T HE N EW T ESTAMENT C HURCH T HE G RANDEUR AND G LORY OF THE C HURCH.
Richard Deem, Paradoxes Class, March 16, 2014. Nucleus A A T T G G C C A A T T A A G G T T G G C C C...
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Transcript of Richard Deem, Paradoxes Class, March 16, 2014. Nucleus A A T T G G C C A A T T A A G G T T G G C C C...
Nucleus
DNA
mRNA
Protein
Transcription
Translation
ER
U
C
A G
Designs in DNARichard Deem,
Paradoxes Class, March 16, 2014
The problem with biology…
…is you need to know things
Nucleus
Central Dogma of Biology
AT
GC
ATAG
TG
C C
A T
T
GC
ATA
TG
C
TA G
C A T CTA
GC
ATA
TG
C G
GC
ATA
DNA
mRNA
Protein
Transcription
Translation
GAGG A U C A CA UU A G
G
U C
AU
A
C
A U
ERG A
G G A U CA
CA
U
UA G
G
U C
AU
A
C
AU
GAG
G A U C A CA UU A G
G
U C
AU
A
C
A U
ChloroplastMitochondrion
Guanine (G)
Adenine (A)
Purines
C
CHC
CN
N N
N
CH
H
NH2
C
CC
CHN
N
O
H2N N
N
CH
H
Thymine (T)
Cytosine (C)
Pyrimidines
C
CHC
CHN
N
H
O
O
CH3
CH
CHC
CN
N
H
NH2
O
DNA Bases
Nucleotide
Structure of Deoxyadenosine
Glycosidic Bond
Adenine (base)C
C CH
C
N
NN
N
HC
NH2
O
OH
OCH2
Sugar (Deoxyribose)
5’
3’
Nucleoside
O- P
O
O-
HC
HC C
CN
N
NH
O
H
OCH2O- P
O
O
O
C
CHC
CN
N N
N
CH
HN
H
O H2C O-P
O
O-
O
O
C
HC C
CNH
N O
H3C
O
OH
H2CO- P
O
O
O
C
C CH
CN
NN
N
HC
NH
H
OCH2O- P
O
O-
O
Adenine
Cytosine
Guanine
Thymine
5’
3’
C
CHC
CHN
N
O
O
CH3
O
OH
H2C O-P
O
O
O
C
C
C
C
NH
N
O
NH
N
NHC
H
OCH2O- P
O
O
O
C C
C
C
HN
N
O
HN
N
NCH
H
O H2C O-P
O
O
O
CH
CHC
CN
N
HN
O
H
O H2C O-P
O
O
O
Adenine
Cytosine
Guanine
Thymine3’
5’
HydrogenBond DNA Structure
A T
G
C
A
TAG
T
GC
C
A
T T
G
C
A
TAT
GC
T
A
GC A T
CTA
G
C
A
TA
TG
C
G
G
C
ATA
DNA Double Helix
Chromosome
Nucleosome
DNA Structure
DNA
Histone H1
4 Histone protein pairs
Human ChromosomesElectron
MicrographKaryotype
Telomere
Centromere
DNA Structure: ChromatinHeterochromatin(condensed DNA)
Euchromatin(actively transcribed DNA)
Nucleus
C
C
C
C
N
N
O
O
CH3
H H
Deoxyribose
C
C
C
C
N
N
O
O
H
H H
Ribose
Bases Found in DNA vs. RNA
Thymine Uracil
DNA RNAAdenine Adenine
Cytosine Cytosine
Guanine Guanine
G A
GG
A U CA
CA
U
UA G
G
U C
AU
A
CA
U
G A
GG
A U CA
CA
U
UA G
G
U C
AU
A
CA
U
Transfer RNA (tRNA)Transfer RNA
Anti-codon
Mesenger RNA (mRNA)
G
A
G
C
U A
U
U
C
GG C
CC
UA
G
C
UC
G
CA U
C
A
C
G
CG
A U
AC G
UA
C
GC
GC
G
C
G
C
GC
GU
A
C
G
A
AU
UCodon
Methionine
Electron Micrograph of Translation Process
Ribosomes Protein chainsmRNA
+
Codon AA Codon AA Codon AA Codon AAUUU Phe UCU
Ser
UAU Tyr UGU CysUUC UCC UAC UGCUUA Leu UCA UAA Stop UGA StopUUG UCG UAG UGG Trp
CUULeu
CCUPro
CAU His CGUArgCUC CCC CAC CGC
CUA CCA CAA Gln CGACUG CCG CAG CGGAUU Ile ACU
ThrAAU Asn AGU SerAUC ACC AAC AGC
AUA Met ACA AAA Lys AGA ArgAUG ACG AAG AGGGUU
ValGCU
AlaGAU Asp GGU
GlyGUC GCC GAC GGCGUA GCA GAA Glu GGAGUG GCG GAG GGG
The Genetic Code
DNA as a Language
• Four “letters” ( bases A, U, G, C)• 64 three letter “words” (codons)• “Redundant” – Many “words” have
the identical “meaning”• 20 unique “words” (amino acids)• Unlimited “sentences” (proteins)
Nucleus
Transcription/Translation
AT
GC
ATAG
TG
C C
A T
T
GC
ATA
TG
C
TA G
C A T CTA
GC
ATA
TG
C G
GC
ATA
DNA
mRNA
Protein
Transcription
Translation
GAGG A U C A CA UU A G
G
U C
AU
A
C
A U
ERG A
G G A U CA
CA
U
UA G
G
U C
AU
A
C
AU
GAG
G A U C A CA UU A G
G
U C
AU
A
C
A U
DNA Design:Alternative Splicing of RNA
Multiple proteins from one gene
DNA
Exons
Genes to Proteins: DNAmRNA
introns (between exons)
5’ 3’mRNA
Translated regionProtein
Transcribed regionPre-mRNA
UTR UTR
Exon5Exon5Exon4Int4Exon4Int3Exon3Exon3Int2Exon2Exon2Int1Exon1Exon1
Alternative Splicing of RNAExon5Exon4Exon2Exon1
Protein isoform A Protein isoform B
mRNA
Pre-mRNA
Types of Alternative Splicing
AS Pattern Type AcronymCassette exon (skipped exon) CEIntron retention IRMutually exclusive exons MXEAlternative 3' sites A3SSAlternative 5' sites A5SSAlternative first exon AFEAlternative last exon ALE
DNA Design: Duons
Overlapping regulatory and protein codes
Promoter region
Transcription Factors
DNA
NFAT Y2 Y1 NFATAP-1 AP-1AP-2
-200 -150 -100-300 -250-6800
NFAT
-800
NFkB
Exons
Genome-Wide Transcription Factor Binding Sites
• Used enzyme DNase I• Digested DNA from 81 different cell
lines• Sequenced and mapped the location
of all TF binding sites
»»»NRSF
«««USF
«««SP1
«««SP1
DNase I cleavageper nucleotide(PLBD2 gene)
Duon Sequences
• 86% of genes expressed at least one duon sequence
• Duons comprise 14% of all exonic coding
• Over 12 million base pairs
Andrew B. Stergachis et al. 2013. Exonic Transcription Factor Binding Directs Codon Choice and Affects Protein Evolution. Science 342, 1367.
Protein Sequence
Example of Duon in DNA
Leu Gln Gln Ile Thr Arg Gly Arg Ser ThrCTGCAGGCCATCACCAGGGGGCGCAGCACC
CCACCAGGGGGCGCA
DNA Sequence
CTCF Binding Sequence
CELSR2 Gene: Chr1:109806358-109806387
Andrew B. Stergachis et al. 2013. Exonic Transcription Factor Binding Directs Codon Choice and Affects Protein Evolution. Science 342, 1367.
Duons are Functional
Andrew B. Stergachis et al. 2013. Exonic Transcription Factor Binding Directs Codon Choice and Affects Protein Evolution. Science 342, 1367.
Firstexon
Internalexons
Finalexon
Non-codingbases
0
1
2
3
4 Dnase I Footprint Density
Dna
se I
foot
prin
ts/k
b p < 10-15
DNA Design: Dual Coding Genes
Multiple proteins from alternative reading frames
Reading Frames
Leu Gln Gln Ile Thr Arg Gly Arg Ser ThrCTGCAGGCCATCACCAGGGGGCGCAGCACC
Cys Arg Pro Ser Pro Gly Gly Ala AlaAla Gly His His Gln Gly Ala Gln His
GACGTCCGGTAGTGGTCCCCCGCGTCGTGGGln Leu Gly Asp Ala Pro Gly Arg Arg Gly
Ala Ala Met Val Leu Pro Arg Leu ValAla Pro TrpStopTrp Pro Ala Cys Cys
Dual-Coding Genes
Coding of multiple proteins by overlapping reading frames is not a feature one would associate with eukaryotic genes. Indeed, codependency between codons of overlapping protein-coding regions imposes a unique set of evolutionary constraints, making it a costly arrangement. Yet in cases of tightly coexpressed interacting proteins, dual coding may be advantageous. Here we show that although dual coding is nearly impossible by chance, a number of human transcripts contain overlapping coding regions.
Wen-Yu Chung, et al. A First Look at ARFome: Dual-Coding Genes in Mammalian Genomes. PLoS Computational Biology 3 (5) e91.
Finding Dual Coding Genes
• Evolutionary assumptions underestimate true numbers of dual coding genes
• 9% of human and 7% of mouse• Less than 30% shared: mouse:human• 90% of genes on opposite strands• 1259 human alternative proteins
detected by mass spectrometryChaitanya R Sanna, et al. Overlapping genes in the human and mouse genomes. BMC Genomics 2008, 9:169.
Benoıt Vanderperre, et al. 2013. Direct Detection of Alternative Open Reading Frames Translation… PLoS ONE 8(8): e70698.
“Sentences” from Two Directions
• A man, a plan, a canal: Panama• Live not on evil• Was it a car or a cat I saw?
Dual Coding Gene: EIF6 (ITGB4BP)
GACAGAAGAAATTCTGGCAGATGTGCTCAAGGTGGAAGTCTTCAGACAGACAGTGGC GAC CCAGGTGCTAGTAGGAAGCTACTGTGTCTTCAGCAATCALeu GlnGln Thr Asp GlySerArgSer Ser Gly Ala Leu GlnGly SerGlnGlyAsp Asn ProGly ArgArgAla CysArg Leu SerLys LeuCysArg
Frame 1108 aa
Han Liang and Laura F. Landweber. 2006. A genome-wide study of dual coding regions in human alternatively spliced genes. Genome Research 16:190–196.
Frame 2226 aa
2851824313486107566
27510182177312566
Phe AsnGln Gln Thr ValValAspLeu Val Ala Leu Tyr SerVal ValArgAlaThr Ile GlnGlu GlyAspLeu ValGlu Phe GlnSer CysValGlu
Dual Coding Gene: Ncaph2
Pro GluHis ArgAsp TrpGlnArgGlu LeuThr Glu Ala GlyLeu ValIleValMet Val AsnLeu AspLysAla GlnGlu Leu GluVal AlaPheAsp
Angelo Theodoratos, et al. Splice variants of the condensin II gene Ncaph2 include alternative reading frame... FEBS Journal 279 (2012) 1422–1432.
Exon 2Long
GlyCys LeuArg GlySerLeuAla ThrGly Arg ArgSer TrpIleLeuMet Cys ThrPro ThrArgSer SerTrp TrpTrp HisThrArg
Exon 2ShortGluMet ValGluAsp
Exon 2IntermediateLeu IleAsp Gln
Leu IleAsp Gln
Leu IleAsp Gln
Exon 1 Alternative Transcripts
Alternative Reading Frame
Protein Products of Ncaph2
50 bp
200 bpLa
dder
Thym
us
Mus
cle
Brain
Bone
Mar
row
Kidney
Testi
s
Heart
Spleen
Liver
Lung
Int 215 bpLong 232 bp
Short 140 bp
Conclusions
• At least three independent examples of design in DNA–Alternative splicing producing multiple
proteins from one gene–Duons–overlapping sequences of
coding and transcription factor binding–Dual coding genes