DNA, RNA,

1
DNA, RNA, Flow of Genetic Information
DNA RNA are long linear polymers, called
nucleic acids. Genetic information is stored in
a sequence of 4 kinds of bases along the chain,
and is passed from one generation to the next
Chapter 5 Outline
5.1 A nucleic acid consists of 4 kinds of bases
linked to a sugar-phosphate backbone 5.2 A
pair of nucleic acid chains with complimentary
sequences can form a double-helical
structure 5.3 DNA is replicated by polymerases
that take instructions from templates 5.4
Gene expression is the transformation of DNA
information into functional molecules 5.5
Amino acids are encoded by groups of three bases
starting from a fixed point 5.6 Most
eucaryotic genes are mosaics of introns exons
2
Polymeric structure of nucleic acids
Linear polymers of covalent structures, built
from similar units
Sequence of bases uniquely characterizes nucleic
acids Represents a form of linear information
Backbone is constant repeating units of
sugar-phosphate
3
Different pentose sugars in RNA DNA
RNA
Sugar carbons have prime numbers, to distinguish
them from atoms in bases
DNA
4
Backbone of DNA RNA
3-to-5 phosphodiester linkages
Sugar, red. Phosphate, blue
5
Purines Pyrimidines
DNA
Note ring atom s
RNA
6
Sugar - base linkage
Base above plane of sugar, linkage is ?
Nucleoside
RNA adenosine, guanosine, cytidine,
uridine DNA deoxyadenosine, deoxyguanosine,
deoxycytidine, thymidine
7
Nucleotides monomeric units of nucleic acids
Deoxyguanosine 3 monophosphate
Adenosine 5-triphosphate
5 nucleotide - most common 3
nucleotide
Nucleotide nucleoside joined to one or more
phosphate groups by an ester linkage
8
Adenosine 5-triphosphate
Adenosine linked to sugar C1
Triphosphate linked to sugar C5
9
Deoxyguanosine 3-monophosphate
10
Structure of DNA chain
5 end, phosphate attached
3 end, free hydroxyl group
11
EM - part of E. coli genome
We can quantify DNA information Each position
has 1 of 4 bases 2 bits of (binary) information
(22 4) Thus, 5100 nucleotides of polyoma DNA
is 2 x 5100 10,200 bits, or 1275 bytes (1 byte
8 bits) E. coli genome has 4.6 million
nucleotides 9.2 million bits or 1.15 megabytes
of information
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Indian muntjak (Asiatic deer)- 3 chromosomes
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3 pairs of chromosomes - Indian muntjak
Genome, nearly as large as human Pair of
human chromosomes green
14
X-ray diffraction of DNA hydrated fiber
Shows double-helix structure
Meridian arcs - stack of nucleotide bases, 3.4 A
apart
Central X - indicates helical structure
R. Franklin M. Wilkins photograph
15
Watson-Crick model - DNA double helix
Axial view
Features
Bases separated by 3.4 Å 10 bases /
turn Rotation 36 degrees / base Helix pitch
34 Å Helix diameter 20 Å
Two helical polynucleotide chains, coiled around
common axis, run in opposite directions Sugar-pho
sphate backbones outside, bases inside Bases
nearly perpendicular to helix axis
16
DNA double helix - radial view
Looking down the helix axis
17
Watson and Crick base pairs
Essentially the same shape
18
A T G C ratios
1950, Erwin Chargaff found AT and GC ratios
nearly the same in all species studied
consistent with Watson-Crick base pairing
19
Axial view of DNA
Base pairs stacked on top of one-another, contrib
utes stability to double helix in 2 ways Base
attraction van der Walls forces Hydrophobic
effect of base stacking, exposure of
polar surfaces to surrounding water
20
14N DNA 15N DNA
1958, Meselson Stahl experiment, resolution by
density gradient centrifugation. UV absorption
photo of cell, 2 distinct DNA bands
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Densitometer tracing of UV photograph
22
Semiconservative replication of DNA
Generation
Position of DNA band depends on its content of
14N and 15N
100 15N
0
1
50 15N 14N in hybrid helix
2 bands, 14N(100) hybrid helix
1.9
UV absorption photo of DNA density gradients, DNA
replicated in E. coli
23
Densitometer tracing of UV photographs
100 15N in both strands
50 15N 14N in hybrid helix
2 bands, 14N(100) hybrid helix
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Semiconservative replication of DNA
Parental DNA, blue
Newly synthesized DNA, red
25
Hypochromism of DNA
Used to detect separation of single strands, DNA
melting
26
DNA melting
At Tm ,50 of helix is separated Below Tm, DNA
is renatured or annealed Separation by by
helicases inside cells
27
EM of circular DNA, mitochondria
Relaxed form
28
EM of circular DNA, mitochondria
Supercoiled form
29
Single stranded nucleic acids elaborate
structures
Stem loop structures
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DNA stem loop
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RNA stem loop
32
RNA complex structure
Base pairing loops
Long-rang interaction
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Long-range interaction
W C base pairing, dashed black lines Other base
pairing, dashed green lines
34
DNA polymerization reaction
By DNA polymerase
Step by step addition of deoxyribonucleotide
units to a DNA chain
New DNA chain assembled directly on a preexisting
DNA template
Primer template required
Activated precursors required dATP, dGTP, TTP,
dCTP
Also required Mg2 ion
35
DNA replication, phosphodiester bridge
Nucleophilic attack by 3 -hydroxyl group of
primer on innermost phosphorus atom of
deoxynucleotide triphosphate (dNTP)
Elongation proceeds, 5 -to- 3
Hydrolysis of pyrophosphate (PPi) helps drive
polymerization
36
Retroviruses reverse flow of information
Reverse transcriptase brought into cell by the
virus (eg. HIV-1)
ssRNA genome
Incorporated into host DNA
37
Roles of RNA in gene expression
Messenger RNA template for translation (protein
synthesis) Transfer RNA carriers of activated
AAs to ribosomes (at least one kind for each of
20 AAs) Ribosomal RNA major component of
ribosomes (play structural and catalytic roles)
38
RNA polymerase
claw shape to hold DNA to be transcribed
Mg2 ion at active site
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Transcription reaction - RNA polymerase
Nucleophilic attack by 3 hydroxyl group
Requirements a template, activated precursors
(NTPs), Divalent metal ion, Mg2 or Mn2
40
RNA polymerase instructions from DNA templates
41
mRNA DNA complementarity
mRNA sequence is the compliment of that of the
DNA template is the same as that of the coding
DNA strand, except for T in place of U
42
Prokaryotic promoter
Consensus sequences centered at -10, -35
Promoter sites specifically binds RNA polymerase,
determine where transcription begins
43
Eukaryotic promoter
Consensus sequences centered at -25 -75
Eukaryotic promoters are further stimulated
by enhancer sequences (can be at a distance of
several kb from start site on either its 5 or 3
side
44
Termination signal in E. coli
Sequence at 3 end of mRNA Hairpin
loop followed by a string of uridines (U)
Alternatively, transcription ended by action of
Rho protein
45
mRNA modification in eukaryotes
(Less known about transcription termination in
eukaryotes)
mRNA is modified after transcription
A cap structure is attached to 5 end a
sequence of adenylates, the poly(A) tail, is
added to the 3 end
46
Amino acid attached to tRNA
Amino acid esterified to 3-hydroxyl group of
terminal adenosine of tRNA Amino acid is in an
activated form Whole molecule is, aminoacyl -
tRNA
47
Aminoacyl - tRNA, symbolic diagram
1961, Crick Brenner, the genetic code 3
nucleotides encode an amino acid, Code is
nonoverlapping, Code has no punctuation, Code
is degenerate, (some AAs encoded by more than one
codon)
The anticodon is the template-recognition site
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Genetic code, nonoverlapping
49
Genetic code, no punctuation
Sequence of bases is read in blocks of 3 bases
from a fixed starting point
50
Genetic code, degenerate (64 codons, 20 aas)
Trp Met, one codon each, other 18 aas, two
or more codons, Leu, Arg, Ser, six codons
each, Synonyms, codons for same aa, Synonyms
differ in last base, 3 stop codons, designate
translation termination
51
Translation initiation start codon
Messenger RNA is translated into proteins on
ribosomes, large molecular complexes of proteins
ribosomal RNA
Initiator tRNA carries fMet (formylmethionine)
to AUG ( sometimes GUG) in prokaryotes, but
initiation is more complex
52
Prokaryotic translation start
53
Eukaryotic translation start
54
Genitic code, universal, except
Nearly but not absolutely universal
Ciliated protozoa read UAA UAG as codons for
aas instead of stop signals. UGA is their only
stop
55
Eukaryotic genes mosaic of introns exons
Introns (intervening sequences), brown
Exons (expressed sequences), blue
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Detecting introns by EM
mRNA hybridized to corresponding genomic
DNA Single loop indicates gene is continuous
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Detecting introns by EM
Two loops of ssDNA (blue), a loop of dsDNA
(blue green) indicate an intron is present
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Transcription processing beta-globin gene
59
Splicing at consensus sequences
Introns excised by spliceosomes (assemblies of
proteins small RNAs)
60
Exon shuffling
Shuffling expands genetic repertoire
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Alternative splicing, 1st variation
62
Alternative splicing, 2nd variation