the molecular biology of genes and gene expression

The Molecular Biology of Genes and Gene Expression

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

2

DNA

RNA

Protein

replication (mutation!)

transcription

translation

(amino acids)

(nucleotides: A,T,G,C)

“software” ~ DNA, RNA

“hardware” ~ proteins

How Genes Work: A PrimerHow Genes Work: A Primer

genes

(nucleotides: A,U,G,C)

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

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5

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

6

• 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

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

• 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

13

• 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

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16

Prokaryotic transcription

• Single RNA polymerase

• Initiation of mRNA synthesis does not require a primer

• Requires– Promoter – Start site Transcription unit– Termination site

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• 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

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Upstream

Downstream

׳5 ׳3

b.

Codingstrand

Templatestrand

׳5׳3

Start site (+1)

TATAAT– Promoter (–10 sequence)

TTGACA–Promoter (–35 sequence)

Holoenzyme

9

a.

Prokaryotic RNA polymerase

Coreenzyme

binds to DNA

dissociates

ATP

Start site RNAsynthesis begins

Transcriptionbubble

RNA polymerase boundto unwound DNA

Helix opens at–10 sequence

׳5 ׳3

׳5׳3

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

• 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

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21

• 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

• 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

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25

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

26

• 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

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Other transcription factors RNA polymerase II

TATA box

Transcriptionfactor

EukaryoticDNA

Initiationcomplex

Enhancers determine the temporal and spatial transcription patterns of genes

(drawing modified from Tijan, R., Molecular Machines That Control Genes, Scientific American 272, Feb, 1995)

Activators = + Auxiliary Transcription FactorsEnhancers

Silencer

Repressor = -auxiliary transcription

factor

Co-activatorsTATA BOX

REPRESSOR

CORE PROMOTER

transcription

Basal Transcription Factors

RNA Polymerase

H

E

F

BA

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• 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

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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

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32


E1 I1 E2 I2 E3 I3 E4 I4

Transcription

Introns are removed

a.

Exons

Introns

Mature mRNA

cap ׳poly-A tail5 ׳33

cap ׳5 poly-A tail ׳3

Primary RNA transcript

DNA template

33


E1 I1 E2 I2 E3 I3 E4 I4

Transcription

Introns are removed

Intron

Exon

DNA

1

2 34

5 67

a.

b. c.

Exons

Introns

mRNA

Mature mRNA

cap ׳poly-A tail5 ׳3

cap ׳5 poly-A tail ׳3


DNA template

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

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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

35


snRNPs

Exon 2Exon 1 Intron

Branch point A

snRNA

Spliceosome

5´

5´

3´

3´

2. snRNPs associate with other factors to form spliceosome.


A

A

36


snRNPs

Exon 2Exon 1 Intron

Branch point A

snRNA

Spliceosome

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.

A

A

A

37


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

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DNADNA

hnRNAhnRNA

mRNA 1mRNA 1

mRNA 2mRNA 2

protein 1protein 1

protein 2protein 2

translationtranslation

translationtranslation

RNA splicingRNA splicing

RNA splicingRNA splicing

Differential RNA splicingcan lead to different protein productsDifferential RNA splicingcan lead to different protein products

5’ ut 3’ utexon 1exon 1 exon 2exon 2 exon 3exon 3intron 1intron 1 intron 2intron 2

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

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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 fi nanced by grant GM63208

2D “Cloverleaf” Model

Acceptor 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 tRNA

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44


PiPi

Aminogroup

Carboxylgroup

Trp

ATP

Aminoacid site

Aminoacyl-tRNAsynthetase

tRNAsite

NH3+ C

O–

O

45


tRNA

PiPi

NH3+

O–

C OC

O CO

OH

OAMP O

OH

AMP

Aminogroup

Carboxylgroup

TrpNH

3+ NH

3+

Trp Trp

ATPAminoacid site

Acceptingsite

Anticodonspecific to tryptophan


tRNAsite

46


tRNA

PiPi

NH3+

O–

C OC OC

O CO

CO

OH

OAMP O

OH

AMP

AMP

O

O

Charged tRNA travels to ribosomeAminogroup

Carboxylgroup

TrpNH

3+ NH

3+

NH3

+

NH3+Trp Trp

TrpATP

Aminoacid site

Acceptingsite

Anticodonspecific to tryptophan


tRNAsite

ChargedtRNA

dissociates

Trp

47

• 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

48


mRNA

90°

0°

3´

5´

Largesubunit

Smallsubunit

Largesubunit

Smallsubunit

Largesubunit

Smallsubunit

49

• 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

50

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

• 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

52

53

• 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

56

• 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

57

• Termination– Elongation continues until the ribosome

encounters a stop codon– Stop codons are recognized by release

factors which release the polypeptide from the ribosome

59

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

61


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´

62



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´

63



Poly-A tail


5´ cap



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´

64


Cut intron

5´ cap





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.


RNA polymerase II


Cut intron

5´ cap

Largesubunit

mRNA 5´ cap


tRNA arrivesin A site Amino acids

mRNA

A siteP site

E site

Cytoplasm

RNA polymerase II


3´

5´

5´

3´

3´

Poly-A tail

Mature mRNA

Poly-A tail

Mature mRNA

65


Poly-A tail


5´ cap






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.


RNA polymerase II


Poly-A tail


5´ cap

Largesubunit

mRNA



mRNA

A siteP site

E site

Lengtheningpolypeptide chain

EmptytRNA

Cytoplasm

RNA polymerase II


3´

5´

5´

3´

3´

5´

3´

5´ cap

66






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.

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.


RNA polymerase II

mRNA



mRNA

A siteP site

E site

Lengtheningpolypeptide chain

EmptytRNA

Empty tRNA moves intoE site and is ejected

Cytoplasm

RNA polymerase II


3´

5´

5´

3´

3´

5´

3´

5´

3´


Poly-A tail


5´ cap


Poly-A tail


5´ cap

Largesubunit

5´ cap

68

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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 codon69

72


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 GAA

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

73

• 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

74

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

77

• 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

the molecular biology of genes and gene expression

Documents

dna rna translation

rna proteinretroviruses

rna genome

rna polymerasefound

rna hardware

dna template

dna nucleotides

site of initiation