Molgen-4.2

1 Introduction

• Plasmid:

  • Definition: Circular, extrachromosomal DNA.

  • Characteristics: It is autonomous and can replicate itself.

• Lysogeny:

  • Definition: The ability of a phage to survive in a bacterium as a stable prophage component of the bacterial genome.

• Episome:

  • Definition: A plasmid that is able to integrate into bacterial DNA.

• Immunity:

  • In plasmids, it refers to the ability of a plasmid to prevent another of the same type from becoming established in a cell.

12.2 The Ends of Linear DNA Are a Problem for Replication

• Special arrangements are necessary to replicate the DNA strand with a 5' end.

• Figure Reference: Figure 12.1 shows that replication could run off the 3' end of a newly synthesized linear strand, but questioning whether it could initiate at a 5' end arises.

12.3 Terminal Proteins Enable Initiation at the Ends of Viral DNAs

• Strand Displacement:

  • Definition: A mode of replication of some viruses where a new DNA strand grows by displacing the previous (homologous) strand of the duplex.

  • Figure Reference: Figure 12.2 illustrates that adenovirus DNA replication is initiated separately at both ends of the molecule, proceeding by strand displacement.

• Terminal Protein:

  • Function: Binds to the 5′ end of DNA, providing a cytidine nucleotide with a 3′–OH end that primes replication.

  • Figure Reference: Figure 12.4 depicts adenovirus terminal protein binding to the 5' end of DNA to prime synthesis of a new DNA strand.

12.4 Rolling Circles Produce Multimers of a Replicon

• Definition: A rolling circle generates single-stranded multimers of the original sequence.

• Figure Reference: Figure 12.5 shows the rolling circle producing a multimeric single-stranded tail.

• Note: Figure 12.7 explains that the fate of the displaced tail determines the products generated by rolling circles.

12.5 Rolling Circles Are Used to Replicate Phage Genomes

• φX174 A Protein:

  • Type: A cis-acting relaxase.

  • Function: Generates single-stranded circles from the tail produced by rolling circle replication.

  • Figure Reference: Figure 12.8 illustrates that ΦX174 RF DNA serves as a template for synthesizing single-stranded viral circles.

12.6 The F Plasmid Is Transferred by Conjugation Between Bacteria

• Conjugation:

  • Definition: A process where two cells come in contact and transfer genetic material.

  • In bacteria, DNA is transferred from a donor to a recipient cell.

• Free F Plasmid:

  • It is a replicon maintained at the level of one plasmid per bacterial chromosome.

• Transfer Region:

  • Definition: A segment on the F plasmid necessary for bacterial conjugation.

• Note: An F plasmid can integrate into the bacterial chromosome, suppressing its own replication.

  • Figure Reference: Figure 12.9 details that the tra region of the F plasmid contains the genes required for bacterial conjugation.

12.7 Conjugation Transfers Single-Stranded DNA

• Process Initiation: Transfer of an F plasmid begins when rolling circle replication initiates at oriT.

• Relaxosome Formation: Initiates the transfer of DNA into the recipient bacterium.

• Conversion: Transferred DNA is converted into double-stranded form in the recipient bacterium.

• F Plasmid Integration:

  • When an F plasmid is integrated, conjugation leads to the transfer of the bacterial chromosome until interrupted by random breakage of the contact between donor and recipient.

  • Hfr Cell:

  • Definition: A bacterium with an integrated F plasmid within its chromosome.

  • Significance: Hfr stands for high frequency recombination, indicating chromosomal genes transfer from an Hfr cell to an F– cell occurs more frequently than from an F+ cell.

• Figure Reference: Figure 12.11 shows that DNA transfer occurs when the F plasmid is nicked at oriT, leading to a single strand being led by the 5' end bound to TraI into the recipient.

• Figure Reference: Figure 12.12 shows that chromosomal DNA transfer happens when an integrated F plasmid is nicked at oriT.

12.8 Single-Copy Plasmids Have a Partitioning System

• Copy Number:

  • Definition: The number of copies of a plasmid maintained in a bacterium relative to the origin of the bacterial chromosome.

  • Single-Copy Plasmids: Exist at one plasmid copy per bacterial chromosome origin.

  • Multicopy Plasmids: Exist at more than one plasmid copy per bacterial chromosome origin.

• Partition Systems: Ensure duplicated plasmids are segregated to different daughter cells during division.

  • Figure Reference: Figure 12.14 illustrates the partitioning of plasmid R1 involving polymerization of the ParM ATPase between plasmids.

• Addiction System:

  • Definition: A survival mechanism used by plasmids that kills the bacterium if the plasmid is lost.

  • Figure Reference: Figure 12.15 demonstrates that plasmids may prevent bacterial survival without them by synthesizing a long-lived killer and a short-lived antidote.

12.9 Plasmid Incompatibility Is Determined by the Replicon

• Definition: Plasmids within a single compatibility group have origins regulated by a common control system.

• Figure Reference: Figure 12.16 explains that two plasmids are incompatible if they belong to the same compatibility group and cannot be distinguished at initiation.

12.10 The ColE1 Compatibility System Is Controlled by an RNA Regulator

• Replication of ColE1:

  • Requires transcription to pass through the origin, where the transcript is cleaved by RNAase H, generating a primer end.

  • Figure Reference: Figure 12.17 shows that replication is initiated by cleaving the primer RNA to provide a 3'–OH end.

• Regulator RNA I:

  • Characteristic: A short antisense RNA pairing with the transcript, preventing the cleavage that generates the priming end.

  • Rom Protein: Enhances pairing between RNA I and the transcript.

  • Figure References:

  • Figure 12.18 shows the complementary sequence of RNA I to the 5' region of primer RNA.

  • Figure 12.19 explains that base pairing with RNA I may change the secondary structure of the primer RNA, thereby preventing the generation of a 3'–OH end.

• Countertranscript:

  • Definition: An RNA molecule that prevents an RNA primer from initiating transcription through base pairing with the primer.

  • Figure Reference: Figure 12.20 clarifies that mutations in the region coding for RNA I and the primer precursor do not affect their pairing ability.

12.11 How Do Mitochondria Replicate and Segregate?

• mtDNA Replication and Segregation:

  • Process is stochastic.

• Heteroplasmy:

  • Definition: Presence of more than one mitochondrial allelic variant within a cell.

• Mitochondrial segregation to daughter cells occurs stochastically as well.

  • Figure Reference: Figure 12.21 indicates that mitochondrial DNA replicates by increasing the number of genomes proportionate to mitochondrial mass.

12.12 D Loops Maintain Mitochondrial Origins

• Mitochondria utilize different origin sequences to initiate replication of each DNA strand.

• Replication of the H strand is initiated within a D loop.

• Replication of the L strand is launched when its origin is revealed by the movement of the first replication fork.

  • Figure Reference: Figure 12.22 shows the D loop maintaining an opening in mammalian mitochondrial DNA with separate origins for each strand's replication.

12.13 The Bacterial Ti Plasmid Causes Crown Gall Disease in Plants

• Crown Gall Disease:

  • Result of infection with the bacterium Agrobacterium tumefaciens, leading to the transformation of plant cells into tumors.

• Ti Plasmid:

  • The infectious agent responsible for the disease, carrying genes for the synthesis and metabolism of opines (arginine derivatives) utilized by the bacterium.

• Nopaline Plasmids:

  • Type of Ti plasmids carrying genes for synthesizing the opine nopaline, retaining the ability to differentiate into early embryonic structures.

• Octopine Plasmids:

  • Plasmids that carry genes coding for synthesizing octopine type opines.

  • Consequently, the tumors formed are undifferentiated.

• Agropine Plasmids:

  • These carry genes for synthesizing agropine type opines, with tumors usually dying early.

• Ri Plasmid:

  • Found in A. tumefaciens and contains disease-causing genes, leading to either hairy root or crown gall disease in plants.

12.14 T-DNA Carries Genes Required for Infection

• T-DNA:

  • Part of Ti plasmid DNA that is transferred to the plant cell nucleus.

  • Figure Reference: Figure 12.24 illustrates T-DNA transfer from Agrobacterium carrying a Ti plasmid into a plant cell.

• Vir Genes:

  • Located outside the transferred region and essential for the transfer process.

  • Induced by phenolic compounds released by plants in response to wounding.

  • Figure Reference: Figure 12.25 indicates that nopaline and octopine Ti plasmids contain a variety of genes including T-regions with overlapping functions.

  • Acetosyringone: Produced by N. tabacum upon wounding, inducing T-DNA transfer from Agrobacterium.

• VirA Protein:

  • A membrane protein that autophosphorylates on histidine upon binding an inducer, subsequently activating VirG by transferring a phosphate group to it.

  • VirA-VirG: Part of several bacterial two-component systems using a phosphohistidine relay.

12.15 Transfer of T-DNA Resembles Bacterial Conjugation

• T-DNA Generation:

  • Occurs when a nick at the right boundary creates a primer for synthesizing a new DNA strand.

• The preexisting single strand displaced by the new synthesis gets transferred to the plant cell nucleus.

• Transfer completes when DNA synthesis meets a nick at the left boundary.

  • Figure Reference: Figure 12.30 shows T-DNA created by displacement when synthesis starts at a right nick.

• Transfer Complex:

  • T-DNA transferred as a complex with the VirE2 single strand-binding protein.

• Integration Mechanism:

  • Converts single-stranded T-DNA into double-stranded DNA integrated into the plant genome, but the mechanism of integration is not fully understood.

• Application: T-DNA can be utilized for gene transfer into plant nuclei.