Nucleic Acids and Protein Synthesis. What are nucleic acids?
Chemistry 19.2-Nucleic-acids-–-part-2
Transcript of Chemistry 19.2-Nucleic-acids-–-part-2
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Chapter 19 (part 2)
Nucleic Acids
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DNA• 1o Structure - Linear array of
nucleotides• 2o Structure – double helix• 3o Structure - Super-coiling,
stem-loop formation• 4o Structure – Packaging into
chromatin
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Determination of the DNA 1o Structure (DNA Sequencing)• Can determine the sequence of
DNA base pairs in any DNA molecule
• Chain-termination method developed by Sanger
• Involves in vitro replication of target DNA
• Technology led to the sequencing of the human genome
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DNA Replication• DNA is a double-helical molecule • Each strand of the helix must be
copied in complementary fashion by DNA polymerase
• Each strand is a template for copying • DNA polymerase requires template
and primer • Primer: an oligonucleotide that pairs
with the end of the template molecule to form dsDNA
• DNA polymerases add nucleotides in 5'-3' direction
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Chain Termination Method
• Based on DNA polymerase reaction • 4 separate rxns• Each reaction mixture contains dATP,
dGTP, dCTP and dTTP• Each reaction also contains a small
amount of one dideoxynucleotide (ddATP, ddGTP, ddCTP and ddTTP).
• Each of the 4 dideoxynucleotides are labeled with a different fluorescent dye.
• Dideoxynucleotides missing 3’-OH group. Once incorporated into the DNA chain, chain elongation stops)
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Chain Termination Method• Most of the time, the polymerase
uses normal nucleotides and DNA molecules grow normally
• Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule
• Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide
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N
NN
N
NH2
O
H
HH
HH
NH
N
N
O
NH2N
O
H
HH
HHO
PO
O
HO
O-
N
NN
N
NH2
O
HO
HH
HH
PO
O
O-
NH
N
N
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NH2N
O
H
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HHO
PO
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O-
NH
N
N
O
NH2N
O
H
HH
HHOH
OH
OH
PHO
O
O-
NH
N
N
O
NH2N
O
H
HH
HHOH
OH
PO
O
PO
O
O-
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N
NN
N
NH2
O
H
OH
HH
HH
PHO
O
O-
NH
N
N
O
NH2N
O
H
HH
HHO
PO
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NH
N
N
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NH2N
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NO CHAIN ELONGATION
OH
PO
O
PO
O
O-
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Chain Termination Method
• Run each reaction mixture on electrophoresis gel
• Short fragments go to bottom, long fragments on top
• Read the "sequence" from bottom of gel to top
• Convert this "sequence" to the complementary sequence
• Now read from the other end and you have the sequence you wanted - read 5' to 3'
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DNA Secondary structure
• DNA is double stranded with antiparallel strands
• Right hand double helix• Three different helical forms
(A, B and Z DNA.
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Comparison of A, B, Z DNA
• A: right-handed, short and broad, 2.3 A, 11 bp per turn
• B: right-handed, longer, thinner, 3.32 A, 10 bp per turn
• Z: left-handed, longest, thinnest, 3.8 A, 12 bp per turn
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A-DNA B-DNA Z-DNA
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Z-DNA• Found in
G:C-rich regions of DNA
• G goes to syn conformation
• C stays anti but whole C nucleoside (base and sugar) flips 180 degrees
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DNA sequence Determines Melting Point
• Double Strand DNA can be denatured by heat (get strand separation)
• Can determine degree of denturation by measuring absorbance at 260 nm.
• Conjugated double bonds in bases absorb light at 260 nm.
• Base stacking causes less absorbance.
• Increased single strandedness causes increase in absorbance
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DNA sequence Determines Melting Point
• Melting temperature related to G:C and A:T content.
• 3 H-bonds of G:C pair require higher temperatures to denture than 2 H-bonds of A:T pair.
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DNA 3o Structure•Super coiling •Cruciform structures
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Supercoils• In duplex DNA, ten bp per turn of helix
(relaxed form)• DNA helix can be over-wound.• Over winding of DNA helix can be
compensated by supercoiling.• Supercoiling prevalent in circular DNA
molecules and within local regions of long linear DNA strands
• Enzymes called topoisomerases or gyrases can introduce or remove supercoils
• In vivo most DNA is negatively supercoiled.• Therefore, it is easy to unwind short
regions of the molecule to allow access for enzymes
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Each super coil compensates for one + or – turn of the double helix
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•Cruciforms occur in palindromic regions of DNA •Can form intrachain base pairing•Negative supercoiling may promote cruciforms
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DNA and Nanotechnology
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DNA and Nanotechnology
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DNA 4o Structure• In chromosomes, DNA is tightly
associated with proteins
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Chromosome Structure• Human DNA’s total length is ~2
meters!• This must be packaged into a
nucleus that is about 5 micrometers in diameter
• This represents a compression of more than 100,000!
• It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments
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Nucleosome Structure• Chromatin, the nucleoprotein
complex, consists of histones and nonhistone chromosomal proteins
• % major histone proteins: H1, H2A, H2B, H3 and H4
• Histone octamers are major part of the “protein spools”
• Nonhistone proteins are regulators of gene expression
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•4 major histone (H2A, H2B, H3, H4) proteins for octomer•200 base pair long DNA strand winds around the octomer•146 base pair DNA “spacer separates individual nucleosomes•H1 protein involved in higher-order chromatin structure.•W/O H1, Chromatin looks like beads on string
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Solenoid Structure of Chromatin
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RNA• Single stranded molecule• Chemically less stable than DNA• presence of 2’-OH makes RNA more
susceptible to hydrolytic attack (especially form bases)
• Prone to degradation by Ribonucleases (Rnases)
• Has secondary structure. Can form intrachain base pairing (i.e.cruciform structures).
• Multiple functions
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Type of RNA• Ribosomal RNA (rRNA) – integral part
of ribosomes (very abundant)• Transfer RNA (tRNA) – carries
activated amino acids to ribosomes.• Messenger RNA (mRNA) – endcodes
sequences of amino acids in proteins.• Catalytic RNA (Ribozymes) – catalzye
cleavage of specific RNA species.
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RNA can have extensive 2o structure