Use of Baculovirus As

Expression Vector
• Viruses in this class have a large circular
DNA genome, 130 kb
• Major viral structural protein is made in
huge amounts in infected cells
– Promoter for this protein, polyhedrin, is very
active
– These vectors can produce up to 0.5 g of
protein per liter of medium
– Nonrecombinant viral DNA entering cells
cannot result in infectious virus as it lacks an
essential gene supplied by the vector 4-1
Baculovirus Expression

Provides the genes

needed for infection

4-2
 Animal Cell Transfection
• Calcium phosphate
– Mix cells with DNA in a phosphate buffer
– Then solution of calcium salt added to form a
precipitate
– Cells take up the calcium phosphate crystals
which include some DNA
• Liposomes
– DNA mixed with lipid to form liposomes, small
vesicles with some of the DNA inside
– DNA-bearing liposomes fuse with cell membrane
carrying DNA inside the cell
4-3
 Red, very important

Summary
• Foreign genes can be expressed in
eukaryotic cells
• These eukaryotic systems have advantages
over prokaryotic ones
– Made in eukaryotic cells tend to fold properly
and are then soluble rather than aggregated
into insoluble inclusion bodies
– Posttranslational modifications are made in a
eukaryotic manner
4-4
Using the Ti Plasmid to Transfer
Genes to Plants
• Genes can be introduced into plants with
vectors that can replicate in plant cells
• Common bacterial vector promoters
and replication origins are not
recognized by plant cells
• Plasmids are used containing T-DNA
– T-DNA is derived from a plasmid known as
tumor-inducing (Ti)
– Ti plasmid comes from bacteria that cause
plant tumors called crown galls
4-5
 Ti Plasmid Infection
• Bacterium infects plant, transfers Ti
plasmid to host cells
• T-DNA integrates into the plant DNA
causing abnormal proliferation of plant
cells
• T-DNA genes direct the synthesis of
unusual organic acids, opines which can
serve as an energy source to the infecting
bacteria but are useless to the plant
4-6
Ti Plasmid Transfers Crown Gall

4-7
Use of the T-DNA Plasmid

4-8
Lecture PowerPoint to accompany

Molecular Biology
Fourth Edition

Robert F. Weaver

Chapter 6
The Mechanism of
Transcription in
Bacteria
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 6.1 RNA Polymerase Structure
By 1969 SDS-PAGE of RNA polymerase from E.
coli had shown several subunits
– 2 very large subunits are b (150 kD) and b’ (160
kD)
– Sigma (s) at 70 kD
– Alpha (a) at 40 kD – 2 copies present in
holoenzyme
– Omega (w) at 10 kD
• Was not clearly visible in SDS-PAGE, but seen in
other experiments
• Not required for cell viability or in vivo enzyme activity
• Appears to play a role in enzyme assembly
6-10
 Sigma as a Specificity Factor
• Core enzyme without the s subunit could not
transcribe viral DNA, yet had no problems with
highly nicked calf thymus DNA
• With s subunit, the holoenzyme worked equally
well on both types of DNA

6-11
 Testing Transcription
• Core enzyme transcribes both DNA strands
• Without s-subunit the core enzyme has basic
transcribing ability but lacks specificity

6-12
 6.2 Promoters
• Nicks and gaps are good sites for RNA
polymerase to bind nonspecifically
• Presence of the s-subunit permitted
recognition of authentic RNA polymerase
binding sites
• Polymerase binding sites are called
promoters
• Transcription that begins at promoters is
specific, directed by the s-subunit
6-13
 Binding of RNA Polymerase to
Promoters
• How tightly does core
enzyme v. holoenzyme
bind DNA?
• Experiment measures
binding of DNA to enzyme
using nitrocellulose filters
– Holoenzyme binds filters
tightly
– Core enzyme binding is
more transient
6-14
 Temperature and RNA
Polymerase Binding
• As temperature is
lowered, the binding
of RNA polymerase to
DNA decreases
dramatically
• Higher temperature
promotes DNA
melting

6-15
 RNA Polymerase Binding
Hinkle and Chamberlin proposed:
• RNA polymerase holoenzyme binds DNA
loosely at first
– Binds at promoter initially
– Scans along the DNA until it finds one
• Complex with holoenzyme loosely bound at the
promoter is a closed promoter complex as DNA
is in a closed ds form
• Holoenzyme can then melt a short DNA region
at the promoter to form an open promoter
complex with polymerase bound tightly to DNA
6-16
 Polymerase/Promoter Binding
• Holoenzyme binds DNA
loosely at first
• Complex loosely bound at
promoter = closed
promoter complex,
dsDNA in closed form
• Holoenzyme melts DNA
at promoter forming open
promoter complex -
polymerase tightly bound

6-17
 Core Promoter Elements
• There is a region common to bacterial promoters
described as 6-7 bp centered about 10 bp upstream of
the start of transcription = -10 box
• Another short sequence centered 35 bp upstream is
known as the -35 box
• Comparison of thousands of promoters has produced a
consensus sequence for each of these boxes

6-18
 Promoter Strength
• Consensus sequences:
– -10 box sequence approximates TAtAaT
– -35 box sequence approximates TTGACa
• Mutations that weaken promoter binding:
– Down mutations
– Increase deviation from the consensus
sequence
• Mutations that strengthen promoter binding:
– Up mutations
– Decrease deviation from the consensus
sequence 6-19
 UP Element
• UP element is a promoter, stimulating
transcription by a factor of 30
• UP is associated with 3 “Fis” sites which
are binding sites for transcription-activator
protein Fis, not for the polymerase itself
• Transcription from the rrn promoters
respond
– Positively to increased concentration of iNTP
– Negatively to the alarmone ppGpp
6-20
The rrnB P1 Promoter

6-21
 6.3 Transcription Initiation
• Transcription initiation was assumed to
end as RNA polymerase formed 1st
phosphodiester bond
• Carpousis and Gralla found that very small
oligonucleotides (2-6 nt long) are made
without RNA polymerase leaving the DNA
• Abortive transcripts such as these have
been found up to 10 nt
6-22
Stages of Transcription Initiation
• Formation of a closed
promoter complex
• Conversion of the closed
promoter complex to an
open promoter complex
• Polymerizing the early
nucleotides – polymerase
at the promoter
• Promoter clearance –
transcript becomes long
enough to form a stable
hybrid with template
6-23
 The Functions of s
• Gene selection for transcription by s
causes tight binding between RNA
polymerase and promoters
• Tight binding depends on local melting of
DNA that permits open promoter complex
• Dissociation of s from core after
sponsoring polymerase-promoter binding

6-24