Download Nucleic Acids-Structure, Central Dogma

Nucleic Acids: Cell
Overview and Core Topics
Outline
I.Cellular Overview
II.Anatomy of the Nucleic Acids
1. Building blocks
2. Structure (DNA, RNA)
III.Looking at the Central Dogma
1. DNA Replication
2. RNA Transcription
3. Protein Synthesis
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 ?
Properties of purines and pyrimidines:
1.keto – enol tautomerism
2.strong UV absorbance
Pentoses of Nucleic Acids
This difference in structure affects secondary structure
and stability.
Which is more stable?
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.
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
Major and minor grooves are lined
with sequence-specific H-bonding.
Tertiary Structure
Supercoiling
supercoiled DNA
relaxed DNA
Topoisomerase I – relaxation of supercoiled structures
Topoisomerase II – add negative supercoils to 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
Transfer RNA
 Extensive H-bonding creates four double helical
domains, three capped by loops, one by a stem
 Only one tRNA structure (alone) is known
 Many non-canonical base pairs found in tRNA
ribosomal RNA (rRNA) : Makes up the ribosomes, together
with ribosomal proteins.
Ribosomes synthesize
proteins
 All ribosomes contain large
and small subunits
 rRNA molecules make up
about 2/3 of ribosome
 Secondary structure
features seem to be
conserved, whereas
sequence is not
 There must be common
designs and functions that
must be conserved
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 semi-conservative.
DNA replication is bidirectional.
 involves two replication forks that move in opposite direction
DNA Replication
 Begins at specific start sites
• in E. coli, origin of replication, oriC locus
• binding site for dnaA, initiation protein
• rich in A-T
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
Overall: each of the two DNA duplexes contain one “old” and one
“new” DNA strand (semi-conservative) and half of the new strand was
formed by leading strand and the other half by lagging strand.
DNA Polymerase
= enzymes that replicate DNA
All DNA Polymerases share the following:
1.Incoming base selected in the active site (base-complementarity)
2.Chain growth 5’  3’ direction (antiparallel to template)
3.Cannot initiate DNA synthesis de novo (requires primer)
First DNA Polymerase discovered – E.coli DNA Polymerase I (by Arthur
Kornberg and colleagues)
Arthur Kornberg
1959 Nobel Prize in Physiology and
Medicine
Roger D. Kornberg
2006 Nobel Prize in Chemistry
http://www.nobelprize.org
DNA Polymerase
 specificity dictated by H-bonding
and shape complementarity
between bases
• binding of correct base is
favorable (more stable)
• interaction of residues in the
enzyme to the minor groove of
DNA
• close down around the
incoming NTP
Mechanism of DNA
linkage:
3’  5’ exonuclease activity
- removes incorrect nucleotides from the 3’-end of the
growing chain (proofreader and editor)
- polymerase cannot elongate an improperly base-paired
terminus
 proofreading mechanisms
• Klenow fragment – removes mismatched
nucleotides from the 3’’ end of DNA
(exonuclease activity)
• detection of incorrect base
- incorrect pairing with the template
(weak H-bonding)
- unable to interact with the minor
groove (enzyme stalls)
Exonuclease activity
5’  3’ exonuclease activity
- remove distorted segments lying in the path of the
advancing polymerase
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
Telomeres
The Ends of Linear DNA Possess Telomeres
 Present because DNA is shortened after each
round of replication
 Contains hundreds of tandem repeats of a
hexanucleotide sequence (AGGGTT in humans)
 Telomeres at the 3’ end is G rich and is slightly
longer
 May form large loops to protect chromosome
ends
DNA Recombination =
natural process of genetic rearrangement
 recombinases
 Holliday junction – crosslike
structure
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
UV Light Causes Pyrimidine Dimerization
 Replication and gene expression are blocked
Chemical mutagens
• 5-bromouracil and 2-aminopurine can be
incorporated into DNA
 Deaminating agents
 Ex: Nitrous acid (HNO2)
 Converts adenine to hypoxanthine, cytosine to uracil, and guanine
to xanthine
 Causes A-T to G-C transitions
 Alkylating agents
 Intercalating agents
 Acridines
 Intercalate in DNA, leading to insertion or deletion
 The reading frame during translation is changed
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)
Do we has a quiz?
QUIZ
1. Draw the structure of any nitrogenous base of your picking. (1 pt)
2.
What is the difference between the glycosidic bond and the
phosphodiester bond? (2 pts)
3. Give the reason why DNA utilizes the deoxyribose while RNA uses
the ribose. (2 pts)
4. Enumerate all the enzymes and proteins involved in DNA
replication and briefly state their importance/function. A short
concise answer will suffice. (4 pts)
5. Give the partner strand of this piece of DNA:
5-ACTCATGATTAGCAG-3  (1 pt)
RNA Transcription
Central Dogma
Process of Transcription has four stages:
1. Binding of RNA polymerase at promoter sites
2. Initiation of 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
Reece R. Analysis of Genes and Genomes.2004. p47.
Limitations of RNAP II:
1. It can’t recognize its target promoter and gene. (BLIND)
2. It is unable to regulate mRNA production in response to
developmental and environmental signals. (INSENSITIVE)
Start of Transcription
 Promoter Sites
 Where RNA Polymerase can indirectly bind
Preinitiation Complex (PIC)
TATA box – a DNA sequence (5’—TATAA—3’) found in the promoter
region of most eukaryotic genes.
Abeles F, et al. Biochemistry. 1992. p391.
Transcription Factors (TF):
TFIID
binds to TATA; promotes TFIIB binding
TFIIA
stabilizes TBP binding
TFIIB
promotes TFIIF-pol II binding
TFIIF
targets pol II to promoter
TFIIE
stimulates TFIIH kinase and ATPase
actiivities
TFII H
helicase, ATPase, CTD kinase activities
Hampsey M. Molecular Genetics of RNAP. Microbiology and Molecular Biology Reviews. 1998. p7.
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!
Reverse Transcription
RNA-Directed DNA Polymerase
 1964: Howard Temin notices that DNA
synthesis inhibitors prevent infection of
cells in culture by RNA tumor viruses.
Temin predicts that DNA is an intermediate
in RNA tumor virus replication
 1970: Temin and David Baltimore
(separately) discover the RNA-directed
DNA polymerase - aka "reverse
transcriptase"
Reverse Transcriptase
 Primer required, but a strange one - a tRNA
molecule that the virus captures from the host
 RT transcribes the RNA template into a
complementary DNA (cDNA) to form a DNA:RNA
hybrid
 All RNA tumor viruses contain a reverse
transcriptase
RT II
 Three enzyme activities
 RNA-directed DNA polymerase
 RNase H activity - degrades RNA in the DNA:RNA hybrids
 DNA-directed DNA polymerase - which makes a DNA
duplex after RNase H activity destroys the viral genome
• HIV RT: very error-prone (1 bp /2000 to 4000 bp)
 HIV therapy: AZT (or 3'-azido-2',3'- dideoxythymidine)
specifically inhibits RT
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
Practice

Encoded sequences.
 (a) Write the sequence of the mRNA molecule
synthesized from a DNA template strand having the
sequence
 (b) What amino acid sequence is encoded by the
following base sequence of an mRNA molecule? Assume
that the reading frame starts at the 5 end.
Answers
 (a) 5’ -UAACGGUACGAU-3’ .
 (b) Leu-Pro-Ser-Asp-Trp-Met.
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.