Download Intro to Nucleic Acids-Structure, Central Dogma

Nucleic Acids: Cell
Overview and Core Topics
DNA and RNA in the Cell
Cellular Overview
Classes of Nucleic Acids: DNA
 DNA is usually found in the nucleus
 Small amounts are also found in:
• mitochondria of eukaryotes
• chloroplasts of plants
 Packing of DNA:
• 2-3 meters long
• histones
 genome = complete collection of
hereditary information of an organism
Classes of Nucleic Acids: RNA
FOUR TYPES OF RNA
• mRNA - Messenger RNA
• tRNA - Transfer RNA
• rRNA - Ribosomal RNA
• snRNA - Small nuclear RNA
THE BUILDING BLOCKS
Anatomy of Nucleic Acids
Nucleic acids are linear polymers.
Each monomer consists of:
1. a sugar
2. a phosphate
3. a nitrogenous base
Nitrogenous Bases
Nitrogenous Bases
DNA (deoxyribonucleic acid):
adenine (A)
guanine (G)
cytosine (C)
thymine (T)
RNA (ribonucleic acid):
adenine (A)
guanine (G)
cytosine (C)
uracil (U)
Why ?
Pentoses of Nucleic Acids
This difference in structure affects secondary structure
and stability.
Nucleosides
linkage of a base and a sugar.
Nucleotides
- nucleoside + phosphate
- monomers of nucleic acids
- NA are formed by 3’-to-5’ phosphodiester linkages
Shorthand notation:
- sequence is read from 5’ to 3’
- corresponds to the N to C terminal of proteins
DNA
Nucleic Acids: Structure
Primary Structure
• nucleotide sequences
Secondary Structure
DNA Double Helix
• Maurice Wilkins and Rosalind Franklin
• James Watson and Francis Crick
Features:
• two helical polynucleotides coiled
around an axis
• chains run in opposite directions
• sugar-phosphate backbone on the
outside, bases on the inside
• bases nearly perpendicular to the axis
• repeats every 34 Å
• 10 bases per turn of the helix
• diameter of the helix is 20 Å
Double helix
stabilized by
hydrogen
bonds.
ATCTGGCAT
TAGACCGTA
Which is more stable?
Axial view of DNA
A and B forms are
both right-handed
double helix.
A-DNA has different
characteristics from
the more common
B-DNA.
Z-DNA
• left-handed
• backbone phosphates zigzag
Comparison Between A, B, and Z DNA:
 A-DNA: right-handed, short and broad, 11 bp per turn
 B-DNA: right-handed, longer, thinner, 10 bp per turn
 Z-DNA: left-handed, longest, thinnest, 12 bp per turn
Tertiary Structure
Supercoiling
supercoiled DNA
relaxed DNA
Consequences of double helical structure:
1. Facilitates accurate hereditary information transmission
2.Reversible melting
• melting: dissociation of the double helix
• melting temperature (Tm)
• hypochromism
• annealing
Structure of Single-stranded DNA
Stem Loop
RNA
Nucleic Acids: Structure
Secondary Structure
transfer RNA (tRNA) :
Brings amino acids to
ribosomes during
translation
ribosomal RNA (rRNA) : Makes up the ribosomes, together
with ribosomal proteins.
messenger RNA (mRNA) : Encodes amino acid sequence
of a polypeptide
small nuclear RNA (snRNA) :With
proteins, forms complexes that are
used in RNA processing in
eukaryotes. (Not found in
prokaryotes.)
DNA Replication, Recombination, and Repair
Central Dogma
Central Dogma
DNA Replication – process of producing identical
copies of original DNA
•
strand separation followed by copying of each
strand
• fixed by base-pairing rules
DNA replication is bidirectional.
 involves two replication forks that move in opposite direction
DNA replication requires unwinding of the DNA helix.
 expose single-stranded templates
 DNA gyrase – acts to overcome torsional stress
imposed upon unwinding
 helicases – catalyze unwinding of double helix
-disrupts H-bonding of the two strands
 SSB (single-stranded DNA-binding proteins) –
binds to the unwound strands, preventing re-annealing
Primer
RNA primes the
synthesis of DNA.
Primase synthesizes
short RNA.
DNA replication is semidiscontinuous
 DNA polymerase synthesizes the new DNA strand
only in a 5’3’ direction. Dilemma: how is 5’ 
3’ copied?
 The leading strand
copies continuously
 The lagging strand
copies in segments
called Okazaki
fragments (about
1000 nucleotides at a
time) which will then
be joined by DNA
ligase
DNA Ligase

= seals the nicks between Okazaki fragments
DNA ligase seals breaks in the double stranded
DNA
 DNA ligases use an energy source (ATP in
eukaryotes and archaea, NAD+ in bacteria) to
form a phosphodiester bond between the 3’
hydroxyl group at the end of one DNA chain and
5’-phosphate group at the end of the other.
DNA replication terminates at the Ter region.
• the oppositely moving replication forks meet here and
replication is terminated
• contain core elements 5’-GTGTGTTGT
• binds termination protein (Tus protein)
Eukaryotic DNA Replication
Like E. coli, but more complex
 Human cell: 6 billion base pairs of DNA to copy
 Multiple origins of replication: 1 per 3000-30000
base pairs

E.coli
 Human

E.coli
 Human
1 chromosome
23
circular chromosome;
linear
Mutations
1. Substitution of base pair
a. transition
b. transversion
2. Deletion of base pair/s
3. Insertion/Addition of base
pair/s
Macrolesions: Mutations involving changes in large portions of the
genome
DNA replication error rate: 3 bp during
copying of 6 billion bp
Agents of Mutations
1. Physical Agents
a) UV Light
b) Ionizing Radiation
2. Chemical Agents
Some chemical agents can be
classified further into
a) Alkylating
b) Intercalating
c) Deaminating
3. Viral
DNA Repair
 Direct repair
 Photolyase cleave pyrimidine dimers
 Base excision repair
 E. coli enzyme AlkA removes modified bases such as 3methyladenine (glycosylase activity is present)
 Nucleotide excision repair
 Excision of pyrimidine dimers (need different enzymes
for detection, excision, and repair synthesis)
RNA Transcription
Central Dogma
Process of Transcription has four stages:
1. Binding of RNA polymerase at promoter sites
2. Initiation of RNA polymerization
3. Chain elongation
4. Chain termination
Transcription (RNA Synthesis)
 RNA Polymerases
 Template (DNA)
 Activated precursors (NTP)
 Divalent metal ion (Mg2+ or Mn2+)
 Mechanism is similar to DNA Synthesis
Start of Transcription
 Promoter Sites
 Where RNA Polymerase can indirectly bind
Termination of Transcription
1. Intrinsic termination = termination sites
 Terminator Sequence
 Encodes the
termination signal
 In E. coli – base paired
hair pin (rich in GC)
followed by UUU…
causes the
RNAP to
pause
causes the RNA strand to detach
from the DNA template
Termination of Transcription
2. Rho termination = Rho protein, ρ
prokaryotes: transcription and translation happen in cytoplasm
eukaryotes: transcription (nucleus); translation (ribosome in cytoplasm)
 In eukaryotes, mRNA is modified after transcription
 Capping, methylation
 Poly-(A) tail, splicing
capping: guanylyl residue
capping and methylation ensure stability
of the mRNA template; resistance to
exonuclease activity
Eukaryotic genes are split genes: coding regions (exons) and
noncoding regions (introns)
Introns & Exons
 Introns
 Intervening
sequences
 Exons
 Expressed
sequences
Splicing
Spliceosome: multicomponent complex of small
nuclear ribonucleoproteins (snRNPs)
splicing occurs in
the spliceosome!
Translation: Protein Synthesis
Central Dogma
Translation
Starring three types of RNA
1. mRNA
2. tRNA
3.rRNA
Properties of mRNA
1. In translation, mRNA is read in groups of bases called “codons”
2. One codon is made up of 3 nucleotides from 5’ to 3’ of mRNA
3. There are 64 possible codons
4. Each codon stands for a specific amino acid, corresponding to the
genetic code
5. However, one amino acid has many possible codons. This property is
termed degeneracy
6. 3 of the 64 codons are terminator codons, which signal the end of
translation
Genetic Code
 3 nucleotides (codon) encode an amino acid
nonoverlapping
 The code has no punctuation
 The code is
Synonyms
 Different codons, same amino acid
 Most differ by the last base
 XYC & XYU
 XYG & XYA
 Minimizes the deleterious effect of mutation
tRNA as Adaptor Molecules
 Amino acid attachment
site
 Template recognition
site
 Anticodon
 Recognizes codon
in mRNA
tRNA as Adaptor Molecules
Mechanics of Protein Synthesis
 All protein synthesis involves three phases:
initiation, elongation, termination
 Initiation involves binding of mRNA and
initiator aminoacyl-tRNA to small subunit(30S),
followed by binding of large subunit (50S) of the
ribosome
 Elongation: synthesis of all peptide bonds with tRNAs bound to acceptor (A) and
peptidyl (P) sites.
 Termination occurs when "stop codon"
reached
Translation
 Occurs in the ribosome
 Prokaryote START
 fMet (formylmethionine) bound to
initiator tRNA
 Recognizes AUG and sometimes
GUG (but they also code for Met
and Val respectively)
 AUG (or GUG) only part of the initiation signal;
preceded by a purine-rich sequence
Translation
 Eukaryote START
 AUG nearest the 5’ end is usually the start signal
Termination
Stop signals (UAA, UGA, UAG):
• recognized by release factors (RFs)
• hydrolysis of ester bond between polypeptide and
tRNA
Reference:
Garrett, R. and C. Grisham. Biochemistry. 3rd edition. 2005.
Berg, JM, Tymoczko, JL and L. Stryer. Biochemistry. 5th
edition. 2002.