What’s So Special About DNA?

 DNA is one of the most boring

macromolecules imaginable; it is made
of only four building blocks and has a
perfectly monotonous structure.
 Worse yet, DNA just sits there - it doesn’t
catalyze reactions or build the cell or
organism.
 So, what’s so good about DNA?
 The answer lies in DNA’s ability to store
and copy information.
 How Can DNA Store &
Copy Information?

 Key properties that allow these neat

tricks are that DNA is a double
stranded molecule held together by
complementary bases that pair
through simple rules.
 DNA is also capable of occasional
change, and occasionally, change is
good.
 What’s a Gene?
 Such a simple question, such a
complicated answer.
 In part, the answer depends on what level
(molecular, cellular, organismal) we’re
interested in.
 A working definition (at the molecular
level) - A gene is a sequence of DNA
capable of producing some element of
biological function.
 Biological function can refer to many
things. It may, for example be a readily
observable trait, like skin color, a cellular
property, the length of the cell cycle, or a
molecular biological property, like the
Eukaryotes
 Mammalian genome contains at least
35000 individual genes, which
together account for less than 20%
of the DNA
 Function of the remaining DNA
( junk ) sequences is not yet clear
 Anatomy of a Gene

 Prokaryotic
 circular chromosomes

 polycistronic- one gene can code for

more than one protein

 genes can be transcribed together

 Eukaryotic
 on many chromosomes

 monocistronic

 made of exons & introns

 Variability in point mutation inβ
globin gene

Promote Nonsense &

r frameshift
defects mutations

EXON 1 EXON 2 EXON 3

CACCC
ATAA
CCAAT
IVS I IVS II

RNA processing Poly A

defects consensus
mutation
DNA
strands
 Each gene ( segment of DNA ) is
made of two complementary
strands ( A=T, G≡C )
 One strand ( sense strand ) contains the
specific base sequence which code for
protein sequence
 The other ( template ) is complementary
to the sense strand & us used to
synthesize the mRNA
Anatomy of a Gene
Promoters
 Promoters are characteristic sequences of
DNA, usually located in front ( upstream)
of the gene that is to be transcribed.
 In prokaryotes , simplest promoter found
in which two general sequences are found:
 One (6-8bp) located at -35bp upstream &
function in initial binding of RNA polymerase
 Second sequence (6-8 ) rich in A-T bases and
located at -10bp upstream ( TATA box 0r
pribnow box) helps in dissociation of DNA
strands
 In eukaryotes promoters are more
complicated, in addition there are
additional sequences which are known as
Middle Repetitive DNA
))1
Some middle repetitive DNA
consists of genes that specify
transfer of RNAs, or histone
proteins that are required in large
amounts in the cell. Other middle
repetitive DNA sequences have no
known useful function, but as they
represent a significant portion of
the genome, they may participate
in chromosomal rearrangements.
Middle Repetitive DNA
))2
The best characterized repetitive sequence in
humans is known as the Alu sequence.
Between 300,000 and 500,000 Alu 1 repeats
of about 300 base pairs each exist in the
human genome, comprising 3-6% of the total
genome. Individual repeats of the Alu
sequence may vary by 10-20% in identity. Alu
sequences also occur in monkeys and
rodents, and similar sequences occur in slime
molds and other animal phyla. In addition to
the Alu family, there are several other
families of middle repetitive DNA in the
 RNA
 Contains the bases G, A, C & U
 Single stranded
 Many types
1. mRNA which is direct carrier of genetic
information (constitutes 3% of total
cellular RNA)
2. rRNA, many types,5S,18S,5.8S
(constitutes 80% of total cellular RNA)
3. tRNA, carrier of activated amino acids
& has an anticodon for binding to
mRNA
4. Other types include hnRNA, snRNA
RNA polymerases (Eukaryotic)
 RNA poly 1- synthesis of rRNA
 RNA poly 11- synthesis of mRNA
 RNA poly 111- synthesis of rRNA & tRNA
 All have the following characteristics:
 Synthesize in 5’ – 3’ direction

 Template dependent & no requirement

for primer
 Large multisubunit enzymes

 Initiates polymerization at the promoter

region
 Hollo enzyme = core enzyme + sigma
factor
Base pairing in transcription
Basic & Not so Basic Transcription
 The coding strand (sense) carries the
code, while the template strand
(antisense) is the strand read by RNA
polymerase to make the 5’-3’
transcript.
 Hollo enzyme is the entire enzyme (5
subunits) including sigma factor.
 Sigma factor enables the polymerase
to recognize the promoter regions &
will be released following polymerase
Transcription

nucleus
Transcription

nucleus

cytosol
Transcription
 Requires the following
1. DNA directed RNA
polymerases
2. Transcription factors
( sigma factor, TF11D,
TF11A ….)
3. dNTP
Mechanism of Transcription
 Initiation
 Recognition of promoter region
 Binding at the promoter region
 Elongation
 Bond formation
 Translocation
 Termination
 Rho independent termination
 Rho dependent termination
Transcription
Initiation
 Involves the interaction of the RNA
polymerase with DNA at a site-
specific (promoters ) fashion,so that
a correct sequence of DNA can be
used as a template.
 Initiation in eukaryotes is far more
complicated.
 Transcription

Binding 
 RNA polymerase binds
to promoter  = specific
DNA sequence that
determines where RNA
polymerase binds and
starts transcription 
  Transcription

Binding 
RNA polymerase binds
to promoter  = upstream
(5 ') of transcription unit
Determines which DNA
strand is template 
Elongation
 Once RNA polymerase has bound to a
promoter, it begins selecting appropriate
complementary ribonucleotide and
forming phosphodiester bridges between
the nucleotide and the nascent chain
 Elongation is very rapid, occurring at the
rate of 40nt per second.
 The double stranded DNA must be
continually unwound, so that the template
strand is accessible
 Termination
 In prokaryotes, occurs by one of two
mechanisms:
 Rho-independent: A hairpin loop is formed
just before a sequence of six to eight uridine
(u) residues near the 3’ end of the newly
synthesized RNA. This secondary structure
dislodges the RNA polymerase from DNA
template, resulting in termination.
 Rho-dependent: Requires the action of a
protein factor called Rho, which has an ATP-
dependent helicase activity. The Rho protein
is believed to travel along the newly
synthesized RNA, chasing the RNA
polymerase and dislodges the RNA
Post-Transcriptional
Modification
 In Eukaryotes large primary transcripts
must be processed to a smaller size before
leaving the nucleus.
 In prokaryotes processing may involve
either removal of sequences from primary
transcript or removal and rejoining of
segments of the transcript.
 This process requires endoribonucleases
and exoribonucleases.
 In Eukaryotes, spliceosomes are required.
RNA Processing
mRNA Processing - Capping

 atypical nucleotide
added to the 5’ end
of the transcript
 7’ methylguanosine
 linked by 5’-5’

triphosphate bridge
to the 5’ end of the
RNA
mRNA Processing - Splicing
 mRNA Processing -
Tailing
 PolyA Polymerase Polyadenylation
PolyA Polymerase Polyadenylation
)eukaryotes)
Eukaryotic transcripts have polyA
tails. These arise due to cleavage
and addition of AAAAs. The signal for
cleavage and polyadenylation is
AAUAAA.
Factors involved:
 Cleavage and Polyadenylation
Specificity Factor CPSF
 Cleavage Stimulatory Factor CStF
mRNA Processing - Tailing
Replication vs Transcription
Similar 
 Complementary template 

TEMPLATE STRAND 
 
Replication vs Transcription
Similar 
 Complementary template 

 Synthesis 5 ' to 3 ' (add only to 3 ' end) 

 
Replication vs Transcription
Similar 
 Complementary template used 

 Synthesis 5 ' to 3 ' (add only to 3 ' end) 

 Triphosphate precursors provide energy for 

polymerization 
 Replication vs Transcription
Different 
 Deoxyribonucleotide precursors vs ribonucleotide 

 
 Replication vs Transcription
Different
 Deoxyribonucleotide precursors vs ribonucleotide 

 DNA polymerases proofread 
 Replication vs Transcription
Different 
 Deoxyribonucleotide precursors vs ribonucleotide 

 DNA polymerases proofread 

 DNA synthesis is discontinuous on the lagging 

strand 
 Replication vs Transcription
Different
 All DNA is duplicated once/ cell cycle during 

DNA synthesis 
 
Ribozymes RNAs that act like enzymes (1)
In some instances, RNAs have a
catalytic ability similar to the type of
activities previously ascribed only to
proteins. These special molecules,
known as ribozymes, possess a catalytic
activity and a substrate specificity
similar to those of proteinaceous
enzymes. The substrate specificity of a
ribozyme is determined via nucleotide
base pairing between complementary
sequences contained within the enzyme
Ribozymes RNAs that act like enzymes (2)
Just like enzymes that are proteins, the
ribozyme will cleave its substrate RNA at a
specific site and then release it, without
itself being consumed in the
reaction.Ribozymes are being considered as
possible therapeutic agents for diseases that
are caused by the inappropriate expression
of an RNA or the expression of a mutated
RNA. In these cases, the development of a
ribozyme that had specificty for a particular
RNA could result in the seletive degradation
of the substrate, eliminating it from the cell
End Of
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