the molecular biology of genes and gene expression
TRANSCRIPT
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
4
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
7
8
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
12
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
14
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
15
16
Prokaryotic transcription
• Single RNA polymerase
• Initiation of mRNA synthesis does not require a primer
• Requires– Promoter – Start site Transcription unit– Termination site
17
• 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
18
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
19
20
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
22
• 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
23
24
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
27
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
29
• 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
30
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
31
32
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Primary RNA transcript
DNA template
b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine
34
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
snRNPs
Exon 2Exon 1 Intron
Branch point A
snRNA
Spliceosome
5´
5´
3´
3´
2. snRNPs associate with other factors to form spliceosome.
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
A
A
36
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
snRNPs
Exon 2Exon 1 Intron
Branch point A
snRNA
Spliceosome
Lariat
5´
5´
3´
3´
5´ 3´
2. snRNPs associate with other factors to form spliceosome.
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
snRNPs
Exon 2Exon 1 Intron
Branch point A
snRNA
Exon 1 Exon 2
Lariat
5´
5´
3´
3´
5´
5´
3´
3´
2. snRNPs associate with other factors to form spliceosome.
4. Exons are joined; spliceosome disassembles.
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
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
38
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
40
41
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
42
43
44
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PiPi
Aminogroup
Carboxylgroup
Trp
ATP
Aminoacid site
Aminoacyl-tRNAsynthetase
tRNAsite
NH3+ C
O–
O
45
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Aminoacyl-tRNAsynthetase
tRNAsite
46
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Aminoacyl-tRNAsynthetase
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
51
• 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
54
55
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
58
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
60
61
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript.
Primary RNA transcript
RNA polymerase IIRNA polymerase II
Primary RNA transcript5´
3´
5´
3´
62
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Primary RNA transcript
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.
Primary RNA transcript
RNA polymerase II
Primary RNA transcript
Poly-A tail
Mature mRNACut intron
5´ cap
RNA polymerase II
Primary RNA transcript5´
3´
5´
3´
63
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Primary RNA transcript
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.
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.
Primary RNA transcript
RNA polymerase II
Primary RNA transcript
Poly-A tail
Mature mRNACut intron
5´ cap
Largesubunit
mRNA 5´ cap
Smallsubunit Cytoplasm
RNA polymerase II
Primary RNA transcript5´
3´
5´
3´
64
Primary RNA transcript
Cut intron
5´ cap
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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.
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.
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.
Primary RNA transcript
RNA polymerase II
Primary RNA transcript
Cut intron
5´ cap
Largesubunit
mRNA 5´ cap
Smallsubunit Cytoplasm
tRNA arrivesin A site Amino acids
mRNA
A siteP site
E site
Cytoplasm
RNA polymerase II
Primary RNA transcript5´
3´
5´
5´
3´
3´
Poly-A tail
Mature mRNA
Poly-A tail
Mature mRNA
65
Primary RNA transcript
Poly-A tail
Mature mRNACut intron
5´ cap
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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.
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.
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.
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.
Primary RNA transcript
RNA polymerase II
Primary RNA transcript
Poly-A tail
Mature mRNACut intron
5´ cap
Largesubunit
mRNA
Smallsubunit Cytoplasm
tRNA arrivesin A site Amino acids
mRNA
A siteP site
E site
Lengtheningpolypeptide chain
EmptytRNA
Cytoplasm
RNA polymerase II
Primary RNA transcript5´
3´
5´
5´
3´
3´
5´
3´
5´ cap
66
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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.
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.
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.
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.
Primary RNA transcript
RNA polymerase II
mRNA
Smallsubunit Cytoplasm
tRNA arrivesin A site Amino acids
mRNA
A siteP site
E site
Lengtheningpolypeptide chain
EmptytRNA
Empty tRNA moves intoE site and is ejected
Cytoplasm
RNA polymerase II
Primary RNA transcript5´
3´
5´
5´
3´
3´
5´
3´
5´
3´
Primary RNA transcript
Poly-A tail
Mature mRNACut intron
5´ cap
Primary RNA transcript
Poly-A tail
Mature mRNACut intron
5´ cap
Largesubunit
5´ cap
67
68
Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer.
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
70
71
72
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
75
76
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