Bioorganic Chemistry

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Bioorganic Chemistry

• Part I.
• "Overview of Biopolymers and theTransfer of
  Genetic Information."

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The Central Dogma
DNA
Protein
RNA
Images adapted from RCSB PDB molecule of the
month http//www.rcsb.org
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The Central Dogma
DNA
Protein
RNA

• Post-translational modification
• Intiens
• Stability/proteolysis

• DNA modification
• Histone code
• Recombianation

• Catalytic RNA
• RNAi
• Translation control
• Non-standard bases

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Guanosine
Adenosine
Thymidine
Cytosine
5

• The ends of DNA are labeled as 3 or 5 to
  designate the free hydroxyl.
• DNA lines up in an antiparallel fashion
• Base pairing defined as A-T, CG,

Images adapted from RCSB PDB molecule of the
month http//www.rcsb.org
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DNA polymerase templated synthesis

• Note Synthesis is 5 ?3
• DNA Polymerase requires a primer

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Why do we care what the mechanism is?

• 3?5 Synthesis makes DNA replication is much
  more complicated.
• The need for primers requires a variety of
  auxillary proteins (DNA primase and Pol I
  exonuclease, DNA ligase)
• PCR requires the use of primers. Practically
  speaking we will need to know the sequence of DNA
  (or at least the ends) to amplify or sequence it
  by PCR!

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Thermus Aquatidcus DNAPolymerase
E. Coli DNAPolymerase
From RCSB Protein Databank, Molecule of the Month
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5

• 5 to 3 synthesis requires that the lagging
  strand be synthesized discontinuously. (Okazaki
  fragments)
• DNA synthesis requires RNA primers (red)

Direction of Replicaiton
New Leading Strand
3
New lagging strand
5
5
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Life, the Science of Biology, by Purves, Orians,
Heller, 5th ed., 1997
Adapted from http//www.uccs.edu/rmelamed/MicroFa
ll2002/
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Packaging of DNA

• NOT just for packaging any more!
• Play an integral role in transcription control.
• Must be manipulated during DNA replication/Cell
  cylce progression

Nucleasome
Images adapted from RCSB PDB molecule of the
month http//www.rcsb.org
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Chromatin Structure
http//sgi.bls.umkc.edu/waterborg/chromat/chroma09
.html
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The Central Dogma
You are here
DNA
Protein
RNA
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Transcription

• No primer required
• Promotor brings in many sequences
• All have TATTA box/TATTA binding protein.
• Polymerase is one of many proteins
• Chromatin remodeling
• Ubiquitination/degredation
• DNA not rigid/linear.

TAFs
Pol II
transcription
TBP


TATTA
Gene
Transcription Start Site
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Transcription DNA?RNA
Template Strand.
Uridine
Thymidine
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DNA
3
5
3
5
Sense strand
5
3
RNA
5
3
Coding Strand
5
RNA Polymerase (RCSB PDB)
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The Structural Differences between RNA and DNA
are subtle but significantly effect secondary
structure and stability. RNA has an
intramolecular nucleophile to catalyze
hydrolysis.
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It is possible to connect RNA by 3-5 phosphates
or by 2-5 phosphodiester likages
Interferon is linked 2-5!
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RNA secondary structures
The Ribose twist adopted by RNA ribose is
different from that of DNA RNA is not able to
adopt long extended regular helices like DNA.
From Blackburn and Gait, Nucleic Acids in
Chemistry and Biology 2nd ed.
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As a result of RNAs unique (different from DNA)
conformational preferences and by virtue of the
fact that it is usually found as a single
stranded species, RNA can adopt complex
topological structures.
Some of these seconday structures can be quite
complex.
From Blackburn and Gait, Nucleic Acids in
Chemistry and Biology 2nd ed.
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Types of RNA

• mRNA Messenger RNA carries the DNA-based
  information to the ribosome for protein
  synthesis.
• tRNA transfer RNA carries the amino acid to the
  ribosome. The anti-codon loop of the tRNA
  matches the mRNA codon with the correct amino
  acid.
• rRNA ribosomal RNAs are integral parts of the
  synthesizing machinery the ribo in ribosome.
• snRNA Small nuclear RNA are involved in several
  processes associated with m-RNA processing.

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RNA is difficult to sequence Most common way to
find the sequence of RNA is to convert it
(reverse transcribe it) to DNA with
reversetranscriptase and to perform DNA
sequencing. ? however, many mRNAs undergo
post-translational modifications (eg. Splicing of
introns and exons) therefore reverse
transcription generally yields cDNA
(complementary DNA) ? Many RNAs, especially tRNAs
are composed of modified base pairs.
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Some of the more than 80 modified bases found in
tRNA.
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5' - AGGUAAGU intron YNCUR AC Y nNAGG
- 3'
Figures from http//www.blc.arizona.edu/marty/411/
411access.html
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Lariat
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Catalytic degradation of mRNA.
The hammer head ribozyme is part of a
self-cleaving intron. Conserved regions shown
in boxes
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Antisense RNA can be used to catalytically target
any mRNA with a GU sequence. Now you only need
to make one antisense molecule to wipe-out many
mRNAs
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Anti-sense technology
Antisense RNA complements DNA to block
transcription
DNA
mRNA
Anti-sense
Sense
Antisense RNA complements mRNA to block
translation.
Protein
This is a powerful tool to learn the function of
specific genes and to validate drug targets.
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New RNAi
Shuey, D.J., McCallus, D.E. and Giordano, T..
(2002). Drug Discovery Today 7. Pg 1040.
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Practical Applications
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The Central Dogma
You are here
DNA
Protein
RNA
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The Ribosome 3 RNA fragments 31Proteins ITS
BIG
2.6 million daltons!
Grey RNA Gold protein
Structure of the 5 half of the large ribosomal
subunit.
Structure by T. Steitz and P. Moore (Yale)
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http//www.rcsb.org/pdb/
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(No Transcript)
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-A-U-G - C-C-U - U-A-C - C-C-G -
A-U-C-C-C-U-
mRNA
-A-U-G - C-C-U - U-A-C - C-C-G - A-U-C -
C-C-U-
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(No Transcript)
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Three tRNAs in modeled in the core of the ribosome
The peptide tunnel Exit -gt
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(No Transcript)
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Post-transcriptional/Translational modifications

• Many non-mRNAs are contain modified (non-standard
  base pairs)
• Many some organisms post-translationally splice
  their proteins (inteins)
• Some organisms use more than 20 amino acids.
• Many proteins are post-translationally modified
• Phosphorylation
• Glycosylation
• Farasylation Geranylation
• Sulfurylation/sulfation

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Inteins (Prokaryotic)
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S. Kent Native Chemical Ligation Reaction of
thioester A with peptide B is highly selective.
Why does molecule A only react here?

Rxn is run at pH 7.4
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T. Muir Expressed Chemical Ligation with inteins
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(No Transcript)
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Tools of the trade A brief guide to manipulation
of genetic information
DNA
Protein
RNA
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Enzymatic manipulation of DNA.
Note template directed RNA synthesis is
similar--REQUIRES A PRIMER!
Replication/Extension
Primer
Nucleotide triphosphates
RNA?cDNA (reverse transcriptase)
DNA?RNA
No Primer
No Reaction


Nucleotide triphosphates
Exonuclease
Nucleotide monophosphates
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Restriction Endonuclease
Most Restriction Endonucleases cut palindromic
sites with to leave an overhang
Bam H1 restriction site
5 ... GGA T C C ... 3 3 ... C C T A GG ...
5
overhang
blunt end
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Ligation
over hang
Ligase
P
ATP
P
DNA must have compatible ends and 3-phosphates
If your DNA lacks 5 phosphates, you can add one
with polynucleotide kinase (PNK)
P
ATP
Blunt ends can also be ligated
P
ATP
P
Non-compatible ends can be made blunt by
restricting or filling
P
P
mung bean nuclease
Klenow Polym.
P
P
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DNA Sequencing
Primer
Nucleotide triphosphates
dideoxynucleotide triphosphate ddNTP
DNTPs are used by polymerases but DNA synthesis
stops at this base and cannot continue!
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dATP dGTP ddCTP dTTP dCTP
Primer
Polymerase
ATCCTAATTACGTTGC
ATCCTAATTACGTTGC
Run Gel
ATCCTAATTACGTTGC
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RUN four Separate RXNs
dATP dGTP ddATP dTTP dCTP
Polymerase
ATCCTAATTACGTTGC
G
A
T
C
dATP dGTP ddGTP dTTP dCTP
ATCCTAATTACGTTGC
Polymerase
dATP dGTP ddTTP dTTP dCTP
ATCCTAATTACGTTGC
Polymerase
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A dye labeled dideoxynucleotide triphosphate
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(No Transcript)
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Dont you wish that you could amplify synthetic
organic molecules this easily!
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The Central Dogma
DNA
Protein
RNA