Download Biochemistry 6/e

Biochemistry I
(CHE 418
β / 5418
Reading Assignment
Berg et. al (2012) Chapter 4
Central Dogma of Molecular Biology
Replication
DNA
Transcription
 RNA
Translation
 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
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)
Nucleotide
• Pentose sugar
– Carbons are numbered with primes to differentiate between carbons
/ nitrogens of nitrogenous bases
β-D-2-Deoxyribose
Ribose
DNA Only
RNA Only
Nucleotide
• phosphate group
Pyrimidine Bases
Pyrimidine
Thymine
(T)
5-methyl-2,4-dioxypyrimidine
Cytosine
(C )
4-amino-2-oxypyrimidine
Uracil
(U)
2,4-dioxypyrimidine
DNA ONLY
DNA and RNA
RNA ONLY
Purine Bases
Purine
Adenine
(A)
6 – aminopurine
Guanine
(G)
2- amino-6-oxypurine
DNA and RNA
DNA and RNA
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.
Adenosine
(A nucleoside)
Adenosine monophosphate
(A nucleotide)
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
G C
A T
Nucleotide content determines
melting 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 Form
Chromatin 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
Single Stranded Nucleic Acids can form
complex structures
• Hydrogen bonding stabilizes more
complex structures.
• Often observed in ribosomal RNA
molecules
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
• Grow E. coli in media
containing 15NH4Cl and
14NH Cl.
4
• Purify DNA
• Separate DNA using density
gradient equilibrium
sedimentation
– CsCl gradient from 1.66 – 1.76 g
/ cm
15N
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
One prokaryotic RNA Pol
Three eukaryotic RNA Pol
RNA Pol I
RNA Pol II
• RNA molecules are complementary
mRNA
to the DNA template.
RNA 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!!!