DNA,RNA,

and
Gene Expression
 Learning Objectives
• Define and use correctly the following terms: Central Dogma,
cell cycle, mitosis, template, DNA polymerase,
semiconservative replication, replisome, primer, primase,
helicase, topoisomerase, DNA ligase, ssDNA-binding protein,
nuclease, DNA proofreading, exonuclease, chain catenation,
leading strand, lagging strand, Okazaki fragment, replication
fork, telomeres, telomerase, transcription, RNA polymerase,
promoter, operator, operon, initiation, elongation, termination,
transcription bubble, intron, exon, RNA splicing, spliceosome,
alternative splicing, ribozyme, 5’- cap, poly (A) tail, translation,
ribosome, codon, reading frame, anticodon, aminoacyl –tRNA,
aminoacyl –tRNA synthetase, mutation, substitution, deletion,
insertion, transition, transversion nonsense mutation, silent
mutation, mutagen, xeroderma pigmentosum, and reverse
transcriptase.
 Learning Objectives
• Describe the four stages of the cell cycle, relating
each stage to protein and nucleic acid synthesis.
• Explain why the DNA helix must be unwound for
its faithful replication. Describe in general terms
how the cell accomplishes this task.
• Describe the structure of eukaryotic genes in
terms of introns and exons.
• Relate the sequence of telomeres to their
structure and presumed biological function
 Learning Objectives
• Review how DNA polymerase achieves high
fidelity in DNA replication
• Describe the process of DNA replication, explain
how it is semiconservative, and compare synthesis
on leading and lagging strand
• Describe the process of transcription, including
how RNA polymerase functions. Identify the roles
of initiation and elongation factors. Describe the
processing of a transcript to produce a mature
mRNA.
 Learning Objectives
• Describe the process of translation, including how
the ribosome functions. State the roles of tRNA
and the tRNA synthetases in this process. Note
the special role of fMet-tRNAfMet
• Describe important structural features of the
ribosome and of tRNA.
• Describe what is meant by “degeneracy” in the
genetic code, and give examples of it.
• Describe how alternative splicing of a primary
RNA transcript can produce different
polypeptide chains from the same gene.
 Learning Objectives
• Compare the process of translation in eukaryotes
to that in prokaryotes, noting similarities and
differences.
• List the three basic types of mutation. Describe
common causes of mutations in a gene. Relate
this phenomena to errors made in DNA
replication and to chemical damage to DNA.
• List drugs that act to block action by DNA
polymerase, by RNA polymerase, and by the
ribosome.
 OUTLINE
• DNA Structure and Function
• RNA Structure and Function
• Organization in the Cell
• Prokaryotic Nucleic Acid
• Eukaryotic Nucleic Acid
• Central Dogma of Genetics
• DNA Replication
• DNA Mutation and Repair
• Transcription
• Translation
 DNA STRUCTURE
• Primary
• Secondary
• Tertiary
 DNA STRUCTURE
• Primary

Garett &Grisham. Biochemistry

 DNA STRUCTURE
• Primary
– Shorthand notation
 DNA STRUCTURE
• Primary
• Secondary
 DNA STRUCTURE
• Primary
• Secondary • Double-stranded, helical
conformation
• Anti-parallel
• Complementary base pairing
• Stability due to intermolecular
forces:
– Hydrogen Bonding
– Ionic interaction
– Hydrophobic interaction, pi-pi
complexation
• Sugar-phosphate group are outside
the helix.
 DNA STRUCTURE
• Primary
• Secondary
 DNA STRUCTURE
• Primary
• Secondary
 DNA STRUCTURE
• Primary
• Secondary
 DNA STRUCTURE
• Primary
• Secondary
 DNA STRUCTURE
A B Z
Overall proportion Short and broad Longer and thinner Elongated and slim

Rise per bp 2.3 Ao 3.320.19 Ao 3.8 Ao

Helix packing 25.5 Ao 23.7 Ao 18.4 Ao
diameter
Helix rotation sense Right-handed Right-handed Left-handed

Bp per turn 11 10 12

Helix axis location Major groove Through bp Minor groove
Major groove Extremely narrow Wide and with Flattened out on
proportion but very deep intermediate depth helix surface
Minor groove Very broad but Narrow and with Extremely narrow
proportion shallow intermediate depth but very deep
 DNA STRUCTURE
• Primary • Circular DNA
• Secondary – Don’t have 5’ & 3’ ends
• Tertiary • Supercoil DNA
– Twisting and coiling
of the double helix.
 DNA STRUCTURE
• Primary
• Secondary
• Tertiary
 Function of DNA

• Storage of genetic information

–Dictated by the sequence of
nucleotides
• Replication
–Provides mechanism for heredity
 RNA STRUCTURE
• Primary
• Secondary
• Tertiary
 RNA FORMS
• mRNA
• tRNA
• rRNA
 RNA FORMS
• mRNA
 RNA FORMS
• tRNA
A AMINO
ACID
C
C

mI Methylinosine
5'p end A Yeast alanine-tRNA
G C
I Inosine G C
UH2 Dihydrouridine G U
T Ribothymidine C G
G C
Ps Pseudouridine
U U
mG Methylguanosine G A UG G C U UA
m2G Dimethylguanosine UH2 C G C G mGU A G G C C
G
C U C C G G
G A G C G C m2G C T PsC
G UH2 C G A UH2
G
U A
Anticodon C G
• the 3 bases are specific for C G
the attached amino acid C G
U Ps
• base pair to the U mI
complementary triplet code I
G
C
on mRNA (the codon)
 RNA FORMS
• rRNA
 Nucleic Acid Organization
• Virus
• Prokaryotic Cell
• Eukaryotic Cell
 Nucleic Acid Organization
Virus
• Only one type of nucleic acid can exist
• Can be single or double-stranded
• Can be linear or circular
• Packaged in the head of the virus
 Nucleic Acid Organization
Prokaryotic

• DNA is the genetic material

• Double stranded DNA, circular or linear
chromosome
• Packaged into nucleoid- no nuclear membrane,
main function is to code for protein synthesis
• Each chromosome is associated with histone-like
proteins and RNA
 Nucleic Acid Organization
Eukaryotic
• DNA is the genetic material and it is linear and
double stranded
• They are packaged into nucleosomes- DNA +
histones
• Histones-small proteins, (+) charged at physiological
pH, thus they form ionic bonds with nucleic acids
– High in Lysine and Arginine
• Together with Mg2+ help neutralize DNA phosphate
groups
The Eukaryotic Cell Cycle
 Cyclin-Dependent Kinases
• Family of protein kinases
• Controls timing of cell cycle
• Associated with regulatory subunit- cyclin
• CDK targets:
• Laminin- protein that forms the filaments that
maintain the structure of the cell
• Falls apart when phosphorylated by CDK
• http://bcs.whfreeman.com/lodish5e/content/cat_0
10/21010-01.htm?v=chapter&i=21010.01&s=2
 Cyclin-Dependent Kinases Targets
Laminin
• protein that forms the filaments that maintain
the structure of the cell
• Falls apart when phosphorylated by CDK
Myosin
• When phosphorylated- contraction of actin-
myosin is halted
• When dephosphorylated – reactivation of actin-
myosin
THE CENTRAL DOGMA
 DNA REPLICATION
• ORIGIN OF REPLICATION
• REPLICATION FORK
• SEMICONSERVATIVE REPLICATION
• DIRECTION OF REPLICATION
• TOPOISOMERASES
• PROKARYOTIC DNA REPLICATION
• EUKARYOTIC DNA REPLICATION
DNA REPLICATION
• Origin of Replication
– region of DNA where
DNA starts to separate in
preparation for its
replication
– single in prokaryotic
DNA
– multiple in eukaryotic
DNA: consensus
sequence
• Replication Fork
– Where active synthesis
occurs
– Move in both directions as
DNA replication occurs
 DNA REPLICATION
• Semi-
conservative
Replication
– demonstrated
by Matthew
Meselson and
Franklin Stahl
 DNA REPLICATION
• Direction of Replication
– 5’3’ end
– the 2 parent strands are replicated in different
ways
– occurs semidiscontinuously
– leading strand-the strand that is being copied in
the direction of the advancing replication fork
• synthesized almost continuously
– lagging strand-the strand which is being copied
against the direction of the advancing fork
• synthesized discontinuously as Okazaki
fragment
DNA REPLICATION
• Topoisomerases
– Relieves supercoiling
– Topoisomerase I
– Topoisomerase II
 DNA REPLICATION
• Drug Inhibitors

genistein

Podophyllinic acid-2
ethylhydrazide

bacterial gyrase inhibitors eukaryotic topoisomerase

inhibitors
 Prokaryotic DNA Replication
• STEPS:
– Initiation
– Elongation
– Termination
 Prokaryotic DNA Replication
INITIATION
Two requirements for replication initiation are as
follows:
1. A nucleotide sequence that specifically binds
initiation proteins, and

2. A mechanism that generates a primer terminus

for DNA polymerase to extend.
 Prokaryotic DNA Replication
INITIATION
• Ori c - DNA replication origin of E. coli
– 245 bp long, with 4 repeats of a 9-bp sequence that
binds to an initiation protein
– three direct repeats of a 13-base-pair sequence
• rich in A and T
• contains binding sites for HU and IHF that facilitate DNA
bending
 Prokaryotic DNA Replication
INITIATION
1. Binding of 10-20 molecules of a DnaA/ATP complex.
2. The DnaC/DnaB protein complex binds to both forks of
the opened loop.
Helicase (DnaB) - open the structure further
3. DnaG (primase) binds and synthesizes RNA primers.
RNA polymerase may also synthesize some primers.
4. DNA polymerase III holoenzyme extends both leading
and lagging strands.
Prokaryotic DNA Replication
 Prokaryotic DNA Replication
ELONGATION
• Elongation of DNA strand

• Excision of RNA Primer and its Replacement with

DNA

• Sealing the Gap

Prokaryotic DNA Replication
ELONGATION OF DNA STRAND

DNA polymerase III

•polymerase function
– 5’3’
•proofreading function
–3’5’ Exonuclease activity only

Activity is blocked by proximity to a RNA

primer
 Prokaryotic DNA Replication
• EXCISION OF RNA PRIMER AND ITS
REPLACEMENT WITH DNA
–DNA polymerase I
• Locates space (“nick”) between the 3’ end of the newly
synthesized DNA and the 5’ end of the RNA primer
• Removes the ribonucleotide one at a time in 5’3’
manner
• as the ribonucleotide is removed it is replaced by a
deoxyribonucleotide
• as the DNA is synthesized it is also being proofread
causing a 3’  5’ exonuclease activity
• The whole process continues until all the gap has been
filled with DNA
• The enzyme also has a 5’  3’ exonuclease activity
Prokaryotic DNA Replication
 Prokaryotic DNA Replication

3’ end by DNA 5’ end by DNA

polymerase III polymerase I

• Sealing the Gap

DNA ligase
– DNA Ligase
– Catalyzes the formation of
phosphodiester bond
between the 5’ end of the
DNA synthesized by DNA
polymerase I and 3’ end of
the DNA synthesized by DNA
polymerase III
 Prokaryotic DNA Replication
• TERMINATION
–Collision of opposing replication forks
–Tus protein
• Binds to the terminator (Ter) sites which is flanked by 10
nearly identical non-palindromic sequence
• These termination sites are polar
• Prevents strand displacement by helicases
 Prokaryotic DNA Replication

• TERMINATION
–Unlinking of Newly synthesized
DNA
• DNA topoisomerase IV , a type II
topoisomerase
– Separates catenated chromosomes
– Transiently breaking both DNA
strands of one chromosome and
allows the other chromosome to pass
through the break
 Eukaryotic DNA Replication
• Initiation
• Elongation
• termination
 Eukaryotic DNA Replication
• Initiation
– Same as that in prokaryotes
 Eukaryotic DNA Replication
Elongation
• Elongation of DNA strand
• Excision of RNA Primer and its Replacement with DNA
• Sealing the Gap
Eukaryotic DNA Replication
O
• Elongation of DNA strand
H3C
NH –Pol α - has primase activity:
O O
N O
leading and lagging strands
N3 –Pol δ- elongate the leading
strand
–Pol ε- elongate the lagging strand
• Pol ε and Pol δ have 3’→5’
exonuclease activity
–Pol γ- replicates mitochondrial
DNA
–Reverse Transcriptase (RT)
O
H3C • RNA-directed DNA polymerase
NH
–Acts more like Polymerase I but it has
HO O
N O RNA as the template
–Inhibited by nucleoside analogs
–Resistance to these drugs arises rapidly
since RT lacks proofreading ability-
which makes the DNA replication
process highly error prone
Eukaryotic DNA Replication
• Excision of RNA Primer
and its Replacement with
DNA
– Pol β- same activity as Pol I in
prokaryotes
• Excise primers and carry out repair
• Sealing the Gap
– DNA Ligase
– Catalyzes the formation of
phosphodiester bond
– Requires ATP hydrolysis
 Eukaryotic DNA Replication
• Termination
Telomerase
– A type of DNA polymerase which synthesizes the telomeres
• telomeres - polyG end of DNAs; chromosome end
– When inhibited, the end of chromosomes would be shortened, hence
AGING
– Cancer cells have active telomerase
– Telomerase inhibitors-potential anti-tumor agents
 DNA MUTATION AND REPAIR
• DNA is constantly being subjected to
environmental insults that cause alteration or
removal of nucleotide bases
• chemicals or UV radiation
• bases can also be altered or lost spontaneously at
a rate of ~1000 bases per day
• Repair is necessary to preserve DNA’s integrity to
carry genetic information
 DNA REPAIR MECHANISMS
I. Direct Reversal of Damage
A.Repair of Damage by UV Light
B.Repair of Alkylated Nucleotides
II. Nucleotide Excision Repair
III. Recombination Repair
 DNA REPAIR MECHANISMS
I. Direct Reversal of Damage
A. Repair of Damage by UV Light
UV radiation (200-300 nm) promotes the formation of a
cyclobutyl ring between adjacent thymine residues on the same
DNA strand to form an intrastrand thymine dimer
Causes a disruption of DNA replication as well as transcription
Repair: photoreactivating enzymes (DNA photolyase)
light-absorbing endonucleases present in bacteria
not present in humans
 DNA REPAIR MECHANISMS
I. Direct Reversal of Damage
B. Repair of Alkylated Nucleotides
Exposure of DNA to alkylating agents (e.g.N-methyl-
N’-nitro-N-nitrosoguanidine, MNNG) yield O6-alkyl
guanine residues
H3C
NH
O
H3C
N NH N
N
N +
- N
O O NH N NH2
O
N-methyl-N’-nitro-N-nitrosoguanidine O -alkyl guanine residues
Causes incorporation of thymine
6
instead of cytosine
Repair: O6-methyl guanine-DNA
methyltransferase
– This mechanism is deficient in some cancer patients
 DNA REPAIR MECHANISMS
Nucleotide Excision Repair
– An oligonucleotide containing the lesion
is excised from the DNA and the resulting
single strand gap is filled in
– Carried out by DNA polymerase I in
prokaryotes and by DNA Polymerase β
in eukaryotes
– another mode of repair for pyrimidine
dimers
– Glycosylases - remove deaminated
purine (adenine and guanine) or
pyrimidine (cytosine) bases caused by
alkylating and deaminating agents
(HNO2 coming from nitrosamines, nitrates
and nitrites)
• Leaves an apurinic or apyrimidinic site
(AP)
– Repair of AP: endonuclease excision
from the DNA strand
– defective in individuals with xeroderma
pigmentosum
– Absent also in individuals with Cockayne
Syndrome
• Xeroderma pigmentosum

• Cockayne Syndrome
 DNA REPAIR MECHANISMS
III.Recombination
Repair
– postreplication repair
– Exchanges the
corresponding segments of
sister DNA strands,
thereby placing a gapped
DNA segment in
opposition to the
damaged strand, where
the gap can be filled and
sealed
 TRANSCRIPTION
• PROTEINS NEEDED
• STEPS INVOLVED:
– Initiation
– Elongation
– Termination
• POST-TRANSCRIPTIONAL MODIFICATIONS
– rRNA
– tRNA
– mRNA
• 5’ capping
• Poly-A tail addition
• mRNA splicing
 Prokaryotic Transcription
• PROTEINS NEEDED
– ρ factor
• termination factor,
• not part of RNA polymerase
– RNA Polymerase
• there is only one type in bacteria
• synthesizes all RNAs except RNA primers
• recognizes the promoter region
• also recognizes the termination region
• moves in 5’3’ end
• produces the primary transcript
 Prokaryotic Transcription
– RNA Polymerase
• Core enzyme- 4 subunits: 2 α, 1 β, and 1β’ are responsible for the 5’ 
3’ RNA polymerase activity
• lacks specificity-cannot recognize the promoter region on the DNA
template
• σ factor- σ subunit enables polymerase to recognize the promoter
sequence in the DNA
• Holoenzyme = σ factor + core enzyme
 Prokaryotic Transcription
–RNA Polymerase
–drug inhibitors:
 Prokaryotic Transcription
STEPS INVOLVED
– Initiation
• Binding of RNA polymerase to the promoter region
• Promoter region:
– Pribnow box- 6 nucleotides (TATAAT)
– -35 sequence-TTGACA, is also recognized by the RNA
polymerase
 Prokaryotic Transcription
STEPS INVOLVED
– Elongation
• Binding of RNA polymerase and σ
factor is released
• RNA polymerase does not require a
primer
– no endo- or exonuclease activity
• Use ribonucleotide triphosphates
• Can only occur in 5’3’ direction
• Binding of the enzyme to the DNA
template results in a local
unwinding of the DNA helix
• RNA polymerase creates a (+)
supercoil ahead of the helix and
leaves a (-) supercoil behind it
 Prokaryotic Transcription
STEPS INVOLVED
• Termination
– ρ-independent: requires newly synthesized RNA
to have 2 important structural features:
• formation of hairpin: complementary region in
the DNA is a palindrome, then a sequence rich in G
stabilizes the hairpin
• bonding of a string of U’s to A in the DNA
template: this bond is weak, facilitating the
separation of the newly synthesized RNA from the
DNA template
– ρ-dependent: ρ factor is required by RNA
polymerase to recognize the termination sequence;
this requires ATPase activity
 Eukaryotic Transcription
• Nuclear RNA Polymerases:
– RNA polymerase I-synthesizes the precursor of
large rRNAs in the nucleolus
– RNA polymerase II-synthesizes the precursors of
mRNAs as well as the small nuclear RNA
• used by some virus to synthesize viral RNA
– RNA polymerase III-synthesizes the small RNAs
including tRNAs, the 5S ribosomal RNA and some
snRNAs
• Mitochondrial RNA polymerase:
– resembles the bacterial RNA polymerase
 Eukaryotic Transcription
STEPS INVOLVED
• Initiation
– Binding of RNA polymerase to the promoter
region
• Promoter region:
– TATA box or Hogness Box-consensus sequence
– CAAT box-second consensus sequence
– One or both may serve as recognition site
– Binding of transcription factors to promoter
sequence of DNA, to RNA polymerase II and
to other transcription factors
• Enhancer Sequences: DNA sequences that
increase the rate of initiation of transcription by
RNA polymerase II
• Enhancer-binding proteins can bind with
transcription factors bound to the promoter
 Eukaryotic Transcription
STEPS INVOLVED
• Elongation
– RNA polymerase II synthesizes a transcript of DNA sequence
– RNA polymerase does not require a primer, no endo- or exonuclease
activity
– Uses ribonucleotide triphosphates
– Binding of the enzyme to the DNA template also results in a local
unwinding of the DNA helix
– drug inhibitor: -amanitin

-amanitin
 Eukaryotic Transcription
Termination
• After the end of the gene has been
reached, RNA polymerase II passes
through one or more AATAAA
sequences, which lie beyond the 3' end of
the coding region .
• The pre-mRNA, carrying this signal as
AAUAAA, is then cleaved by a special
endonuclease that recognizes the signal
and cuts at a site 11 to 30 residues to its 3'
side.
• A tail of polyriboadenylic acid, poly(A), as
much as 200 bases long, is added by a
special non-template-directed
polymerase.
Eukaryotic Transcription
POST TRANSCRIPTIONAL
MODIFICATIONS
– rRNA
• synthesized from long precursor molecules
called preribosomal (45S) RNAs
• eukaryotic 5S rRNA is synthesized by RNA
Polymerase III and modified separately

– tRNA
• addition of –CCA sequence by
nucleotidyltransferase on the 3’ terminal
end of tRNAs
• modification of bases at specific positions to
produce “unusual bases”
 Eukaryotic Transcription
POST TRANSCRIPTIONAL MODIFICATIONS
– mRNA
• 5’ capping
• Addition of poly-A tail
• mRNA splicing
 TRANSLATION
• GENETIC CODE
• MUTATIONS
• STEPS INVOLVED:
– Initiation
– Elongation
– Termination
• POST-TRANSLATIONAL MODIFICATIONS
– Trimming
– Covalent alterations
 TRANSLATION
• GENETIC CODE
– Highly degenerate
– Arrangement of the code table is
nonrandom
– commaless and
overlapping
– A triplet code
 TRANSLATION
MUTATIONS
1. Point Mutation
• one base pair replaces another
a. transition
– purine  purine
– pyrimidine  pyrimidine

b. transversion
– purine  pyrimidine
• results to:
– silent mutation
– missense mutation
– nonsense mutation
2. Insertion Mutation
• results to frameshift mutation
 TRANSLATION
• Initiation
– Shine-Dalgarno Sequence-
sequence in the mRNA located
6-10 bases upstream the
initiating codon near its 5’ end
– Initiating Codon- AUG
• Elongation
– translocation-movement of
the ribosome to the next 3
codons towards the 3’ end of
mRNA
– causes the release of the
uncharged tRNA
– movement of the peptidyl-
RNA to the P site
 TRANSLATION
• Termination
– termination codon moves
into the A site
– UAG, UGA, UAA
– these codons are
recognized by release
factors which bind to GTP
 TRANSLATION
• Post-translational Modifications
– Trimming
• Activation of digestive zymogens
• Covalent alterations
– Phosphorylation-carried out by protein kinases
– Glycosylation
» N-linked-carbohydrates are attached to the N of Asn
» O-linked-carbohydrates are attached to the OH of Thr or Ser
» Hydroxylation-Pro and Lys residues of α-chains of collagen are
hydroxylated in the ER
» Other covalent modification-binding of biotin to carboxylase
END OF LECTURE
 QUIZ
• 1. the principle that information in the cell passes
from DNA to RNA and then to protein.
• 2. the 3 contiguous ribonucleotides in a tRNA
molecule that bind to the cognate codon in
mRNA.
• 3. enzyme catalyzing the condensation of
aspartate and carbamoyl phosphate to form N-
carbamoyl aspartate
• 4. site on a chromosome serving as the
attachment point for a spindle in mitosis
• 5. mutation resulting from the removal of one or
more nucleotides in a DNA sequence in a
chromosome
• 6. purine or pyrimidine base covalently joined to
deoxyribose
• 7. Enzyme responsible for the synthesis of dTMP
from dUMP
• 8. synthesis of biochemicals from simple precursors
• 9. the complete set of genes, control sequences
and other genetic information for a cell, an
organism or a virus.
• 10. a hairpin loop is an example of what level of
organization of ribonucleic acids