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Biochemistry I(CHE 418 / 5418
Reading Assignment
Berg et. al (2012) Chapter 4
β
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
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
Nucleic Acids
• Nucleic Acid - linear, non-branched polymer of nucleotides
Classes of Nucleic Acids– RNA = ribonucleic acid– DNA = 2' deoxyribonucleic acid
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
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
Nucleotide
• phosphate group
O-P-O
O-
O
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
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
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
Bases are attached to sugar by Beta Glycosidic linkage
• N-9 of purine and N-1 of pyrimidine
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
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
Erwin Chargaff’s “Rules”• Edwin Chargaff
determined the composition of DNA from many organisms– [A] = [T]
– [G] = [C]
DNA is a Helix• X ray diffraction
photograph– Maurice Wilkins
and Rosalind Franklin
– Two chains that wind in a regular helical structure.
Watson and Crick (Complementary) Base Pairing
A T
G C
Nucleotide content determinesmelting point of DNA.
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.
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)
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
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.
Chromatosomes Pack to FormChromatin Fibers
Histones H1, H2A, H2B, H3, H4
Histones contain (>20%) arg and lys ---basic amino acids
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
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
Properties of DNA
• Melt / Anneal / Reanneal
• Hypochromic effects
• Supercoiled / Relaxed
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
Hypochromic effect (Hypochromism)
• DNA can melt and then re-anneal.
• If sequences are similar, they will reanneal or hybridize.
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
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
• Hydrogen bonding stabilizes more complex structures.
• Often observed in ribosomal RNA molecules
Single Stranded Nucleic Acids can form complex structures
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).
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
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
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
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)
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
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
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”
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)
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.
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
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
Transcriptional Termination• Rho dependent
– Involves protein called Rho
• Rho independent– Involves stem loop structure in
mRNA
– Stem loop is followed by UUUs
mRNA
• Prokaryotic mRNA are polycistronic– May encode two or more proteins
• Eukaryotic mRNA are monocistronic– Encode only one protein
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
Amino acids are attached to 3’ end of tRNA
• Aminoacyl-tRNA synthestase– attaches amino acid to tRNA
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
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
Codon Usage Table
• mRNA are “read” three nucleotides (codon) at a time starting from a fixed point.
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
Mitochondrial Genetic Code differs from the Universal Code
Eukaryotic mRNA contain Exons and Introns
Philip Sharp and Richard Roberts (1977)• Exons – coding regions• Introns –noncoding regions
– intervening sequences
Introns were discovered by hydridizing mRNA to genomic DNA
Splicing• Removal of introns• Spliceosome – specific proteins and small nuclear
RNA.• Most introns start with GU and end with AG
WHY UNDERSTAND TRANSLATION?• Many Antibiotics kill bacteria by inhibiting
prokaryotic translation!!!