biochemistry i (che 418 / 5418 reading assignment berg et. al (2012) chapter 4 β
TRANSCRIPT
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Biochemistry I(CHE 418 / 5418
Reading Assignment
Berg et. al (2012) Chapter 4
β
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Central Dogma of Molecular Biology
DNA RNA Protein mRNA. tRNA, rRNA, snRNA Nucleus
– Replication - DNA directed DNA synthesis
– Transcription - DNA directed RNA synthesis· Processing of mRNA capping, polyadenylation, splicing
• Cytoplasm– Translation - RNA directed Protein synthesis
Transcription Translation
Replication
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Functions of Nucleic Acids
Building blocks of DNA and RNA– DNA = Genetic material– RNA = Adapter molecule between DNA and protein
Transport chemical energy within the cell– ATP
Signal molecule cyclic AMP
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Nucleic Acids
• Nucleic Acid - linear, non-branched polymer of nucleotides
Classes of Nucleic Acids– RNA = ribonucleic acid– DNA = 2' deoxyribonucleic acid
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Nucleotide• Nucleotide contains:
– Pentose sugar– Nitrogenous base– Phosphate
– One or more
Adenosine Triphosphate (ATP)
N
NN
N
NH2
O
OHOH
HH
HH
OPO
O-
O
PO
O-
O
P-O
O-
O
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Nucleotide
• Pentose sugar– Carbons are numbered with primes to differentiate between
carbons / nitrogens of nitrogenous bases
β-D-2-DeoxyriboseDNA Only
RiboseRNA Only
OH
3' 2'
1'O
4'
OHOH
HHH
5'
H
HOOH
3' 2'
1'O
4'
HOH
HHH
5'
H
HO
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Nucleotide
• phosphate group
O-P-O
O-
O
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Pyrimidine Bases
Cytosine(C )
4-amino-2-oxypyrimidineDNA and RNA
Thymine(T)
5-methyl-2,4-dioxypyrimidineDNA ONLY
Uracil(U)
2,4-dioxypyrimidineRNA ONLY
Pyrimidine
N
NH
NH2
O
N3
2
N1
6
5
4
HN
NH
O
O
HN
NH
O
O
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Purine Bases
Adenine (A)
6 – aminopurineDNA and RNA
Guanine(G)
2- amino-6-oxypurineDNA and RNA
N1
2
N3
4
56
NH
9
8
N7
N
N NH
N
NH2
HN
N NH
N
O
H2N
Purine
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Sugar Phosphate Backbone
• Nucleotides connected by 3’ to 5’ phosphodiester bond– Imparts uniform negative charge to DNA / RNA
• Negative charge repels nucleophilic species (e.g. hydroxyl) thus the phosphodiester bond resists hydrolytic attack.
• Separation by agarose gel electrophoresis
– Creates 3’ and 5’ end (directionality) • Convention: Nucleotide sequences are written 5’ to 3,’ L to R
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Bases are attached to sugar by Beta Glycosidic linkage
• N-9 of purine and N-1 of pyrimidine
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Nucleotide
Nucleoside = sugar + nitrogenous base,
Nucleotide = sugar + nitrogenous base + phosphate.
N
NN
N
NH2
O
OHOH
HH
HH
OPO
O-
O
Adenosine monophosphate(A nucleotide)
Adenosine(A nucleoside)
N
NN
N
NH2
O
OHOH
HH
HH
OH
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What data did Watson and Crick use to determine the structure of DNA
• X ray diffraction photograph of DNA crystals
• Chargaff’s rules
• Bond angles from reference books
• Built models
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Erwin Chargaff’s “Rules”• Edwin Chargaff
determined the composition of DNA from many organisms– [A] = [T]
– [G] = [C]
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DNA is a Helix• X ray diffraction
photograph– Maurice Wilkins
and Rosalind Franklin
– Two chains that wind in a regular helical structure.
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Watson and Crick (Complementary) Base Pairing
A T
G C
Nucleotide content determinesmelting point of DNA.
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Double Helix• B form
– Diameter of helix = 20.0 Å (2.00 nm)
– 10.4 base pairs / turn; 34 Å (3.4 nm)
– 1 base pair 3.4 Å (0.34 nm)
• Note– Complementary base pairing
– Major grove
– Minor grove
– Antiparallel
– Hydrogen bonding between complementary base pairs.
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DNA held together by hydrogen bonding and Hydrophobic interactions
• Hydrogen bonding between base pairs– 4 – 21 kJ / mol (1 – 5 kcal/mol)
• Hydrophobic interactions (van der Walls) due to base stacking.– 2 – 4 kJ / mol (0.5 – 1.0 kcal / mol)
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Forms of DNA• B form
– “Normal” form
– Watson and Crick form
• A form– “dehydrated” B form
– nucleotide tilted 20o relative to helical axis
• Z form (“zig zag”)– stretches of alternating purine /
pyrimidines
– base pairs flip 180o
– Left handed helix
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DNA is Organized into Genes• Gene
– discrete, functional unit of DNA – when expressed, (transcribed) yields a
functional product • rRNA, tRNA, snRNA• mRNA - translated into a polypeptide sequence.
– Open reading frame - long stretch of nucleotides that can encode polypeptide due to absence of stop codons.
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Chromatosomes Pack to FormChromatin Fibers
Histones H1, H2A, H2B, H3, H4
Histones contain (>20%) arg and lys ---basic amino acids
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Karyotype
• Photograph of chromosomes from a single organism
• Arranged by size (largest to smallest)
• Homo sapiens – 46 chromosomes
– 23 pairs
• 3 billion base pairs (hapliod)
• 25,000 genes
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Chromosome Contains• Centromere site that
connects sister chromatids
• Kinetochore attachment site of spindle to chromosome
• Telomere - nucleotide repeat at end of linear chromosome– TTAGGG x 1000
– synthesized by telomerase
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Properties of DNA
• Melt / Anneal / Reanneal
• Hypochromic effects
• Supercoiled / Relaxed
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dsDNA can Reversibly Melt• Heating DNA breaks
hydrogen bonding between base pairs.– Acid or base also works
• Tm = melting temperature– Half the helical structure is
lost
• Single stranded DNA absorbs light more efficiently than double stranded DNA
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Hypochromic effect (Hypochromism)
• DNA can melt and then re-anneal.
• If sequences are similar, they will reanneal or hybridize.
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DNA exist as Linear or Circular Molecules
• Prokaryotic, Mitochondrial and Chloroplast genomes are circular– Circular molecules may exist in
topological isomers • Relaxed
• Supercoiled
• Eukaryotic genomes are linear molecules
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Single Stranded Nucleic Acids can form complex structures
• Stem Loops are produced by H-bonding between complementary regions in DNA and RNA.
• Mismatches are observed
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• Hydrogen bonding stabilizes more complex structures.
• Often observed in ribosomal RNA molecules
Single Stranded Nucleic Acids can form complex structures
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Replication
"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”
Watson, J.D. and Crick, F.H.C., Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid, Nature, 171, pp. 737-738, (1953).
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DNA may be labeled with 15N
• Grow E. coli in media containing 15NH4Cl and 14NH4Cl.
• Purify DNA• Separate DNA using density
gradient equilibrium sedimentation– CsCl gradient from 1.66 – 1.76 g
/ cm
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Testing the Semiconservative Replication Hypothesis
• Matthew Meselson and Franklin Stahl (1958)– Grew E. coli in 15NH4Cl until DNA was completely labeled.
– Transferred E. coli to 14NH4Cl containing media.
– Followed labeling pattern of DNA through several generations using density gradient equilibrium sedimentation
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DNA Replication• DNA directed DNA synthesis• DNA Polymerase
– adds deoxyribonucleotide units to an existing DNA molecule in a template directed fashion in the 5’ to 3’ direction.
• E. coli DNA Pol I isolated by Author Kornberg (1958)
– DNA Polymerase requires• Four dNTPs (dA,T,dG,dC)• Divalent cation (Mg2+)• Template DNA• Primer provides 3’ OH
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DNA Polymerase Reaction Mechanism• Nucleophilic attack by the 3’ OH on the alpha phosphate group of
dNTP
• PPi (pyrophophosphate) is hydrolyzed to Pi + Pi (orthophosphate)
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Types of RNA• Types of RNA
– Ribosomal RNA (rRNA)– part of the ribosome
– Transfer RNA (tRNA) -
– Messenger RNA (mRNA)– sequence translated into protein sequence.
– Small nuclear RNA (snRNA) – involved in splicing (spliceosome)
– Micro RNA (mi RNA) – small RNA complementary to mRNA that inhibits translation of the mRNA
– Small interfering RNA (siRNA) – small RNA that binds to mRNA causing destruction of mRNA
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Transcription
• DNA directed RNA synthesis
• RNA Polymerase– adds ribonucleoside triphosphate units
to an existing DNA molecule in a template directed fashion in the 5’ to 3’ direction.
– RNA Polymerase requires:• Four NTPs (A,U,G,C)
• Divalent cation (Mg2+)
• Template DNA
• NO primer required
• Lacks endo and exo nuclease activity
• RNA molecules are complementary to the DNA template.
One prokaryotic RNA PolThree eukaryotic RNA Pol
RNA Pol IRNA Pol II
mRNARNA Pol III
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Structure of RNA
• Genes may or may not be transcribed depending on the needs of particular cell type.
• gene is a functional region of DNA
– expressed genes are “TURNED ON”– unexpressed genes are “TURNED OFF”
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RNA Polymerase Reaction Mechanism• Nucleophilic attack by the 3’ OH on the alpha phosphate group of
NTP (ribonucleoside triphosphates)
• PPi (pyrophophosphate) is hydrolyzed to Pi + Pi (orthophosphate)
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RNA molecules are complementary to the DNA template.
• mRNA is complementary to template strand• mRNA is identical (except for U to T changes) to the
coding strand.
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Prokaryotic Promotor
– Pribnow box (also called TATA box)• 5’TATAAT 3’ centered at -9/-10
– designated by the 5’ to 3’ sequence on the NONtemplate strand» 8 to 10 nucleotides left (5’ or upstream) of transcriptional start site
(designated +1 --- there is no 0 nucleotde)
– -35 sequence • 5’TTGACA3’ centered -35 from
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Eukaryotic Promotor
• Class II genes– those synthesized by RNA Pol II.
• Pre mRNA and snRNA
• Parts– TATA or Hogness box– GC box (GGGCGG)– CAAT box
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Transcriptional Termination• Rho dependent
– Involves protein called Rho
• Rho independent– Involves stem loop structure in
mRNA
– Stem loop is followed by UUUs
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mRNA
• Prokaryotic mRNA are polycistronic– May encode two or more proteins
• Eukaryotic mRNA are monocistronic– Encode only one protein
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Eukaryotic mRNA are Posttranscriptionally Modified
• Capping– attachment of 7-methylguansine using 5’ to 5’triphosphate linkage
• Polyadenylation– attachment of 40 to several hundred adenine nucleotides to 3’ end of mRNA
• Splicing– removal of introns
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Amino acids are attached to 3’ end of tRNA
• Aminoacyl-tRNA synthestase– attaches amino acid to tRNA
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Translation• Stages of Translation
– Initiation• assemble and align ribosome, mRNA,
and tRNAfMet
– Elongation• template directed synthesis of proteins
– Termination• termination factors halt protein
synthesis• ribosome, mRNA and new protein
dissociate
• Orientation of Translation– Ribosomes move 5’ to 3’
along mRNA– Protein is synthesized N to C
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Genetic Code• Marshall Nirenberg, Har Gobind Khorana, Frances Crick
• Specific- Unambiguous– specific codon always codes for SAME amino acid– Three nucleotides (codon) = one amino acid
• 61 codons encode amino acids– Codons encoding one amino acid usually differ in the last base.
• 3 codons encode stop codons (UAA, UAG, UGA)
• Universal– conserved from species to species
• main exception = mitochondria
• Redundant (also called degenerate)– amino acid may have more than one codon
• Nonoverlapping and comma less (no puctuation)– read from fixed starting point (AUG)– lacks punctuation between codons
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Codon Usage Table
• mRNA are “read” three nucleotides (codon) at a time starting from a fixed point.
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Translational Start Site• AUG encodes Met (n-terminal amino acid).
– Prokaryotes use a Shine-Dalgarno sequence to align a ribosome on the mRNA upstream ( 5’) of AUG
– Eukaryotes use the 5’Cap to align the ribosome on the mRNA
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Mitochondrial Genetic Code differs from the Universal Code
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Eukaryotic mRNA contain Exons and Introns
Philip Sharp and Richard Roberts (1977)• Exons – coding regions• Introns –noncoding regions
– intervening sequences
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Introns were discovered by hydridizing mRNA to genomic DNA
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Splicing• Removal of introns• Spliceosome – specific proteins and small nuclear
RNA.• Most introns start with GU and end with AG
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WHY UNDERSTAND TRANSLATION?• Many Antibiotics kill bacteria by inhibiting
prokaryotic translation!!!