Transcript Key Concepts

Chapter 16
Extrachromosomal Replication
16.1 Introduction
 Bacterial extrachromosomal genomes fall into two general types:
plasmids and bacteriophages (phages).
 Some plasmids, and all phages, have the ability to transfer from a
donor bacterium to a recipient by an infective process.
- plasmids: self-replicating circular molecules of DNA that are
maintained in the cell in a stable and characteristic number of
copies, that is, the number remains constant from generation to
generation.
- phages that are found as part of the bacterial chromoosome are aid
to show lysogeny; plasmids that have the ability to behave like this
are called episomes.
16.2 The Ends of Linear DNA Are a Problem for Replication
Key Concepts
 Special arrangements must be made to replicate the DNA strand with a
5’ end.
 Figure 16.1: replication of a 5’ end is a problem.
 Special mechanism must be employed for replication at the ends of
linear replicons:
- The problem may be circumvented by converting a linear replicon
into a circular or multimeric molecule. Phages such as T4 or lambda
use such mechanisms (see Section 16.4).
- The DNA may form an unusual structure—for example, by creating
a hairpin at the terminus, so that there is no free end.
- Instead of being precisely determined, the end may be variable.
Eukaryotic chromosomes may adopt this solution, in which the
number of copies of a short repeating unit at the end of the DNA
changes (see Section 28.18).
- A protein may intervene to make initiation possible at the actual
terminus. Several linear viral nucleic acids have proteins that are
covalently linked to the 5’ terminal base. The best characterized
examples are adenovirus DNA, phage φ29 DNA, and poliovirus
RNA.
Figure 16.1. Replication could run off the 3’ end of a newly
synthesized linear strand, but could it initiate at a 5’ end?
16.3 Terminal Proteins Enable Initiation at the Ends of Viral DNAs
Key Concepts
 A terminal protein binds to the 5’ end of DNA and provides a cytidine
nucleotide with a 3’-OH end that primes replication.
 Figure 16.2: Adenovirus DNA replicates by strand displacement.
 In several viruses that use such mechanisms, a protein is found
covalently attached to each 5’ end, In the case of adenovirus, a
terminal protein is linked to the mature viral DNA via a
phosphodiester bond to serine, as indicated in Figure 16.3.
 The terminal protein carries a cytidine nucleotide that provides the
primer, and it is associated with DNA polymerase (Figure 16.4).
Figure 16.2. Adenovirus DNA
replication is initiated separately at the
two ends of the molecule and proceeds
by strand displacement.
Figure 16.3. The 5’ terminal phosphate at each end of adenovirus DNA
is covalently linked to serine in the 55 kD Ad-binding protein.
Figure 16.4. Adenovirus terminal
protein binds to the 5’ end of DNA and
provides a C-OH end to prime
synthesis of a new DNA strand.
16.4 Rolling Circles Produce Multimers of a Replicon
Key Concepts
 A rolling circle generates single-stranded multimers of the original
sequence.
 Replication of only one strand is used to generate copies of some
circular molecules. A nick opens one strand, and then the free 3’-OH
end generated by the nick is extended by the DNA polymerase.
 Figure 16.5: The rolling circle replicates DNA.
 Figure 16.6: Rolling circles show in microscopy.
 Figure 16.7: Rolling circle products are versatile.
Figure 16.5. The rolling circle
generates a multimeric singlestranded tail.
Figure 16.6. A rolling circle appears as a circular molecule with
a linear tail by electron microscopy.
Figure 16.7. The fate of the
displaced tail determines the types
of products generated by rolling
circles. Cleavage at unit length
generates monomers, which can be
converted to duplex and circular
forms. Cleavage of multimers
generates a series of tandemly
repeated copies of the original unit.
Note that the conversion to doublestranded form could occur earlier,
before the tail is cleaved from the
rolling circle.
16.5 Rolling Circles Are Used to Replicate Phage Genomes
Key Concepts
 The φX A protein is a cis-acting relaxase that generates singlestranded circles from the tail produced by rolling circle replication.
 Replication by rolling circles is common among bacteriophages.
Unit genomes can be cleaved from the displaced tail, generating
monomers that can be packaged into phage particles or used for
further replication cycles (Figure 16.8).
 Phage φX174 consists of a single-stranded circular DNA known as
the plus (+) strand. A complementary strand, called the minus (-)
strand, is synthesized. This action generates the duplex circle shown
at the top of the figure, which is then replicated by a rolling circle
mechanism.
 The A protein (called relaxase) coded by the phage genome nicks
the (+) strand of the duplex DNA at a specific site that defines the
origin for replication.
 The structure of the DNA plays an important role in this reaction,
for the DNA can be nicked only when it is negatively supercoiled.
Figure 16.8. φX174 RF DNA is a
template for synthesizing singlestranded viral circles. The A protein
remains attached to the same genome
through indefinite revolutions, each
time nicking the origin on the viral (+)
strand and transferring to the new 5’
end. At the same time, the released viral
strand is circularized..
15.6 The F Plasmid Is Transferred by Conjugation between
Bacteria
Key Concepts
 A free F factor is a replicon that is maintained at the level of one
plasmid per bacterial chromosome.
 An F factor can integrate into the bacterial chromosome, in which case
its own replication system is suppressed.
 The F factor codes for specific pili that form on the surface of the
bacterium.
 An F pilus enables an F-positive bacterium to contact an F-negative
bacterium and to initiate conjugation.
 Bacterial conjugation: a plasmid genome or host chromosome is
transferred from one bacterium to another.
 Conjugation is mediated by the F plasmid, which is the classic
example of an episome—an element that may exist as a free circular
plasmid, or that may become integrated into the bacterial
chromosome as a linear sequence. The F plasmid is a large circular
DNA ~100 kb in length.
 In its free (plasmid) form, the F plasmid utilizes its own replication
origin (oriV) and control system, and is maintained at a level of one
copy per bacterial chromosome, this system is suppressed, and F
DNA is replicated as a part of the chromosome.
 A large (~33 kb) region of the F plasmid called the transfer region
is required for conjugation. It contains ~40 genes that are required
for the transmission of DNA (Figure 16.9).
 F-positive bacteria possess surface appendages called pili (pilus)
that are coded by the F factor. The gene traA codes for the single
subunit protein, pilin.
 Mating is initiated when the tip of the F-pilus contacts the surface of
the recipient cell (Figure 16.10).
Figure 16.9. The tra region of the F plasmid contains the genes
needed for bacterial conjugation.
Figure 16.10. Mating bacteria are initially connected
when donor F pili contact the recipient bacterium.
16.7 Conjugation Transfers Single-Stranded DNA
Key Concepts
 Transfer of an F factor is initiated when rolling circle replication
begins at oriT.
 The free 5’ end initiates transfer into the recipient bacterium.
 The transferred DNA is converted into double-stranded form in the
recipient bacterium.
 When an F factor is free, conjugation “infects” the recipient bacterium
with a copy of the F factor.
 When an F factor is integrated, conjugation causes transfer of the
bacterial chromosome until the process is interrupted by (random)
breakage of the contact between donor and recipient bacteria.
 Transfer of the F factor is initiated at a site called oriT, the origin of
transfer, which is located at one end of the transfer region.
 Figure 16.11 shows that the freed 5’ end leads the way into the
recipient bacterium.
 When an integrated F plasmid initiates conjugation, the orientation
of transfer is directed away from the transfer region and into the
bacterial chromosome; such strains are described as Hfr (for high
frequency recombination).
 Figure 16.12 shows that, following a short leading sequence of F
DNA, bacterial DNA is transferred.
Figure 16.11. Transfer of DNA occurs
when the F factor is nicked at oriT and
a single strand is led by the 5’ end into
the recipient. Only one unit length is
transferred. Complementary strands
are synthesized to the single strand
remaining in the donor and to the
strand transferred into the recipient.
Figure 16.12. Transfer of chromosomal
DNA occurs when an integrated F factor
is nicked at oriT. Transfer of DNA starts
with a short sequence of F DNA and
continues until prevented by loss of
contact between the bacteria.
16.8 The Bacterial Ti Plasmid Causes Crown Gall
Disease in Plants
Key Concepts
 Infection with the bacterium A. tumefaciens can transform plant cells
into tumors.
 The infectious agent is a plasmid carried by the bacterium.
 The plasmid also carries genes for synthesizing and metabolizing
opines (arginine derivatives) that are used by the tumor cell.
 Most events in which DNA is rearranged or amplified occur within a
genome, but the interaction between bacteria and certain plants
involves the transfer of DNA from the bacterial genome to the plant
genome.
 Crown gall disease, shown in Figure 16.13, can be induced in most
dicotyledonous plants by the soil bacterium Agrobacterium
tumefaciens.
 The tumor-inducing principle of Agrobacterium resides in the Ti
plasmid.
 Ti plasmids can be divided into four groups, according to the types
of opine (novel derivatives of arginine) that are made; Nopaline,
Octopine, Agropine, Ri plasmids
 The types of genes carried by a Ti plasmid are summarized in
Figure 16.14.
Figure 16.13. An Agrobacterium carrying a Ti plasmid of
the nopaline type induces a teratoma, in which
differentiated structures develop.
Figure 16.14. Ti plasmids carry genes involved in
both plant and bacterial functions.
16.9 T-DNA Carries Genes Required for Infection
Key Concepts
 Part of the DNA of the Ti plasmid is transferred to the plant cell
nucleus.
 The vir genes of the Ti plasmid are located outside the transferred
region and are required for the transfer process.
 The vir genes are induced by phenolic compounds released by plants
in response to wounding.
 The membrane protein VirA is autophosphorylated on histidine when it
binds an inducer.
 VirA activates VirG by transferring the phosphate group to it.
 The VirA-VirG is one of several bacterial two component systems that
use a phosphohistidine relay.
 The interaction between Agrobacterium and a plant cell is illustrated
in Figure 16.15.
 The bacterium does not enter the plant cell, but rather transfers part
of the Ti plasmid to the plant nucleus. The transferred part of the Ti
genome is called T-DNA. It becomes integrated into the plant
genome.
 Transformation of plant cells requires three types of function carried
in the Agrobacterium:
- Three loci on the Agrobacterium chromosome, chvA, chvB, and
pscA, are required for the initial stage of binding the bacterium to
the plant cell. They are responsible for synthesizing a
polysaccharide on the bacterial cell surface.
- The vir region carried by the Ti plasmid outside the T-DNA region
is required to release and initiate transfer of the T-DNA.
- The T-DNA is required to transform the plant cell.
Figure 16.15. T-DNA is
transferred from Agrobacterium
carrying a Ti plasmid into a plant
cell, where it becomes integrated
into the nuclear genome and
expresses functions that transform
the host cell.
 The organization of the major two types of Ti plasmid is illustrated
in Figure 16.16.
 The T-region occupies ~23 kb.
 The virulence genes code for the functions required for the transfer
process. Six loci (virA, -B, -C, -D, -E, and –G) reside in a 40-kb
region outside the T-DNA. Their organization is summarized in
Figure 16.17.
 We may divide the transforming process into (at least) two stages:
- Agrobacterium contacts a plant cell and the vir genes are induced.
- vir gene products cause T-DNA to be transferred to the plant cell
nucleus, where it is integrated into the genome.
 Figure 16.18: Wounding produces acetosyringone.
 Figure 16.19: VIrA-VirG is a two-component system.
Figure 16.16. Nopaline and octopine Ti plasmids carry a variety of
genes, including T-regions that have overlapping functions.
Figure 16.17. The vir region of the Ti plasmid has six loci that are
responsible for transferring T-DNA to an infected plant.
Figure 16.18. Acetosyringone (4-acetyl-2,6-dimethoxyphenol) is
produced by N. tabacum upon wounding and induces transfer of TDNA from Agrobacterium.
Figure 16.19. The two-component
system of VirA-VirG responds to
phenolic signals by activating
transcription of target genes.
16.10 Transfer of T-DNA Resembles Bacterial Conjugation
Key Concepts
 T-DNA is generated when a nick at the right boundary creates a primer
for synthesis of a new DNA strand.
 The preexisting single strand that is displaced by the new synthesis is
transferred to the plant cell nucleus.
 Transfer is terminated when DNA synthesis reaches a nick at the left
boundary.
 The T-DNA is transferred as a complex of single-stranded DNA with
the VirE2 single strand-binding protein.
 The single stranded T-DNA is converted into double-stranded DNA
and integrated into the plant genome.
 The mechanism of integration is not known. T-DNA can be used to
transfer genes into a plant nucleus.
 Figure 16.20: T-DNA is bounded by direct repeats.
 Figure 16.21: A model for transfer.
Figure 16.20. T-DNA has almost identical repeats of 25 bp at each end in the Ti
plasmid. The right repeat is necessary for transfer and integration to a plant
genome. T-DNA that is integrated in a plant genome has a precise junction that
retains 1 to 2 bp of the right repeat, but the left junction varies and may be up to
100 bp short of the left repeat.
Figure 16.21. T-DNA is generated by
displacement when DNA synthesis starts
at a nick made at the right repeat. The
reaction is terminated by a nick at the left
repeat.