Download Nucleic Acids and DNA

Nucleic Acids, DNA, RNA and
Protein Synthesis
CHM 341
Suroviec Fall 2016
I. Nucleotides, Nucleic Acids and
Bases
A. Bases
• Planar, aromatic,
heterocyclic
• Purine (2 rings)
• Pyrimidine (1 ring)
Adenine (A)
Guanine (G)
Thyamine (T)
Cytosine ( C)
Uracil (U)
B. Nucleosides
• Ribonucleotides
– sugar = ribose
• Deoxyriboneculeotide
– Sugar = 2´-deoxyribose
C. Nucleotides (total molecule)
• Have a phosphate on
carbon #5
• Can have up to 3
phosphates
• Monophosphate (NMP)
• Diphosphate (NDP)
• Triphosphate (NTP)
– Where N is any one of
the nucleic acids
II. Nucleic Acid Structure
• Can be found singly
• Most often found in a
polymer
• DNA (or RNA)
polymerizes 5´
phosphate to 3´ OH
– Makes phosphodiester
bond
• Polymer of non-identical
residues has a property
that individual
monomers do not.
A. Base composition of DNA
•
1940’s Erwin Chargaff
discovered that when
measuring the amount
of each base A = T
and G = C. Lead to
Chargaff’s rules.
• Watson and Crick took
this data and other
material that hinted that
DNA stacked to
propose that DNA was
double stranded.
•
Maurie Wilkins and
Rosalind Franklin
made and X-ray that
indicated that DNA
was helical in nature
• Put the bases together
in such a way so that
the complimentary Hbonds were formed and
the width of the base
pairs would be similar.
Characteristics of DNA model
1.
2.
3.
4.
5.
6.
DNA strands run in opposite
directions (antiparallel)
Sugar phosphate backbone is
found on outside, bases inside
and pair up
Each base is H-bonded with a
base on the opposite strand with
the same number of H-bonds
A complete turn takes 34 Å and
has 10 bases per turn
2 helical polynucleotide chains
coiled around a central axis
(diameter = 20 Å)
DNA strand is quite stiff and will
not bend much around the axis
 - DNA
Helix is right handed
III. Overview of Nucleic Acid Function
• Carries genetic info
• Directs protein
synthesis
• Double stranded
nature allows for
easy replication
A.
•
DNA replication
• W-C model allows each
DNA strand to act as
template for replication
2 hypothesis for replication
came forth:
• Conservative: where the
parental DNA strand
retains both old stands
and creates new ds DNA
• Semi conservative:
where the created DNA
has one old strand and
one new strand. Shown
to be how DNA
replicated
DNA, RNA & Protein Synthesis
• DNA directs its own
replication and is also
transcribed into RNA.
RNA then translates into
proteins.
– CENTRAL DOGMA of
MOLECULAR BIOLOGY
• Transcription: transferring
into from DNA --> RNA
• Translation: transferring
info from RNA --> proteins
IV. Replication
• Involves 20+ proteins
– Helicases: opens the
double strand, splits the
strands apart starting at
replication fork rich in A-T
– SSB: bind to the single
strand DNA stablizing it
– Primase: adds short
stretches of RNA and
allows the the DNA
polymerase to start.
– DNA polymerase I:
catalyzes the addition of
deoxynucleotides to the
chain
– DNA Polymerase I & III: add DNA with
high fidelity to the newly growing DNA
strand.
– Ligase: closes up gaps in the DNA
DNA polymerase I
• DNA polymerase I catalyzes
addition of a addition of
dNTP to chain
• Requires dATP, dGTP, TTp,
dCTP and Mg2+
• Elongation occurs 5´ to 3´
where 3´ hydroxyl bind to
the new deoxyribonucleotide
• DNA polymerase is a
“template directed enzyme”
DNA polymerase III
• Adds nucleotides to the
3´ end of the chain
• New strand reads 5´ to
3´
• Needs a primer with free
3´ hydroxyl group to start
addition of new DNA
– The strand is going to be
started with a RNA primer
that is later removed and
replaced
• The incoming dNTP first
forms an appropriate base
pair and then the DNA
polymerase III links the
incoming bases together
• Binds complementary DNA
nucleotides starting at the
3´ end of the RNA primer
at a rate of 1000/second
• Makes a mistake 1/108
IV. Replication
• Opening of the DNA
– Double stranded DNA is opened by helicase
– Kept open by SSB
– Exposed DNA bind DNA polymerase III and RNA
synthesizing protein primase
• This makes the replication fork
IV. Replication
• Leading strand
synthesis begins with
synthesis of primase
of short RNA primer
• dNTPs are added by
DNA polymerase III
• Continously added to
this strand toward the
fork
Replication
• Lagging strand
synthesis is done in
short bursts
• Needs multiple RNA
primers
• Synthesized in opposite
direction of the fork
• DNA primase moves 5’
to 3’ and makes RNA
primer to which DNA is
then added by DNA
polymerase III
Replication
• Keeping the DNA
sequence correct is
important: 1 mispair per
109 base pairs
• Polymerase reaction
occurs in 2 stages
– Incoming dNTP base
pairs with the template
while enzyme is open
catalytically inactive
– Polymerization only
occurs after polymerase
has closed around base
pair which positions
residues
Transcription
• DNA is in the
nucleus
• Protein synthesis
takes place in the
ribosome
• RNA is the
intermediate
• Cells contain 3
types of RNA
– Ribosomal RNA
(rRNA)
– Transfer RNA (tRNA)
– Messenger RNA
(mRNA)
RNA polymerase
• RNAP couples together
the ribonucleotide
triphosphates on DNA
templates
• Builds RNA in the 5’
 3’ direction (reads the
DNA in the 3’ --> 5’
direction)
RNA polymerase
• 3’ hydroxyl group
attacks the
triphosphate
• Creates
phosphodiester
bond
• Releases PPi
• Does not need a
primer
RNA polymerase
• Initiation of RNA
synthesis occurs only at
promoters
– Usually starts at GTP or
ATP
– New RNA strand base
pairs temporarily with
DNA template to form
DNA/RNA template
– DNA must unwind then
rewind
– Template strand
– Nontemplate strand or
coding strand
RNA polymerase
• RNA polymerase
lacks ability to proof
read
• No 3’--> 5’
exonuclease activity
• One error in 104
ribonucleotides
added
Post transcription of RNA
• In Eukaryotes RNA is
further modified
• mRNA undergoes gene
splicing where introns are
removed and exons
are rejoined
• 5’ obtains a cap
• 3’ gets polyA tail
Characteristics of RNA
• Contains AUGC
• Uracil is less “energy
expensive”
• Normally single stranded
• Has –OH on 2’ carbon of
ribose
•
1.
2.
3.
4.
5.
6.
7.
Seven roles of RNA
mRNA – carries DNA code
to make proteins
rRNA – forms complex of
2/3 RNA, 1/3 protein to
form protein in ribosome
tRNA – carries the amino
acids to the mRNA
snRNA – helps splice
exons
Ribozymes – RNA capable
of catalytic activity
Antisense RNA – act to
bind RNA to stop
translation
Viral RNA – carry
hereditary information
Translation
• mRNA to proteins
– Need mRNA,
ribosome and tRNA
• mRNA is produced
from DNA
• mRNA read from
ribosomes and tRNA
Ribosome
• Large protein/RNA complex
• 2 units (large/small)
• Synthesis begins at start
codon near 5’ end
– Smaller unit (usually has tRNA
bound) binds to AUG codon
on mRNA
– binds to large subunit
– Large unit then binds
– Large unit has 3 tRNA binding
sites (APE)
– A: aminoacyl-tRNA
– P:peptidyl-tRNA
– E: free-tRNA
Initiation
• AUG signals the beginning
of polypeptide chains
• Read the code off of the
mRNA and translate into
amino acids
• One start codon
• 3 stop codon
tRNA
• Read the code on the mRNA
and translate into the correct
amino acid
• Acceptor stem
– 5’ terminal nucleotide and 3’
terminal nucleotide (-OH
group where amino acid
binds)
– 3’end always has CCA
sequence
• Specific linkage is catalyzed by
amino acyl-tRNA synthetase
(tranferase).
• Anticodon recognizes the
complementary codon on the
mRNA
Aminoacylation
• Process of adding an
aminoacyl group to a
compound
– Produces tRNA
molecules with their CCA
3’ ends covalently linked
to an amino acid
• Aminoacyl tRNA
synthetase (one specific
for each amino acid)
• Needs ATP to drive the
reaction
Initiation and Elongation
• mRNA bearing the code for
the polypeptide binds to the
small ribosome unit
• Aminoacyl-tRNA then binds
followed by larger ribosomal
unit
• Aminoacyl-tRNA base-pairs
with mRNA codon AUG to
start the polypeptide
• Chain is elongated by
addition of amino acids
• Added by individual tRNA
• Polypeptides are grown from
amino-terminal end to
carboxyl-end
Elongation
•
•
•
•
•
mRNA passes through ribosome
AUG is held in P site
2nd amino acid binds in the A site
Make peptide bond
Ribosome then moves toward 3’ end using GTP and leaving A site
open