Addition of successive amino acids

occurs as a cycle

59


NH3+

3′

5′

OHO

CO

CO

OC OC

O

N

O

NH3+

Aminoacid 1

Aminoacid 2

Peptidebond

formation

Polypeptidechain

“Empty”tRNA

AminogroupAminoacid 1

Peptidebond

Aminoacid 2

Amino end(N terminus)

Aminoacid 1

Aminoacid 2

Aminoacid 3

Aminoacid 4

Aminoacid 5

Aminoacid 6

Aminoacid 7

Carboxyl end(C terminus)

COO–

P siteA site

NH3+ NH3

+

60


3′

3′

3′ 3′

3′5′

5′

5′

5′

“Ejected” tRNA

E P A

E P A

E P AE P A

E P A

Sectioned ribosome

Pi

GTP

GTP

GDP +

Next round

Elongationfactor

Elongationfactor

+ Pi

Elongationfactor

Elongationfactor Growing

polypeptide GDP

GTP

5′

61

• There are fewer tRNAs than codons• Wobble pairing allows less stringent

pairing between the 3′ base of the codon and the 5′ base of the anticodon

• This allows fewer tRNAs to accommodate all codons

62

• Termination– Elongation continues until the ribosome

encounters a stop codon– Stop codons are recognized by release

factors which release the polypeptide from the ribosome

63


3′

5′

3′

5′

EP

A

Dissociation

GC

GC

U

AAUA

Sectionedribosome

Releasefactor

Polypeptidechain releases

64

Protein targeting

• In eukaryotes, translation may occur in the cytoplasm or the rough endoplasmic reticulum (RER)

• Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition particle (SRP)

• The signal sequence and SRP are recognized by RER receptor proteins

• Docking holds ribosome to RER• Beginning of the protein-trafficking pathway

65


Signal recognitionparticle (SRP)

Signal

Ribosomesynthesizingpeptide

Exit tunnel

Docking

Polypeptideelongationcontinues

Rough endoplasmicreticulum (RER)

Cytoplasm Lumen of the RER

SRP binds to signalpeptide, arresting

elongation

Protein channel

NH2

66


1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript.


RNA polymerase IIRNA polymerase II

Primary RNA transcript5´

3´

5´

3´

67



Poly-A tail

Mature mRNACut intron

5´ cap


2. The primary transcript is processed by addition of a 5′ methyl-G cap, cleavage and polyadenylation of the 3′end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm.


RNA polymerase II


Poly-A tail


5´ cap

RNA polymerase II


3´

5´

3´

68



Poly-A tail


5´ cap


2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm.

3. The 5′cap of the mRNA associates with the small subunit of the ribosome. The initiator tRNA and large subunit are added to form an initiation complex.


RNA polymerase II


Poly-A tail


5´ cap

Largesubunit

mRNA 5´ cap

Smallsubunit Cytoplasm

RNA polymerase II


3´

5´

3´

69





4. The ribosome cycle begins with the growing peptide attached to the tRNA in the P site. The next charged tRNA binds to the A site with its anticodon complementary to the codon in the mRNA in this site.

mRNA


tRNA arrivesin A site Amino acids

mRNA

A siteP site

E site

Cytoplasm

3´

5′

3′


Cut intron

Largesubunit

5′cap

RNA polymerase II


Poly-A tail


5′cap


5´

70






5. Peptide bonds form between the amino terminus of the next amino acid and the carboxyl terminus of the growing peptide. This transfers the growing peptide to the tRNA in the A site, leaving the tRNA in the P site empty.

mRNA



mRNA

A siteP site

E site

Lengtheningpolypeptide chain

EmptytRNA

Cytoplasm

3´

5′

3′

5′

3′


Cut intron

Largesubunit

5′cap

RNA polymerase II


Poly-A tail


5′cap


5´

71






5. Peptide bonds form between the amino terminus of the next amino acid and the carboxyl terminus of the growing peptide. This transfers the growing peptide to the tRNA in the A site, leaving the tRNA in the P site empty.

mRNA



mRNA

A siteP site

E site

Lengtheningpolypeptide chain

EmptytRNA

3´

5′

3′

5′

3′


Cut intron

Largesubunit

5′cap

RNA polymerase II


Poly-A tail


5′cap


5´

6. Ribosome translocation moves the ribosome relative to the mRNA and its bound tRNAs. This moves the growing chain into the P site, leaving the empty tRNA in the E site and the A site ready to bind the next charged tRNA.

Empty tRNA moves intoE site and is ejected

5′

3′

Cytoplasm

72

73

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Mutation: Altered Genes

• Point mutations alter a single base• Base substitution – substitute one base for

another– Silent mutation – same amino acid inserted– Missense mutation – changes amino acid

inserted• Transitions• Transversions

– Nonsense mutations – changed to stop codon74

75

Stop

mRNA

Protein

Coding

CG

AT

AT

Template

Pro Thr ArgMet

a.

5′–ATGCCTTATCGCTGA–3′3′–TACGGAATAGCGACT–5′5′–AUGCCUUAUCGCUGA–3′

Stop

mRNA

Protein

Coding

CG

Template

Pro Thr ArgMet

b.

Silent Mutation

5′–ATGCCCTATCGCTGA–3′3′–TACGGGATAGCGACT–5′5′–AUGCCCUAUCGCUGA–3′


76

Stop

mRNA

Protein

Coding

AT

Template

Pro Thr HisMet

c.

Missense Mutation

5′–ATGCCCTATCACTGA–3′3′–TACGGGATAGTGACT–5′5′–AUGCCCUAUCACUGA–3′


Stop

mRNA

Protein

Coding

AT

Template

ProMet

d.

Nonsense Mutation

5′–ATGCCCTAACGCTGA–3′3′–TACGGGATTGCGACT–5′5′–AUGCCCUAACGCUGA–3′

77


Polar

Normal HBB Sequence

Abormal HBB Sequence

Nonpolar (hydrophobic)

Amino acids

Nucleotides

Amino acids

Nucleotides

Leu

C C C CGT T TA GG A GAA

Thr Pro Glu Glu

CT TGAA

Lys Ser

Leu

C C C CGT T TA GG GAG

Thr Pro val Glu

CTT TGAA

Lys Ser

1

1

NormalDeoxygenated

Tetramer

AbnormalDeoxygenated

Tetramer

Tetramers form long chainswhen deoxygenated. Thisdistorts the normal red bloodcell shape into a sickle shape.

Hemoglobintetramer

"Sticky" non-polar sites

2

2

1

1

2

2

78

• Frameshift mutations– Addition or deletion of a single base– Much more profound consequences– Alter reading frame downstream– Triplet repeat expansion mutation

• Huntington disease• Repeat unit is expanded in the disease allele

relative to the normal

79

Chromosomal mutations

• Change the structure of a chromosome– Deletions – part of chromosome is lost– Duplication – part of chromosome is copied– Inversion – part of chromosome in reverse

order– Translocation – part of chromosome is moved

to a new location

80

A B C D E F G H A E F G HI IJ J

B C D E F G H I J A B C D B C D E F G H I J

Duplication

Deletion

Deleted

Duplicated

a.

A

b.


81

A B C D E F G H I J

A B C D E F G H I J

K L M N O P Q R

K L M D E F G H I J

A B C N O P Q R

A D C B E F G H I J

Inversion

Inverted

c.

d.

Reciprocal Translocation


82

• Mutations are the starting point for evolution

• Too much change, however, is harmful to the individual with a greatly altered genome

• Balance must exist between amount of new variation and health of species

Education

Biology 201 Chapter 15