Download II. The selected examples

微生物遺傳與生物技術
(Microbial Genetics and
Biotechnology)
金門大學
食品科學系
何國傑 教授
Autonomously replicating genetic
entities: (2) Bacterial viruses –
bacteriophages (or phages for short)
Bacteriophages
• Like all viruses, phages are so small that they can be
seen only under the electron microscope, and are
essentially genes (on DNA or RNA genome) wrapped
in a protein coat (capsid) with or without membrane
envelope.
• There are two types of life cycles: lytic or lysogenic
life cycle.
• Phages are usually detected only by the plaques (the
holes) they form on the lawns of susceptible host
bacteria.
• Phages are not alive, they can not multiply outside a
bacterial cell.
• Bacterial colony vs plaque
Bacteriophages
• Icosahedral
capsid
• Filament
capsid
I. The lytic development cycle of
bacteriophage (Fig. 7.2)
1. A phage adsorbs to an actively growing bacterial cell by
binding to a specific receptor on the cell surface.
2. The phage injects its entire DNA into the cell, and transcribes
RNA usually using host RNA polymerase immediately. Those
transcribed soon after infection are called the early genes of
the phage and encode mostly enzymes involved in DNA
synthesis. The early genes have promoters that mimic those of
the host DNA and recognized by host σ factor.
3. Next, mRNAs are transcribed from the rest of the phage genes,
the late genes. These genes have promoters that are unlike
those of host cell and are not recognized by the host RNA
polymerase alone. Most of these genes encode proteins
involved in assembly of the head and tail.
I. The lytic development cycle of
bacteriophage (Fig. 7.2)
4. After phage particle is completed, the DNA is
taken up by the head and the tails are attached.
5. Finally, the cells break open, or lyse and new
phage are released to infect other sensitive
bacteria.
6. Whole process known as lytic cycle, takes less
than 1 hour for many phages and hundreds of
progeny phage can be produced from a single
infecting phage (burst size).
7. Virulent phages - phages that lyse their host
during the reproductive cycle
I. The lytic development cycle of
bacteriophage
I. The lytic development cycle of
bacteriophage (Fig. 7.2)
Transcriptional regulation during development
of a typical large DNA phage
1. The purple arrows indicates activation of gene expression
2. The black bars indicates repression of gene expression
Phage plaques
II. The selected examples
The following examples are selected either because
they are the basis for cloning technologies or because
of the impact they have had on our understanding
regulatory mechanisms in general.
IIa. Phage T7, a phage-encoded RNA polymerase
1. Phage T7 has a relatively simple program of gene
expression after infection, with only two major
classes of genes, the early and late genes .
II. The selected examples - Phage T7
2. Some genes of about 50 T7 genes are shown in Fig. 7.4
(Genetic map of hage T7).
3. After infection, expression of T7 genes proceeds from left to
right, up to and including gene 1.3 are early genes. The
genes to the right of 1.3 are transcribed after few minutes’
delay – the late genes.
4. Nonsense and temperature-sensitive mutations were used
to identify which of the early-gene products is responsible
for turning on the late genes. It turns out the gene 1, which
encodes the T7 RNA polymerase.
II. The selected examples - Phage T7
5. In fact, transcription of the late genes by the gene 1 product
may help pull the DNA into the cell, causing sequential gene
expression.
6. The specificity of phage RNA polymerase for their own
promoter has been exploited in many applications in
molecular genetics:
(1) T7 phage based expression vectors, for example pET
vectors (for plasmid expression T7):
i. Using T7 polymerase and T7 gene10 promoter to express
foreign gene in E. coli. Gene10 promoter is a very strong
promoter. Hundreds of thousands of copies of T7 head
protein must be synthesized in a few minutes after
infection. Some pET vectors have strong translation
initiation regions and His tag.
ii. T7 promoter is made inducible by providing the T7 RNA
polymerase only when the foreign gene is to be
expression. This is important in cases where the fusing
protein is toxic to cell.
II. The selected examples - Phage T7
iii. Fig. 7.5 demonstrates the strategy for regulating the expression
of genes cloned into a pET vector.
(i) To provide a source of inducible T7 RNA polymerase, E. coli
strains has been constructed in which phage gene 1 for
RNA polymerase is cloned downstream of the inducible lac
promoter and integrated into their chromosome. Therefore,
it is expressed only if the inducer IPTG is added. This kind
of strain often has the DE3 suffix.
(ii) The T7 RNA polymerase then transcribes the gene cloned
into the pET vector downstream of the T7 late promoter on
the cloning vector.
Strategy for regulating the expression of genes
cloned into a pET vector
1. Gene 1 (for T7 RNA polymerase) is inserted into the E. coli
chromosome and transcribed from lac promoter. Gene 1 is
expressed only when inducer IPTG is added.
2. The T7 RNA polymerase then transcribes the cloned gene
inserted downstream of the T7 late promoter on pET vector.
3. If the protein product of the cloned gene is toxic, the
transcription of the cloned gene before induction can be
reduced by:
(1) The T7 lysozyme encoded by a compatible plasmid, pLysS
binds to any residual T7 RNA polymerase in absence of
induction and inactivates the RNA polymerase.
(2) The presence of lac operators between the T7 late promoter
and cloned gene also reduce the transcription of the cloned
gene in the absence of IPTG.
Strategy for regulating the expression of
genes cloned into a pET vector
II. The selected examples - Phage T7
[ 6. The specificity of phage RNA polymerase for their own
promoter has been exploited in many applications in
molecular genetics: (slide 12)
(1) T7 phage based expression vectors, for example pET
vectors (for plasmid expression T7):]
(2) Phage display
i. A randomized peptide coding DNA sequence is fused to
the T7 head protein-encoded gene (gene 10). Different
phages display different versions of the peptide on their
surface.
ii. The phage that display a version of the peptide which
binds to the other protein molecule can then be
screened and isolated.
iii. The cloned DNA can then sequenced and protein can be
determined.
II. The selected examples - Phage T4
IIb. Phage T4: Transcription activators, antitermination, a new
sigma factor, and replication-coupled transcription
1. Structure of phage T4
(1) Phage T4 is much larger than T7, with over 200 genes.
(2) Genes are divided into groups according to the time
individual gene expression: immediate-early genes
(express immediately after infection), delayed-early and
middle genes (express only few minutes after infection),
and true-late genes.
i. Immediate-early genes and delayed-early genes are
transcribed from the same normal σ70 promoter, but
delayed-early genes are regulated by an
antitermination mechanism (discuss when phage λ is
talked).
II. The selected examples - Phage T4
Phage T7 and coliphages Todd have short,
non-contractle tails without fibers.
II. The selected examples - Phage T4
ii. Middle genes are transcribed from their own promoter, which
look somehow different fromσ70 promoters in that their - 35
sequence is replaced by a sequence centered at - 30 called
Mot box (Fig. 7.9). These promoters required the phageencoded MotA and AsiA proteins, the products of delay-early
genes. AsiA protein binds to region 4 ofσ70 and inhibits its to
the - 35 sequence. AsiA allows MotA to bind to region 4, it can
now recognize the - 30 sequence of the middle T4 promoter.
II. The selected examples - Phage T4
iii. The products of true-late genes are mostly the head, tail, and
tail fiber components of the phage and enzymes and proteins
needed to lyse and release the phage.
(i) The initiation of transcription of true-early genes is coupled
to the replication of DNA.
(ii) The promoters of true-early genes have the sequence
TATAAATA rather than the - 10 sequence of a typicalσ70
promoter, and they lack a - 35 sequence. The product of the
T4 regulatory gene 55 is an alternate sigma factor that binds
to the host RNA polymerase, changing its specificity so that
it recognizes only the promoters of true-early genes. This
altered sigma seems to lack a region 4 to recognize - 35
sequences. This makes it unable to form open complexes
and initiate transcription efficiently.
II. The selected examples - Phage T4
(iii) Protein gp33 which binds to β subunit can
substitute for the region 4 ofσ70 to allow open
complex formation and the initiation of
transcription, only it binds with gp45.
(iv) The same strategy use alternate sigma factors to
activate the late genes is also employed in
bacteria, ex., sporulation of Bacillus subtilis.
* Structure of typicalσ70 factor:
region 1 – preventing itself from binding to DNA;
region 2 – binding to -10
region 3 – involving in both core and DNA binding;
region 4 – binding to -35
II. The selected examples - Phage T4
(3) Replication-coupled transcription
i. In addition to the alternate sigma factor, gp55, and
the accessory protein, gp33, the products of other T4
genes are required to turn on the transcription of late
genes. Some of these products are also required for
replication of phage DNA.
ii. Coupling the transcription of the true-late genes to
the replication of phage DNA makes sense from a
strategic standpoint. Many of the true-late genes
encode parts of the phage particle including the head,
and the phage heads are not needed until phage
DNA is available to be packaged inside them.
II. The selected examples - Phage T4
@ Replication-coupled transcription
A.
1. The normal situation where the clamp loaders, gp44 and gp62,
load the gp45 clamp on the DNA as DNA polymerase (gp43)
begins synthesizing an Okazaki fragment at an RNA primer.
2. The gp41 helicase separates the DNA dtrands.
3. After the Okazaki fragment is synthrsized, the gp43 comes of
the gp45 clamp, which stays on the DNA and slides to a truelate promoter, contacts gp33 on RNA polymerase (RNA pol)
and activates transcription. (gp55, the alternate sigma factor)
4. A new gp45 clamp is loaded onto the DNA as replication
continues.
B.
1. If infected by a DNA ligase-deficient mutant of T4, nicks persist in the
DNA. The gp45 clamp may load at such nicks independent of the
other replication proteins and slide on the DNA until it contacts gp33
on RNA pol at a late promoter.
II. The selected examples - Phage T4
II. The selected examples - Phage T4
III. Phage DNA replication
1. Phage M13 – a filamentous phage with a circular, singlestranded DNA. M13 is a male-specific phage. They do not lyse
the cell when released.
(1) While the phage DNA enters the cytoplasm, the major coat
protein is stripped off into the host inner membrane.
(2) When phage is released, the coat proteins are waiting in the
membrane to be assembled.
III. Phage DNA replication – M13
(3) The steps in the replication of phage M13 DNA are outlined
in Fig. 7.12.
i. An RNA primer is used to synthesize the complementary
minus strand (in black) to form double-stranded
replicative-form (RF) DNA, which is dependent entirely on
host functions. Normally the host RNA polymerase
recognize only double-stranded DNA, but the singlestranded DNA form a hairpin at the origin of replication.
The 5’ exonuclease of DNA polymerase I removes the
RNA primer and the nick is sealed by polymerization
activity of Pol I and ligase.
III. Phage DNA replication – M13
ii. The product of gene II, an endonuclease, nicks the plus
strand of the RF and remains attached to the 5’ phosphate
at the nick. The host Rep, a helicase, help unwind the DNA
at the nick.
iii. More + strands are synthesized via rolling-circle replication,
and their – strand are synthesized to make more RFs.
iv. The gene V product binds to plus strands as they are
synthesized, preventing them from being used as template
for more RF synthesis and helping package them into
phage protein coat. The single-stranded DNA is transferred
to the assembling viral particle in the membrane and leaked
out of the cell.
Phage M13
M13 cloning vector
(4) M13 cloning vector (Fig. 7.13)