Download Nucleic Acids - Rubin Gulaboski

Nucleic Acids
Nucleic Acids
Structures of Nucleic Acids
DNA Replication
RNA and Transcription
1
Nucleotides
Nucleic acids consist of nucleotides that
have a sugar, nitrogen base, and phosphate
Base
PO4
Sugar
nucleoside
2
Nitrogen-Containing Bases
O
NH2
H
N
N
N
N
NH2
N
N
H
guanine (G)
N
H
thymine (T)
NH2
N
N
N
O
H
adenine (A)
O
H
CH3
CH3
N
O
N
H
cytosine (C)
O
H
O
CH3
N
N
H
uracil (U)3
Sugars
HOCH2 O
OH
ribose
OH
OH
HOCH2 O
OH
OH
(no O)
deoxyribose
4
Nucleosides in DNA
Base
Adenine (A)
Guanine (G)
Cytosine (C)
Thymine (T)
Sugar
Deoxyribose
Deoxyribose
Deoxyribose
Deoxyribose
Nucleoside
Adenosine
Guanosine
Cytidine
Thymidine
5
Nucleosides in RNA
Base
Adenine (A)
Guanine (G)
Cytosine (C)
Uracil (U)
Sugar
ribose
ribose
ribose
ribose
Nucleoside
Adenosine
Guanosine
Cytidine
Uridine
6
Example of a Nucleoside
NH2
N
O
O-
O
P O CH2
-
N
O
O
OH
deoxyctyidine monophosphate (dCMP)
7
Nucleotides in DNA and RNA
DNA
dAMP
dGMP
dCMP
dTMP
Deoxyadenosine monophosphate
Deoxyguanosine monophosphate
Deoxycytidine monophosphate
Deoxythymidine monophosphate
RNA
AMP
GMP
CMP
UMP
adenosine monophosphate
guanosine monophosphate
cytidine monophosphate
uridine monophosphate
8
Structure of Nucleic Acids
•
•
•
•
Polymers of four nucleotides
Linked by alternating sugar-phosphate bonds
RNA: ribose and A, G, C, U
DNA: deoxyribose and A,G,C,T
base
P
sugar
nucleotide
base
P
sugar
base
P
sugar
base
P
sugar
nucleotide nucleotide nucleotide
9
Nucleic Acid Structure
NH2
N
CMP
O
O
O-
P O CH2
-
O
N
O
3
NH2
3,5-phosphodiester bond
OH
O
5
O P O CH2
-
N
N
N
O
N
AMP
O
OH
10
Double Helix of DNA
• DNA contains two strands of nucleotides
• H bonds hold the two strands in a doublehelix structure
• A helix structure is like a spiral stair case
• Bases are always paired as A–T and G-C
• Thus the bases along one strand
complement the bases along the other
11
Complementary Base Pairs
•Two H bonds for A-T
•Three H bonds for G-C
12
Double Helix of DNA
13
Learning Check NA1
Write the complementary base sequence
for the matching strand in the following
DNA section:
-A-G-T-C-C-A-A-T-G-C• •
• •
• • • • • • • •
• • • • • • • •
14
Solution NA1
Write the complementary base sequence
for the matching strand in the following
DNA section:
-A-G-T-C-C-A-A-T-G-C• •
• •
• • • • • • • •
• • • • • • • •
-T-C-A-G-G-T-T-A-C-G15
DNA Replication
• DNA in the chromosomes replicates itself
every cell division
• Maintains correct genetic information
• Two strands of DNA unwind
• Each strand acts like a template
• New bases pair with their complementary base
• Two double helixes form that are copies of
original DNA
16
DNA Unwinds
G-C
A-T
C-G
T-A
GACT-
-C
-T
-G
-A
17
DNA Copied with Base Pairs
Two copies of original DNA strand
G-C
A-T
C-G
T-A
G-C
A-T
C-G
G-A
18
Nucleic Acid Chemistry
Where the info is…interpreting the
blueprint
Central Dogma
Replicati
on
DNA ---------------- RNA-------------- protein
transcription
translation
Central Dogma
• Replication
– DNA making a copy of itself
• Making a replica
• Transcription
– DNA being made into RNA
• Still in nucleotide language
• Translation
– RNA being made into protein
• Change to amino acid language
Replication
• Remember that DNA is self
complementary
• Replication is semiconservative
– One strand goes to next generation
– Other is new
• Each strand is a template for the other
– If one strand is 5’ AGCT 3’
– Other is:
3’ TCGA 5’
Replica
• Write the strand complementary to:
3’ ACTAGCCTAAGTCG 5’
Answer
Replication is
Semiconservative
Replication
• Roles of enzymes
– Topoisomerases
– Helicase
– DNA polymerases
– ligase
• DNA binding proteins
– DNA synthesis
• Leading strand
• Lagging strand
Replication
Replication
• Helix opens
– Helicase
• Causes supercoiling upstream
– Topoisomerases (gyrase)
• DNA Binding Proteins
– Prevent reannealing
Replication
Replication
• Leading strand
– 3’ end of template
– As opens up, DNA polymerase binds
– Makes new DNA 5’ - 3’
• Same direction as opening of helix
• Made continuously
Replication
Replication
• Lagging strand
– 5’ end of template
• Can’t be made continuously as direction is
wrong
– RNA primer
– New DNA made 5’  3’
• Opposite direction of replication
• Discontinuous
– Okazaki fragments
• Ligase closes gaps
Transcription
• DNA template made into RNA copy
– Uracil instead of Thymine
• One DNA strand is template
– Sense strand
• Other is just for replication
– Antisense (not to be confused with
nonsense!)
• In nucleus
– nucleoli
Transcription
• From following DNA strand, determine
RNA sequence
3’ GCCTAAGCTCA 5’
Answer
Transcription
Transcription
• DNA opens up
– Enzymes?
• RNA polymerase binds
– Which strand?
– Using DNA template, makes RNA
• 5’-3’
• Raw transcript called hnRNA
Transcription
How does RNA polymerase know where to
start?
upstream promotor sequences
Pribnow Box
TATA box
RNA polymerase starts transcription X
nucleotides downstream of TATA box
Introns and Exons
• Introns
– Intervening sequences
– Not all DNA codes for protein
– Regulatory info, “junk DNA”
• Exons
– Code for protein
Processing of hnRNA into mRNA
• 3 steps
– Introns removed
• Self splicing
– 5’ methyl guanosine cap added
– Poly A tail added
• Moved to cytosol for translation
Processing of hnRNA into mRNA
Translation
• RNA -- Protein
– Change from nucleotide language to amino
acid language
• On ribosomes
• Vectorial nature preserved
– 5’ end of mRNA becomes amino terminus
of protein
– Translation depends on genetic code
Genetic Code
• Nucleotides read in triplet “codons”
– 5’ - 3’
• Each codon translates to an amino acid
• 64 possible codons
– 3 positions and 4 possiblities (AGCU)
makes 43 or 64 possibilities
– Degeneracy or redundancy of code
• Only 20 amino acids
• Implications for mutations
Genetic Code
Genetic Code
• Not everything translated
• AUG is start codon
– Find the start codon
• Also are stop codons
• To determine aa sequence
– Find start codon
– Read in threes
– Continue to stop codon
Translation
• Steps:
– Find start codon (AUG)
– After start codon, read codons, in threes
– Use genetic code to translate
Translate the following:
GCAGUCAUGGGUAGGGAGGCAACCUGAACCGA
C
Answer
Translation Process
• Requires Ribosomes, rRNA, tRNA and,
of course, mRNA
– Ribosome
• Made of protein and rRNA
• 2 subunits
• Has internal sites for 2 transfer RNA molecules
Ribosome
Left is cartoon diagram
Right is actual picture
Transfer RNA
• Mostly double stranded
– Folds back on itself
• Several loops
– Anticodon loop
• Has complementary nucleotides to codons
• 3’ end where aa attach
Transfer RNA
Translation
• Initiation
– Ribosomal subunits assemble on mRNA
– rRNA aids in binding of mRNA
• Elongation
–
–
–
–
tRNAs with appropriate anticodon loops bind to complex
have aa attached (done by other enzymes)
Amino acids transfer form tRNA 2 to tRNA 1
Process repeats
• Termination
– tRNA with stop codon binds into ribosome
– No aa attached to tRNA
– Complex falls apart
Translation
Translation
• Happening of process (circa 1971)
• http://www.youtube.com/watch?v=u9dh
O0iCLww
Mutations
• Changes in nucleotide sequence
• Can cause changes in aa sequence
– Degeneracy in genetic code can prevent
• Two types
– Point mutations
• Single nucleotide changes
– Frame shift
• Insertions or deletions
Point Mutations
• Single nucleotide changes
• Old sequence
AUG GGU AGG GAG GCA ACC UGA ACC GAC
aa:
G
R
E A
T
New sequence
AUG GGU AGU GAG GCA ACC UGA ACC GAC
aa:
G
S
E
A
T
Point mutations
• Depending on change, may not change
aa sequence
• Old sequence
AUG GGU AGG GAG GCA ACC UGA ACC GAC
aa:
G
R
E A
T
New sequence
AUG GGU AGA GAG GCA ACC UGA ACC GAC
aa:
G
R
E
A
T
Point Mutations
• Change could make little difference
– If valine changed to leucine, both nonpolar
• Change could be huge,
– Could erase start codon
• Old sequence
AUG GGU AGG GAG GCA ACC UGA ACC GAC
aa:
G
R
E A
T
New sequence
AUU GGU AGA GAG GCA ACC UGA ACC GAC
aa: no start codon…protein not made
Point Mutations
• Other possibilities,
– Stop codon inserted
• Truncated protein
– Stop codon changed
• Extra long protein
• Bottom line,
– Depends on what change is
Frame Shift mutations
• Insertions or deletions
– Change the reading frame
• Insertion example
Old sequence
AUG GGU AGG GAG GCA ACC UGA ACC GAC
aa:
G
R
E
A
T
New sequence
AUG GGU AGG AGA GGC AAC CUG AAC CGA C
aa:
G
R
R G
N L
N
R
Frame Shift Mutations
• Deletion example
• Old sequence
AUG GGU AGG GAG GCA ACC UGA ACC GAC
aa:
G
R
E A
T
New sequence Delete second A (Underlined above)
AUG GGU GGG AGG CAA CCU GAA CCG AC
aa:
G
G
R Q
P G
P
Complementary DNA Strand
Template:
3’ ACTAGCCTAAGTCG 5’
5’ TGATCGGATTCAGC 3’
Back
RNA Transcript
DNA
RNA
Back
3’ GCCTAAGCTCA 5’
5’ CGGAUUCGAGU 3’
Translation Answer
Find start codon
GCAGUCAUGGGUAGGGAGGCAACCUGAACCGAC
Read in threes after that:
AUG GGU AGG GAG GCA ACC UGA ACC GAC
Using Genetic code
AUG GGU AGG GAG GCA ACC UGA ACC GAC
G
R
E
A
T
After stop codon…rest is garbage
Back
stop