DNA Replication 09-10

Heavenly Father, in whom
we live and move and

have our being: We
humbly pray thee so to
guide and govern us by
thy Holy Spirit, that in all
the cares and occupations
of our life we may not
forget thee, but may
remember that we are
ever walking in thy sight;
through Jesus Christ our
Lord. Amen
DNA Replication and Transcription
a). DNA replication
i). Cell cycle/ semi-conservative replication
ii). Initiation of DNA replication
iii). Discontinuous DNA synthesis
iv). Components of the replication apparatus
b). Transcription
i). Process of RNA transcription
ii). Types of RNA molecules
iii). RNA processing
Central Dogma
Replication DNA Reverse

Transcription
Transcription
RNA
Translation
Protein
DNA: structure
DNA is double stranded
DNA strands are antiparallel
G-C pairs have 3 hydrogen bonds

A-T pairs have 2 hydrogen bonds
Cellular DNA is almost exclusively
B DNA
B-DNA has ~10.5 bp/turn of the

helix
The mammalian cell cycle
DNA synthesis and

Rapid growth and histone synthesis
preparation for
DNA synthesis
S
phase
G0 G1
phase
Quiescent cells
G2
phase
M
phase
Growth and
preparation
for
cell division
Nucleosomes and DNA Replication
• SYNTHESIS OF HISTONES OCCURS DURING
THE S PHASE
• HISTONES ASSEMBLE INTO OCTAMER

STRUCTURES
DNA Replication
 DNA replication is the process by which the
genetic material is copied
 The original DNA strands are used as templates for
the synthesis of new strands
 It occurs very quickly, very accurately and at the

appropriate time in the life of the cell
DNA REPLICATION is SIMILAR BETWEEN
PROKARYOTES & EUKARYOTES :
-however, the E coli genome is a single circular DNA molecule:
- the human genome is on 46 linear molecules (chromosomes)
STRUCTURAL OVERVIEW OF DNA
REPLICATION
 DNA replication relies on the complementarity of
DNA strands
 The AT/GC rule or Chargaff’s rule
 The process can be summarized as such

 The two DNA strands come apart
 Each serves as a template strand for the synthesis of
new strands
 The two newly-made strands = daughter strands

 The two original ones = parental strands
Identical
base
sequences
Figure 11.1
11-4
Models of DNA Replication
 In the late 1950s, three different mechanisms
were proposed for the replication of DNA
 Conservative model
 Both parental strands stay together after DNA replication
 Semiconservative model
 The double-stranded DNA contains one parental and one
daughter strand following replication
 Dispersive model
 Parental and daughter DNA are interspersed in both strands
following replication
Three
Models
 Three
theoretical
models
proposed
 Each had
different
expectations for
products of one
or two rounds
of replications
Matt Meselson and Franklin Stahl
 1958
 E. coli
 Naturally occurring (N14) and heavy (N15)
isotopes of nitrogen
 CsCl
 Ultracentrifuge
Interpreting the Data
After ~ two generations, DNA is of After one generation,

two types: “light” and “half-heavy” DNA is “half-heavy”
This is consistent with only This is consistent with both semi-

the semi-conservative model conservative and dispersive models
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-12
Requirements for DNA Replication
1. TEMPLATE
Both DNA Strands Could Be Expressed
DNA
5’ 3’
3’ 5’
DNA
DNA is synthesized in 5’→ 3’ direction
2. Substrate
3. Enzymes
4. Primers
DNA Polymerases
 DNA polymerases are the enzymes that catalyze
the attachment of nucleotides to make new DNA
 In E. coli there are five proteins with polymerase

activity
 DNA pol I, II, III, IV and V
 DNA pol I and III

 Normal replication
 DNA pol II, IV and V
 DNA repair and replication of damaged DNA
11-21
DNA Polymerases
 DNA pol I
 Composed of a single polypeptide
 Removes the RNA primers and replaces them with DNA
 DNA pol III

 Composed of 10 different subunits (Table 11.2)
 The a subunit synthesizes DNA
 The other 9 fulfill other functions
 The complex of all 10 is referred to as the DNA pol III
holoenzyme
11-23
DNA Polymerase III:
subunit composition
DNA Polymerase III is a Processive
Enzyme
 DNA polymerase III remains attached to the
template as it is synthesizing the daughter strand
 This processive feature is due to several different

subunits in the DNA pol III holoenzyme
 b subunit is in the shape of a ring
 It is termed the clamp protein
 g subunit is needed for b to initially clamp onto the DNA
 It is termed the clamp-loader protein
 d, d’ and y subunits are needed for the optimal function
of the a and b subunits
Moran et al 2006
The Reaction of DNA Polymerase
 DNA polymerases catalyzes a phosphodiester bond
between the
 Innermost phosphate group of the incoming
deoxynucleoside triphosphate
 AND
 3’-OH of the sugar of the previous deoxynucleotide

 In the process, the last two phosphates of the
incoming nucleotide are released
 In the form of pyrophosphate (PPi)
Figure 11.10
Innermost
phosphate
Bacterial Replication
• E. coli chromosome is circular, double-stranded DNA

(4.6x103 kilobase pairs)
• Replication begins at a unique site (origin)
• Proceeds bidirectionally until the two replication complexes
meet (termination site)
• Replisome - protein machinery for replication (one
replisome at each of 2 replication forks)
• The replication forks eventually meet at the opposite side of
the bacterial chromosome
• This ends replication
Figure 11.4
Initiation of Replication
 The origin of replication in E. coli is termed oriC
 origin of Chromosomal replication
 Three types of DNA sequences in oriC are

functionally significant
 AT-rich region
 DnaA boxes
 GATC methylation sites
 Other proteins such as HU and  DNA replication is initiated by the binding of
IHF also bind. DnaA proteins to the DnaA box sequences
 This causes the region to  This binding stimulates the cooperative
wrap around the DnaA binding of an additional 20 to 40 DnaA
proteins and separates the proteins to form a large complex
AT-rich region
11-17
Composed of six subunits
Travels along the DNA in
the 5’ to 3’ direction
Uses energy from ATP
Bidirectional replication
Keep the parental
Breaks the hydrogen strands apart
bonds between the
two strands
Alleviates
supercoiling
Synthesizes an
RNA primer
Figure 11.7
DNA polymerases cannot
initiate DNA synthesis
Problem is overcome by
the RNA primers
synthesized by primase
Problem is overcome by
synthesizing the 3’ to 5’
strands in small fragments
DNA polymerases can

attach nucleotides only in
Unusual features of DNA polymerase function

Diagram of the replication fork
DNA Replication Complexes
 DNA helicase and primase are physically bound to
each other to form a complex called the primosome
 This complex leads the way at the replication fork
 The primosome is physically associated with the

DNA polymerase holoenzyme forming the replisome
Replisome DNA synthesis
 DNA pol I removes the RNA primers and fills the
resulting gap with DNA
 It uses its 5’ to 3’ exonuclease activity to digest the RNA
and its 5’ to 3’ polymerase activity to replace it with DNA
 After the gap is filled a covalent bond is still

missing
 DNA ligase catalyzes a phosphodiester bond

 Thereby connecting the DNA fragments
DNA Replication Complexes
 Lagging strand synthesis is summarized as such:
 The lagging strand is looped

 This allows the attached DNA polymerase to synthesize the
Okazaki fragments in the normal 5’ to 3’ direction
 Upon completion of an Okazaki fragment, the enzyme

releases the lagging template strand
 Another loop is then formed
 This processed is repeated over and over again

Proofreading Mechanisms
 1. Instability of mismatched pairs
 Complementary base pairs have much higher stability
than mismatched pairs
 This feature only accounts for part of the fidelity
 It has an error rate of 1 per 1,000 nucleotides
 2. Configuration of the DNA polymerase active site

 DNA polymerase is unlikely to catalyze bond formation
between mismatched pairs
 This induced-fit phenomenon decreases the error rate to
a range of 1 in 100,000 to 1 million
Proofreading Mechanisms
 3. Proofreading function of DNA polymerase
 DNA polymerases can identify a mismatched nucleotide

and remove it from the daughter strand
 The enzyme uses its 3’ to 5’ exonuclease activity to

remove the incorrect nucleotide
 It then changes direction and resumes DNA synthesis in

Termination of Replication
 Opposite to oriC is a pair of termination sequences
called ter sequences
 These are designated T1 and T2
 The protein tus (termination utilization substance)

binds to these sequences
 It can then stop the movement of the replication forks
E. coli Tus bound to DNA
• Tus binds to specific

sequences at the
termination site of DNA
replication
• Tus blocks movement of
the replisome
Termination of Replication
 DNA replication ends when oppositely advancing
forks meet (usually at T1 or T2)
 Finally DNA ligase covalently links all four DNA
strands
 DNA replication often results in two intertwined
molecules
 Intertwined circular molecules are termed catenanes
 These are separated by the action of topoisomerases
 QUICK REVIEW
The Eukaryotic DNA Replication
 Eukaryotic DNA replication is not as well
understood as bacterial replication
 The two processes do have extensive similarities,

 The bacterial enzymes described in Table 1.1 have also been
found in eukaryotes
 Nevertheless, DNA replication in eukaryotes is more

complex
 Large linear chromosomes
 Tight packaging within nucleosomes
 More complicated cell cycle regulation
Multiple Origins of Replication
 Eukaryotes have long linear chromosomes
 They therefore require multiple origins of replication
 To ensure that the DNA can be replicated in a reasonable time
 In 1968, Huberman and Riggs provided evidence

for the multiple origins of replication
 DNA replication proceeds bidirectionally from many

origins of replication

Bidrectional
DNA synthesis
Replication
forks will
merge
Figure 11.20
Part (b) shows a micrograph of a replicating DNA chromosome
 The origins of replication found in eukaryotes have
some similarities to those of bacteria
 Origins of replication in Saccharomyces cerevisiae are

termed ARS elements (Autonomously Replicating
Sequence)
 They are 100-150 bp in length
 They have a high percentage of A and T
 They have three or four copies of a specific sequence
 Similar to the bacterial DnaA boxes
 Origin recognition complex (ORC)
 A six-subunit complex that acts as the initiator of
eukaryotic DNA replication
 It appears to be found in all eukaryotes
 Requires ATP to bind ARS elements
 Single-stranded DNA stimulates ORC to hydrolyze ATP

Eukaryotes Contain Several
Different DNA Polymerases
 Mammalian cells contain well over a dozen different
DNA polymerases
 Four: alpha (a), delta (d), epsilon (e) and gamma

(g) have the primary function of replicating DNA
 a , d and e  Nuclear DNA
 g  Mitochondrial DNA
11-69
Properties of DNA polymerases
DNA polymerases of humans
a b g d e
Location nucl nucl mito nucl nucl
Replication yes no yes yes yes
Repair no yes no yes yes3
Functions
5’ to 3’ polymerase yes yes yes yes yes
3’ to 5’ exonuclease no no yes yes yes
5’ to 3’ exonuclease1 no no no no no
Primase yes no no no no
Associates with PCNA2 no no no yes yes
Processivity low high
Strand synthesis lagging* repair both leading* lagging*
1
activity present in associated proteins
2
Proliferating Cell Nuclear Antigen – “sliding clamp”
3
involved in transcription-linked DNA repair
 DNA pol a is the only polymerase to associate with
primase
 The DNA pol a /primase complex synthesizes a short
RNA-DNA hybrid
 10 RNA nucleotides followed by 20 to 30 DNA nucleotides
 This is used by DNA pol d or e for the processive
elongation of the leading and lagging strands
 Current evidence suggests a greater role for DNA pol d
 The exchange of DNA pol a for d or e is called a

polymerase switch
 It occurs only after the RNA-DNA hybrid is made
 Refer to Figure 11.21

Figure 11.21
Proteins at the replication fork in humans
helicase
leading strand
3’
PCNA pol d 5’
5’
3’
DNA ligase
5’ to 3’ exo
SSB associated
with the
complex pol a
(or pol d)
pol e
topoisomerases I and II
primase activity
lagging strand associated with pol a
 DNA polymerases also play a role in DNA
repair
 DNA pol b is not involved in DNA replication
 It plays a role in base-excision repair
 Removal of incorrect bases from damaged DNA
 Recently, more DNA polymerases have been

identified
 Lesion-replicating polymerases
 Involved in the replication of damaged DNA
 They can synthesize a complementary strand over the
abnormal region
Nucleosomes and DNA Replication
 Replication doubles the amount of DNA
 Therefore the cell must synthesize more histones to
accommodate this increase
 Synthesis of histones occurs during the S phase

 Histones assemble into octamer structures
 They associate with the newly made DNA very near the
replication fork
 Thus following DNA replication, each daughter

strand has a mixture of “old” and “new” histones
 Refer to Figure 11.22
Telomeres and DNA Replication
 Linear eukaryotic chromosomes have telomeres at
both ends
 The term telomere refers to the complex of

telomeric DNA sequences and bound proteins
 Telomeric sequences consist of
 Moderately repetitive tandem arrays
 3’ overhang that is 12-16 nucleotides long
Figure 11.23
 Telomeric sequences typically consist of

 Several guanine nucleotides
 Often many thymine nucleotides
11-76
11-77
 DNA polymerases possess two unusual features
 1. They synthesize DNA only in the 5’ to 3’ direction
 2. They cannot initiate DNA synthesis
 These two features pose a problem at the 3’ end of
linear chromosomes
Figure 11.24
 Therefore if this problem is not solved
 The linear chromosome becomes progressively shorter
with each round of DNA replication
 Indeed, the cell solves this problem by adding DNA
sequences to the ends of telomeres
 This requires a specialized mechanism catalyzed
by the enzyme telomerase
 Telomerase contains protein and RNA
 The RNA is complementary to the DNA sequence found
in the telomeric repeat
 This allows the telomerase to bind to the 3’ overhang
11-79
Step 1 = Binding
The binding-
polymerization- Step 2 = Polymerization
translocation cycle can
occurs many times
This greatly lengthens

one of the strands
Step 3 = Translocation
The complementary
strand is made by primase,
DNA polymerase and ligase
RNA primer
Figure 11.25
GENE TRANSCRIPTION AND
RNA MODIFICATION
OVERVIEW OF TRANSCRIPTION
 Transcription literally means the act or

process of making a copy
 In genetics, the term refers to the copying

of a DNA sequence into an RNA sequence
What is transcribed?
Not all of the
Sequence of nucleotides in DNA in genes is
human beta-globin gene used to encode the
The DNA sequences proteins that they
highlighted in color show specify; much of
the three regions of the the rest is
gene that specify the concerned with
amino acid sequence. determining when,
and in what
amounts, the
protein encoded is
made – regulatory
regions of genes
CISTRON
• Start codon: specifies the first amino acid in
a
protein sequence, usually a formylmethionine
(in bacteria) or a methionine (in eukaryotes)
Signals the end of • Bacterial mRNA may be polycistronic, which

protein synthesis means it encodes two or more polypeptides
Eukaryotic:monocistronic
Definitions and Conventions
The 3 Stages of Transcription
Initiation
 The promoter functions as a recognition
site for transcription factors
 The transcription factors enable RNA
polymerase to bind to the promoter
forming a closed promoter complex
 Following binding, the DNA is denatured
into a bubble known as the open
promoter complex, or simply an open
complex
Elongation
 RNA polymerase slides along the DNA
in an open complex to synthesize the
RNA transcript
Termination
 A termination signal is reached that
causes RNA polymerase to dissociated
from the DNA
RNA Transcripts Have Different
Functions
 Once they are made, RNA transcripts play different
functional roles
 A structural gene is a one that encodes a

polypeptide
 When such genes are transcribed, the product is an RNA
transcript called messenger RNA (mRNA)
 Well over 90% of all genes are structural genes

Types of RNA % total cellular
RNA mass
Ribosomal RNA (rRNA) 85

-the RNA structural component of the ribosome
-in eukaryotes there are 4 major forms: 28S, 18S and 5.8S and 5S
-in prokaryotes there are only 3: 23S, 16S, and 5S
“S” refers to a Svedberg Unit, which is a measure of size based upon the
molecular sedimentation rate during ultracentrifugation
Messenger RNA (mRNA) 2

-the RNA that transfers genetic information stored in DNA into a form
useable for protein synthesis
Transfer RNA (tRNA) 12

-assists in decoding the information contained within mRNA during
translation by recruiting the correct amino acid to the growing peptide
chain
Other forms (snRNA, snoRNA) 1
-small nuclear RNAs that participate in RNA processing
RNA Transcripts Have Different
Functions
 The RNA transcripts from nonstructural genes are
not translated
 They do have various important cellular functions
 In some cases, the RNA transcript becomes part of a

complex that contains protein subunits
 For example
 Ribosomes
 Spliceosomes
 Signal recognition particles
12-10
TRANSCRIPTION IN BACTERIA
Promoters
 Promoters are DNA sequences that “promote” gene
expression
 More precisely, they direct the exact location for the
initiation of transcription
 Promoters are typically located just upstream of the
site where transcription of a gene actually begins
 The bases in a promoter sequence are numbered in
relation to the transcription start site
12-13
Sequence elements that play
a key role in transcription
Bases preceding
this are numbered
in a negative
direction
There is no base
numbered 0
Bases to the right are

numbered in a
positive direction
Sometimes termed
the Pribnow box,
after its discoverer
Figure 12.3 The conventional numbering system of promoters
The most commonly
occurring bases
Figure 12.4 Examples of –35 and –10 sequences within a

variety of bacterial promoters
Initiation of Transcription
 Binding of the RNA Polymerase to the binding
sites (prokaryotic)
E. coli RNA polymerase
36.5kD
151kD 11kD Core

155kD complex
36.5kD
70kD
HOLOENZYME
Prokaryotic RNA polymerase structure
RNA polymerase of bacteria is a multisubunit protein
Subunit Number Role
a 2 uncertain
b (Rifampicin target) 1 forms phosphodiester bonds
b’ 1 binds DNA template
s 1 recognizes promoter and
facilitates initiation
a2bb’s a2bb’ + s
holoenzyme core polymerase sigma factor
 The binding of the RNA polymerase to the
promoter forms the closed complex
 Then, the open complex is formed when the

TATAAT box is unwound
 A short RNA strand is made within the open

complex
 The sigma factor is released at this point
 This marks the end of initiation
 The core enzyme now slides down the DNA to

synthesize an RNA strand
12-20
Similar to the
synthesis of DNA
via DNA
polymerase
Figure 12.7
Mechanism of RNA synthesis
RNA RNA
A=T A = T
U=A U=A
• RNA synthesis usually initiated with ATP or GTP (the first nucleotide)
• RNA chains are synthesized in a 5’ to 3’ direction
Termination of Bacterial
Transcription
 Termination is the end of RNA synthesis
 It occurs when the short RNA-DNA hybrid of the open
complex is forced to separate
 This releases the newly made RNA as well as the RNA polymerase
12-24
Rho-independent transcriptional termination
5’
Nelson & Cox, 2005, p. 1001

Termination
Rho Dependent
Terminator
RNA
Pol.
5’
RNA
r Help, rho RNA
hit me! Pol.
RNA
5’
r Pol.
r
RNA
5’
Eukaryotic Transcription
Nuclear
Cytoplasm
pores
DNA
Transcription
RNA
RNA
Processing
mRNA G AAAAAA G AAAAAA
Export
Nucleus
TRANSCRIPTION IN
EUKARYOTES
 Many of the basic features of gene
transcription are very similar in bacteria and
eukaryotes
 However, gene transcription in eukaryotes is

more complex
 Larger organisms
 Cellular complexity
 Multicellularity
Eukaryotic RNA Polymerases
 Nuclear DNA is transcribed by three different RNA
polymerases
 RNA pol I
 Transcribes all rRNA genes (except for the 5S rRNA)
 RNA pol II
 Transcribes all structural genes
 Thus, synthesizes all mRNAs
 Transcribes some snRNA genes
 RNA pol III
 Transcribes all tRNA genes
 And the 5S rRNA gene
Sequences of Eukaryotic Structural
Genes
 Eukaryotic promoter sequences are more variable
and often more complex than those of bacteria
 For structural genes, at least three features are

found in most promoters
 Transcriptional start site
 TATA box
 Regulatory elements
Figure 12.11 Usually an
adenine
 The core promoter is relatively short

 It consists of the TATA box
 Important in determining the precise start point for
transcription
 The core promoter by itself produces a low level of

transcription
 This is termed basal transcription
Figure 12.11 Usually an
adenine
 Regulatory elements affect the binding of RNA

polymerase to the promoter
 They are of two types
 Enhancers
 Stimulate transcription
 Silencers
 Inhibit transcription
 They vary in their locations but are often found in the

–50 to –100 region
Sequences of Eukaryotic Structural
Genes
 Factors that control gene expression can be divided
into two types, based on their “location”
 cis-acting elements
 DNA sequences that exert their effect only on nearby
genes
 Example: TATA box, enhancers and silencers
 trans-acting elements
 Regulatory proteins that bind to such DNA sequences
RNA Polymerase II and its
Transcription Factors
 Three categories of proteins are required for basal
transcription to occur at the promoter
 RNA polymerase II
 Five different proteins called general transcription factors
(GTFs)
 A protein complex called mediator
 Figure 12.12 shows the assembly of transcription

factors and RNA polymerase II at the TATA box
12-35
Figure 12.12
12-36
A closed complex
Figure 12.12
 TFIIH plays a major role in the

 One subunit hydrolyzes ATP and phosphorylates a
formation of the open complex domain in RNA pol II known as the carboxyl terminal
 It has several subunits that
domain (CTD)
perform different functions
 This releases the contact between TFIIB and
RNA pol II
 Other subunits act as helicases
 Promote the formation of the open complex
RNA pol II can now

proceed to the
elongation stage
Released after
the open
complex is
formed
Initiation at RNA pol III promoters
Positions of promoter elements in

tRNA and 5S rRNA genes.
Initiation of transcription of a tRNA gene:

1. The TFIIIC transcription factor binds via
recognition of the A and B sites
2. This permits subsequent binding of the
trimeric TFIIIB factor immediately
upstream of the transcription start site.
3. In response to TFIIIB binding, RNA
polymerase III is recruited and initiates
transcription.
In the case of 5S rRNA genes, the process is

similar, except that an additional factor,
TFIIIA, is required. TFIIIA binds the C box,
which permits subsequent binding of TFIIIB
and TFIIIC, then recruitment of RNA pol III.
Initiation at RNA Pol I promoters
One model:
1. Two identical subunits of the
upstream binding factor bind to the
upstream core element and the
core promoter element.
2. Protein:protein interactions
between UBF molecules force
these two DNA sequences to come
into close proximity.
3. This enables subsequent binding of
selectivity factor I, which consists
of four subunits.
4. Ultimately, this stabilized structure
permits binding of other factors
(not shown), and finally RNA pol I.
RNA MODIFICATION
 Analysis of bacterial genes in the 1960s and 1970
revealed the following:
 The sequence of DNA in the coding strand corresponds
to the sequence of nucleotides in the mRNA
 This in turn corresponds to the sequence of amino acid in
the polypeptide
 This is termed the colinearity of gene expression
 Analysis of eukaryotic structural genes in the late

1970s revealed that they are not always colinear
with their functional mRNAs
12.4 RNA MODIFICATION
 Instead, coding sequences, called exons, are
interrupted by intervening sequences or introns
 Transcription produces the entire gene product

 Introns are later removed or excised
 Exons are connected together or spliced
 This phenomenon is termed RNA splicing

 It is a common genetic phenomenon in eukaryotes
 Occurs occasionally in bacteria as well
Processing Eukaryotic mRNA
5’ Untranslated Region 3’ Untranslated Region
Protein Coding Region

5’5’ G Int. 11 Exon
Exon 1Exon Exon22 Exon 3’
Int. 23 Exon 3 AAAAA 3’
5’ Cap .1 .2 3’ Poly A Tail

t t
In In
RNA processing achieves three things:

1 Removal of introns
2 Addition of a 5’ cap
3 Addition of a 3’ tail
 This signals the mRNA is ready to move out
of the nucleus and may control its life span
in the cytoplasm
is then methylated by 2'-O-methyltransferases.
Tailing
 Most mature mRNAs have a string of adenine
nucleotides at their 3’ ends
 This is termed the polyA tail
 The polyA tail is not encoded in the gene sequence

 It is added enzymatically after the gene is completely
transcribed
 The attachment of the polyA tail is shown in

Figure 12.20
Figure 12.20
Consensus sequence in
higher eukaryotes
Appears to be important in the Length varies between

stability of mRNA and the species
translation of the polypeptide From a few dozen adenines
to several hundred
Pre-mRNA Splicing
 The spliceosome is a large complex that splices
pre-mRNA
 It is composed of several subunits known as

snRNPs (pronounced “snurps”)
 Each snRNP contains small nuclear RNA and a set of
proteins
 In eukaryotes, the
transcription of structural
genes, produces a long
transcript known as pre-
mRNA
 Also as heterogeneous nuclear
RNA (hnRNA)
 This RNA is altered by splicing

and other modifications,
before it leaves the nucleus
 Splicing in this case requires

the aid of a multicomponent
structure known as the
spliceosome Figure 12.16
12-62
Pre-mRNA Splicing
 The subunits of a spliceosome carry out several
functions
 1. Bind to an intron sequence and precisely recognize

the intron-exon boundaries
 2. Hold the pre-mRNA in the correct configuration
 3. Catalyze the chemical reactions that remove introns

and covalently link exons
12-71
 Intron RNA is defined by particular sequences within the
intron and at the intron-exon boundaries
 The consensus sequences
Corresponds to the boxed

adenine in Figure 12.22
Sequences shown in bold
are highly conserved
Figure 12.21 Serve as recognition sites for

the binding of the spliceosome
Intron loops out and
exons brought closer
together
Intron will be degraded and
the snRNPs used again
Figure 12.22
DNA Binding Domains
 Transcription factors exhibit a number of
different motifs found in the area known to
bind DNA:
 Zinc finger -First found in TFIIIA
 Helix-turn-helix - First described from phage
receptors
 Amphipathic Helix-loop-helix - Identified in
some development regulators
 Leucine zipper - Held together by
interactions between leucine amino acids
Zinc Fingers
≈6
Amino
acids ≈ 23
Amino
acids
C H C H C H
2- 4
Zn++ Amino Zn++ Zn++
acids
C H C H C H
7 - 8 Amino acid linker

Transcription Inhibitors
actinomycin D, acridine:
-intercalate between successive G=C base
pairs in duplex DNA
-inhibit transcriptional elongation in pro- and
eukaryotes
rifampicin:
-binds the b subunit of bacterial RNA
polymerase
-blocks promoter clearance (elongation)
a-amanitin:
-produced by fungus Amanita phalloides
(death cap mushroom)
-potent inhibitor of RNA pol II and weak Nelson & Cox, 2005, p. 1006
inhibitor of RNA pol III
inhibitors of Replication
 Novobiocin
 Nalidixic acid and ciprofloxacin
 Camptothecin
 2,3 deoxyinosine
 cytosine arabinoside (cytarabin, araC)
Give Us, O Lord, A Steadfast Heart
Give us, O Lord, a steadfast heart, which no unworthy affection

may drag downwards;
Give us an unconquered heart, which no tribulation can wear out;
Give us an upright heart, which no unworthy purpose may tempt

aside.
Bestow upon us also, O Lord our God, understanding to know

you, diligence to seek you, wisdom to find you, and a faithfulness
that may finally embrace you; through Jesus Christ our Lord.
St. Thomas Aquinas

DNA Replication 09-10

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DNA Replication 09-10

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Heavenly Father, in whom

we live and move and

Replication DNA Reverse

DNA is double stranded

DNA strands are antiparallel

G-C pairs have 3 hydrogen bonds

Cellular DNA is almost exclusively

B-DNA has ~10.5 bp/turn of the

DNA synthesis and

• HISTONES ASSEMBLE INTO OCTAMER

 It occurs very quickly, very accurately and at the

 The process can be summarized as such

 The two newly-made strands = daughter strands

After ~ two generations, DNA is of After one generation,

This is consistent with only This is consistent with both semi-

 In E. coli there are five proteins with polymerase

 DNA pol I and III

 DNA pol III

 This processive feature is due to several different

 3’-OH of the sugar of the previous deoxynucleotide

• E. coli chromosome is circular, double-stranded DNA

 Three types of DNA sequences in oriC are

DNA polymerases can

Unusual features of DNA polymerase function

 The primosome is physically associated with the

 After the gap is filled a covalent bond is still

 DNA ligase catalyzes a phosphodiester bond

 The lagging strand is looped

 Upon completion of an Okazaki fragment, the enzyme

 This processed is repeated over and over again

 2. Configuration of the DNA polymerase active site

 DNA polymerases can identify a mismatched nucleotide

 The enzyme uses its 3’ to 5’ exonuclease activity to

 It then changes direction and resumes DNA synthesis in

 The protein tus (termination utilization substance)

• Tus binds to specific

 The two processes do have extensive similarities,

 Nevertheless, DNA replication in eukaryotes is more

 In 1968, Huberman and Riggs provided evidence

 DNA replication proceeds bidirectionally from many

 Origins of replication in Saccharomyces cerevisiae are

 Requires ATP to bind ARS elements

 Single-stranded DNA stimulates ORC to hydrolyze ATP

 Four: alpha (a), delta (d), epsilon (e) and gamma

 The exchange of DNA pol a for d or e is called a

 Refer to Figure 11.21

 Recently, more DNA polymerases have been

 Synthesis of histones occurs during the S phase

 Thus following DNA replication, each daughter

 The term telomere refers to the complex of

 Telomeric sequences typically consist of

This greatly lengthens

 Transcription literally means the act or

 In genetics, the term refers to the copying

Signals the end of • Bacterial mRNA may be polycistronic, which

 A structural gene is a one that encodes a

 Well over 90% of all genes are structural genes

Ribosomal RNA (rRNA) 85

Messenger RNA (mRNA) 2

Transfer RNA (tRNA) 12

 In some cases, the RNA transcript becomes part of a

 Signal recognition particles

Bases to the right are

Figure 12.3 The conventional numbering system of promoters