MBB 407/511

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Nov. 5, 2004
MBB 407/511 Lecture 17 Structures of DNA and
RNA (Part II)
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6Å
3.4Å
34Å
DNA is1) double stranded 2) a
right-handed helix
Helix vs Spiral
Merriam Websters Collegiate Dictionary
Helix (n)a. Something spiral in form. b.
A coil formed by winding wire around a uniform
tube. c. A curve traced on a cylinder
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Examples of helices
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Hydrogen Bonds Hold the Two DNA Strands Together
What are hydrogen bonds?
Notice that
A-T bp have 2 H bonds G-C bp have 3 H bonds ?G-C
bp are more stable
Helix has constant diameter
Why?
1) Purines always pair with pyrimidines
2) The H bonding distance between the A-T and G-C
bp is the same
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DNA Strands are Antiparallel
Why?
1. Strands must run anti-parallel in order to
get H bonding
Phosphodiester bonds link adjacent nucleotides
Phosphomonoester bond
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The Double Helix Has Alternating Major and Minor
Grooves
Why?
Unequal offsetting of the two strands
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How is DNA Synthesized?
Requires dNTPs
Why?
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DNA can exist in 3 different 3-dimensional forms
B-DNA
Base pair spacing 3.4Å/bp
10.5 bp/helix repeat
36 bp
Helix diameter 20Å
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How to Detect DNA and RNA
Nucleic acids absorb UV light Why?
A spectrophotometer
Determine nucleic acid
For dsDNA 1 A260 unit 50 mg/ml of DNA
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Double stranded DNA denatures at high temperature
UV light absorbance increases as DNA goes from
double-stranded to single-stranded
Hyperchromic shift
Melting temperature (Tm)
What is the basis of hyperchromic shift? Stacked
bases absorb less UV light
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Melting temperature Tm increases with GC content
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Cot Curve Analysis of DNA Samples
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Visualizing DNA by Ethidium Bromide Staining
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Nucleic Acids Can Be Precipitated
1. Salt (0.3M NaOAc, 0.5M NH4OAc, 0.4 M LiCl)
Monovalent cations neutralize the
repulsive effects of the phosphate groups
2. Alcohol (70 Ethanol or 50 isopropanol)
To concentrate DNA/RNA by exluding H20
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The Effects of Alkali on DNA and RNA
Alkali hydrolyzes RNA
Alkali denatures dsDNA
The 2OH groups are susceptible to nucleophilic
attack
At high pH, deprotonation of bases disrupts
hydrogen bonding
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Q Does RNA have a secondary structure?
RNA is single stranded and is most stable when
it Engages in intrastrand base pairing
A tRNA
16S rRNA
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How about the 3-D structure of RNAs?
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Roles of DNAs and RNAs
DNA
RNA
Messenger RNAs (mRNAs)
Transfer RNAs (tRNAs)
Ribosomal RNAs (rRNAs)
Small nuclear RNAs (snRNAs)
Catalytic RNAs (Ribozymes) self-splicing
(e.g., group I and group II introns) cleavage
of other RNAs (e.g., RNA component of RNaseP
cleaves tRNAs) hammerhead RNAs (
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Examples of catalytic RNAs
Group II intron (self-splicing)
RNaseP cleavage of tRNA 5 ends
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Hammerhead ribozyme
Ribozymes catalyze 1. Cleavage reactions 2.
Transesterification reactions
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How come only RNAs be catalytic?
They are single-stranded
can adopt complex 3-D structures
They have 2 OH groups
highly reactive
Newest ribozymes the snRNAs in the spliceosme
the 23S rRNA in ribosomes
What are the implications?
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DNA and RNA are Degraded by Nucleases
(phosphodiesterases)
Exonucleases
5?3 exo and 3 ?5 exo
Endonucleases
cut internally

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DNAs are more stable than RNAs
DNA is very stable it stores the genetic
information
There arent many DNases in the cell.
DNA is protected.
RNA is very unstable its turnover is
important for regulating gene expression
Lots of RNases in the cell. RNAs generally not
protected.
Some RNAs more stable than others
rRNAs and tRNA aka stable RNAs
mRNAs have half-lives from minutes to hours
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Answer to last lectures question Why is
thymine found only in DNA, uracil only in RNA?
Deamination of cytosine in DNA is very common in
the cell, but can lead to mutations after DNA
replication.
deamination
CG
UG
DNA replication
DNA replication
CG
UT
CG

?There is a repair system that looks for uracil
attached to deoxyribose sugars and excises the
base then replaces the uracil with a cytosine.
If uracil was a normal base in DNA, the cell
might not detect the UG bps and there would be
too many CG to UT mutations.