Chapter 15mmsalemscienceteacher.weebly.com/uploads/2/3/3/6/... · 2018. 9. 9. · Exon 1 Intron...

Chapter 15

Genes and How They Work

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

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

• 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

Experimental Procedure

Growth on

minimal

medium

plus arginine

Wild-type

Neurospora

crassa

Mutagenize

with X-rays

Grow on

rich medium arg mutants

No growth

on minimal

medium

Results

Mutation

in Enzyme

Ornithine

Citruline

Arginosuccinate

Arginine

HE F G H

Glutamate Ornithine Citruline Arginosuccinate Arginine

Enzymes

encoded

by arg

genesarg 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

3′3′

Transcription

Protein

Eukaryotes

Prokaryotes

Translation

template

strand

• 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

• 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)9

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 frame10

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 not

change after third deletion

All amino acids changed

after deletion

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

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

• 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

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

Prokaryotic transcription

• Single RNA polymerase

• Initiation of mRNA synthesis does not

require a primer

• Requires

– Promoter

– Start site Transcription unit

– Termination site

• 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

Holoenzyme

Prokaryotic RNA polymerase

enzyme

Upstream

Downstream

Coding

strand

Template

strand

Start site (+1)

TATAAT– Promoter (–10 sequence)

TTGACA–Promoter

(–35 sequence)

Holoenzyme

enzyme

TATAAT

TTGACA

Upstream

Downstream

binds to DNA

Helix opens at

–10 sequence

Coding

strand

Template

strand

Start site (+1)

TTGACA–Promoter

(–35 sequence)

Holoenzyme

enzyme

5′ 3′

TATAAT

TTGACA

binds to DNA

dissociates

Start site RNA

synthesis begins

Transcription

bubble

RNA polymerase bound

to unwound DNA

Helix opens at

–10 sequence

5′ 3′

Upstream

Downstream

Coding

strand

Template

strand

Start site (+1)

TTGACA–Promoter

(–35 sequence)

Holoenzyme

enzyme

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

polymerase

Start site

Codingstrand Unwinding

Template strand

Transcription bubble

Upstream

Rewinding

Downstream

• Termination

– Marked by sequence that signals “stop” to

polymerase

• Causes the formation of phosphodiester bonds to

• RNA–DNA hybrid within the transcription bubble

dissociates

• RNA polymerase releases the DNA

• DNA rewinds

– Hairpin

DNA and RNA

Polymerase dissociates

dissociates

from DNA

Cytosine

Guanine

Adenine

Uracil

mRNA hairpin

causes RNA

polymerase to pause

RNA polymerase

Four or more U

ribonucleotides

5′26

• 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

0.25 µm

RNA polymerase DNA

Ribosomes

Polyribosome

Polypeptide

chains

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

• 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

TATA box

Transcription

factor

Eukaryotic

Other transcription factors

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

the core promoter.

Other transcription factors RNA polymerase II

TATA box

Transcription

factor

Eukaryotic

1. A transcription factor recognizes and

binds to the TATA box sequence, which

is part of the core promoter.

2. Other transcription factors are

recruited, and the initiation complex

begins to build.

Other transcription factors RNA polymerase II

TATA box

Transcription

factor

Eukaryotic

Initiation

complex

1. A transcription factor recognizes and

binds to the TATA box sequence, which

is part of the core promoter.

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.

• 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

Methyl group

5′ cap

P mRNA

CH35′

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

E1 I1 E2 I2 E3 I3 E4 I4

Transcription

Introns are removed

Introns

Mature mRNA

׳33 poly-A tail5׳ cap

׳5 cap ׳3 poly-A tail

Primary RNA transcript

DNA template

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

E1 I1 E2 I2 E3 I3 E4 I4

Intron

Introns

Mature mRNA

3' poly-A tail5' cap

5' cap 3' poly-A tail

DNA template

Introns are removed

Transcription

snRNPs

Exon 2Exon 1 Intron

Branch point A

5′ 3′

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

snRNPs

Exon 2Exon 1 Intron

Branch point A

2. snRNPs associate with other factors to form spliceosome.

Spliceosome

snRNPs

Exon 2Exon 1 Intron

Branch point A

Lariat

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.

Spliceosome

snRNPs

Exon 2Exon 1 Intron

Branch point A

Exon 1 Exon 2

Lariat

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

Excised

intron

Spliceosome

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 mRNAs43

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

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” Model

Acceptor end

Anticodon

׳3׳5

3D Ribbon-like Model Acceptor end

Anticodon loop

3D Space-filled Model

Anticodon loop

Acceptor end

Anticodon end

Acceptor end

tRNA charging reaction

• Each aminoacyl-tRNA synthetase

recognizes only 1 amino acid but several

• 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 tRNA46

Carboxyl

acid site

Accepting

Anticodon

specific to tryptophan

Aminoacyl-tRNA

synthetase

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.

Carboxyl

acid site

Aminoacyl-tRNA

synthetase

NH3+ C

Carboxyl

acid site

Accepting

Anticodon

Aminoacyl-tRNA

synthetase

C OC O

Charged tRNA travels to ribosomeAmino

Carboxyl

TrpNH3

acid site

Accepting

Anticodon

Aminoacyl-tRNA

synthetase

Charged

dissociates

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.

• The ribosome has multiple tRNA binding

– 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

subunit

• 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

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

GTP GDP

subunit

Initiation

factor

Initiation

factor

fMet 3′

GTP GDP

E site

Initiation complex Complete ribosome

tRNA in

P site

A site

subunit

5′ 5´

• 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

• 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

acid 1

acid 2

Peptide

formation

Polypeptide

“Empty”

acid 1

Peptide

acid 2

Amino end

(N terminus)

acid 1

acid 2

acid 3

acid 4

acid 5

acid 6

acid 7

Carboxyl end

(C terminus)

COO–

P site

A site

3′3′

“Ejected” tRNA

E P AE P A

Sectioned ribosome

Next round

Elongation

factor

Elongation

factor

Elongation

factor

Elongation

factorGrowing

polypeptide GDP

• 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

• Termination

– Elongation continues until the ribosome

encounters a stop codon

– Stop codons are recognized by release

factors which release the polypeptide from the

ribosome

Dissociation

Sectioned

ribosome

Release

factor

Polypeptide

chain releases

Protein targeting

• In eukaryotes, translation may occur in the

cytoplasm or the rough endoplasmic reticulum

• 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

Signal recognition

particle (SRP)

Signal

Ribosome

synthesizing

peptide

Exit tunnel

Docking

Polypeptide

elongation

continues

Rough endoplasmic

reticulum (RER)Cytoplasm Lumen of the RER

SRP binds to signal

peptide, arresting

elongation

Protein channel

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´

Poly-A tail

Mature mRNACut intron

5´ cap

1. RNA polymerase

II in the nucleus

copies one

strand

of the DNA to

produce the

primary

transcript.

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

Poly-A tail

5´ cap

1. RNA polymerase

II in the nucleus

copies one

strand

of the DNA to

produce the

primary

transcript.

is processed by

addition of a 5´methyl-G cap,

cleavage and

3´ end, and removal of

introns. The mature

mRNA is then

exported through

cytoplasm.

3. The 5′cap of the

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

subunit

mRNA 5´ cap

subunit Cytoplasm

RNA polymerase II

1. RNA polymerase

II in the nucleus

copies one

strand

of the DNA to

produce the

primary

transcript.

is processed by

addition of a 5′

methyl-G cap,

cleavage and

introns. The mature

mRNA is then

exported through

cytoplasm.

associates with

the small subunit

of the ribosome.

The initiator

tRNA and large

subunit are

added to form

an initiation

complex.

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.

subunitCytoplasm

tRNA arrivesin A site Amino acids

A siteP site

E site

Cytoplasm

Cut intron

subunit

5′cap

RNA polymerase II

Poly-A tail

5′cap

1. RNA polymerase

II in the nucleus

copies one

strand

of the DNA to

produce the

primary

transcript.

is processed by

addition of a 5′

methyl-G cap,

cleavage and

introns. The mature

mRNA is then

exported through

cytoplasm.

associates with

the small subunit

of the ribosome.

The initiator

tRNA and large

subunit are

added to form

an initiation

complex.

mRNA in this site.

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.

subunitCytoplasm

A siteP site

E site

Lengthening

polypeptide chain

Emptyt

Cytoplasm

Cut intron

subunit

5′cap

RNA polymerase II

Poly-A tail

5′cap

1. RNA polymerase

II in the nucleus

copies one

strand

of the DNA to

produce the

primary

transcript.

is processed by

addition of a 5′

methyl-G cap,

cleavage and

introns. The mature

mRNA is then

exported through

cytoplasm.

associates with

the small subunit

of the ribosome.

The initiator

tRNA and large

subunit are

added to form

an initiation

complex.

mRNA in this site.

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.

subunitCytoplasm

A siteP site

E site

Lengthening

polypeptide chain

Emptyt

Cut intron

subunit

5′cap

RNA polymerase II

Poly-A tail

5′cap

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 into

E site and is ejected

Cytoplasm

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 codon

Protein

Coding

Template

Pro Thr ArgMet

5′–ATGCCTTATCGCTGA–3′

3′–TACGGAATAGCGACT–5′

5′–AUGCCUUAUCGCUGA–3′

Protein

Coding

Template

Pro Thr ArgMet

Silent Mutation

5′–ATGCCCTATCGCTGA–3′

3′–TACGGGATAGCGACT–5′

5′–AUGCCCUAUCGCUGA–3′

Protein

Coding

Template

Pro Thr HisMet

Missense Mutation

5′–ATGCCCTATCACTGA–3′

3′–TACGGGATAGTGACT–5′

5′–AUGCCCUAUCACUGA–3′

Protein

Coding

Template

ProMet

Nonsense Mutation

5′–ATGCCCTAACGCTGA–3′

3′–TACGGGATTGCGACT–5′

5′–AUGCCCUAACGCUGA–3′

Normal HBB Sequence

Abormal HBB Sequence

Nonpolar (hydrophobic)

Amino acids

Nucleotides

Amino acids

Nucleotides

C C C CGT T TA GG A GAA

Thr Pro Glu Glu

CT TGAA

Lys Ser

C C C CGT T TA GG GAG

Thr Pro val Glu

CTT TGAA

Lys Ser

Normal

Deoxygenated

Tetramer

Abnormal

Deoxygenated

Tetramer

Tetramers form long chains

when deoxygenated. This

distorts the normal red blood

cell shape into a sickle shape.

Hemoglobin

tetramer

"Sticky" non-

polar sites

• 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

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

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 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

Reciprocal Translocation

• 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

Chapter 15mmsalemscienceteacher.weebly.com/uploads/2/3/3/6/... · 2018. 9. 9. · Exon 1 Intron...

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