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Unformatted text preview: Chapter 12 Extrachromosomal Replicons
Genes IX Chapter 16 There are two types of independently replicating genomes in bacterial cells:
1. Plasmids: self-replicating circular genomes that maintain a
relatively constant copy number. A. Low copy: from 1-10 copies per cell; have a segregation mechanism. B. High copy: more than 10 copies, sometimes up to 100 or more. Are distributed between the daughter cells randomly. C. Episome: a plasmid that can integrate into the host chromosome Phages: self-replicating entities that produce infectious particles. May be circular or linear, DNA or RNA. A. Lysogenic: phages that can integrate into the host genome. B. Immunity: the ability of a phage or plasmid to exclude reinfection by a similar entity. 2. Phages and Plasmids sometimes mediate the exchange of host genes. Sometimes when phages and plasmids exit the host genome, host DNA sequences are taken in the process. When this occurs, these sequences can be transferred to the next recipient host genome. For both phages and plasmids, the spread to new hosts is a normal part of the life cycle. For phage this transfer occurs though infectious particles. For plasmids, the transfer occurs directly by a process called "conjugation."
Plasmid conjugation Lambda phage Free plasmids are always circular, but phages may exist as either circular or linear entities. In chapter 11 (The Replicon) we looked at how the circular host genome is replicated. Some phages and plasmids use similar mechanisms; however there are other ways to replicate a circular genome that produce very high numbers of replicated molecules, something that would be beneficial to phages. Phages (and eukaryotic viruses) often have linear genomes which raises a new set of problems for the replication process. The Ends of Linear DNA Are a Problem for Replication
s Special arrangements must be made to replicate the DNA strand with a 5 end. The 3' end can The 3' end can run off the end run off the end of the template. of the template. ...but how does ...but how does the 5' end get the 5' end get replicated? replicated? The problem of linear replications
Since the new strand is always synthesized in the 5' to 3' direction, it is easy to finish the new strand by running off the end of the template, but how does the polymerase initiate at the 3' end of the template strand? (How does it get the 1st base?)
How to How to initiate in aa initiate in way that way that replicates the replicates the first bases at first bases at 5' end? 5' end? 3' 3' 5' DNA pol usually binds region that surrounds origin. It is difficult for the polymerase to start at the end. Strand displacement
Linear template: Adenovirus 1. Bottom strand is used as template and the top strand is displaced.
Replicate each Replicate each strand separately. strand separately. 2. Top strand forms terminal duplex before initiating DNA synthesis.
5' 5' 3' 3' 5' 5' 3'
Free single strand + 3' 3' 5' 5' 3'
Duplex origin formed by base pairing; The problem of linear replications
Solutions: 3. protein intervenes. Often used by viral nucleic acids that have proteins that are covalently linked to the 5' terminal base.
Examples: adenovirus DNA (80 kDa) phage 29 DNA poliovirus RNA (22 amino acids) adenovirus Strand displacement for linear DNA replication:
1.The first synthesis uses a normal doublestranded linear template 2.The displaced strand mimics a linear double strand by forming a hairpin structure. The terminal protein has a single nucleotide primer attached.
Terminal protein dCTP covalently attached. This serves as the 3' end to prime DNA synthesis.
A protein covalently binds adenovirus DNA. Strand displacement: Linear template: Adenovirus
1. Terminal protein-dCTP binds to 5' end of the top strand of adenovirus DNA between 9 & 18 nucleotide 2. Host protein, nuclear factor I, essential for the initiation; binds between 17 & 48 nucleotide 3. Initiation complex forms between positions 9 and 48= at a fixed distance from the actual DNA end
TP 5' 3' NF1 What is the primer? dCTP dCTP Strand displacement: Linear template: Adenovirus
1. Terminal protein-dCTP/DNA polymerase complex binds to 5' end of the top strand of adenovirus DNA 2. 3` OH of dCTP serves as a primer for DNA synthesis (note: deoxyribonucleotide) 3. New strand is covalently linked to the initiating dCTP 4. Old TP is displaced by the new TP for each new replication cycle
TP remains TP remains attached attached 5' 3' 5' 5' 3' The problem of linear replications
Solutions: 2. create unusual structure at the end.
example: hairpin so there is no free end; or linear mitochondrial DNA of Paramecium crosslinks the ends.
3' 5' The problem of linear replications
4. End may be variable by using repetitive sequences.
example: Eukaryotic chromosomes have short sequence repeats at the termini (telomeres). A separate mechanism adds or removes these repeats. It is not necessary to replicate to the end as long as some of the repeats are copied. Rolling Circle Replication
Solutions: 1. convert linear to circular or multimeric molecules (rolling circles)
T4 Examples: lambda ( ) phage is circular and T4 phage multimeric. Rolling Circles Produce Multimers of a Replicon
s A rolling circle generates single-stranded multimers of the original sequence. Another example Another example of each strand of each strand being replicated being replicated independently. independently. A A protein protein Rolling Circles Are Used to Replicate Phage Genomes
s The X A protein is a cis-acting relaxase. It generates single-stranded circles from the tail produced by rolling circle replication.
The A protein has multiple functions: 1. Origin recognition 2. Endonuclease 3. Ligase 174 phage as a simple model for replication: rolling circle
"Lagging strand" synthesis Rolling circle replication (+) (+) + strand Replicative form (RF): ds plasmid (+) + strand packaged to form virion Rolling circle replication is a model for leading strand synthesis. Rolling Circle Replication is used by
plasmids to transfer themselves and host chromosome sequences to other bacteria during conjugation. Click to see animation. McGraw-Hill The F Plasmid Is Transferred by Conjugation between Bacteria
s 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 Its own replication system is suppressed. s s The F factor codes for pili that form on the surface of the bacterium. The F plasmid is transferred by conjugation between bacteria.
1. Bacterial conjugation: a plasmid genome or host chromosome is transferred from one bacterium to another in a mating process mediated by F plasmid. 2. F-plasmid: an example of an episome in E. coli.
(Remember, not all plasmids are episomes.) 3. Episome: an element that may exist as a free circular plasmid, or that may become integrated into the bacterial chromosome as a liner sequence. F-plasmid
1. large circular plasmid (100 kb) 2. only 60% (ca. 60 genes) has been mapped. 3. 32 kb is organized as a unit to transfer its genome to another bacteria (transfer region or tra genes) 4. Three methods of replication: a. oriV as free plasmid (one copy/ bacterial chromosome) b. uses E. coli chromosomal origin when integrated (oriC); oriV is suppressed. c. oriT during conjugation free F-plasmid
Tra: Discrete region that Tra has transfer genes: tra & trb loci (~40 genes) (Origin of transfer) used to initiate replication for transfer tra genes 32 kb 100 kb IS elements (used to insert into host chromosome) oriV used to initiate plasmid replication An F-pilus enables an F-positive bacterium to:
1. Establish contact an F-negative bacterium 2. Initiate conjugation by pulling the cells together F-pili tra region of the F plasmid
Transfer genes traJ
activator Direction of transfer traY/I Transcription unit oriT traM J YALEKBPVRC WU N trbCDE traF trbB traH G ST D I/Z finP tra & trb loci; ~40 genes Expressed coordinately as a part of single transcription unit traY/traI tra region of the F plasmid
Transfer genes activator TraJ Direction of transfer TraY- recruits TraI to 5' end of DNA TraI- covalently attaches to 5' end of DNA & unwinds it (relaxase). Coupling TraY/TraI protein
DNA nicking and unwinding oriT traM J YALEKBPVRC WU N trbCDE traF trbB traH G ST D I/Z finP TraT-outer membrane Surface Senses pilin protein that blocks Negative that exclusion mating pair formation. regulator mating
pair formed transcript TraS-blocks DNA transfer. Conjugation Transfers SingleStranded DNA
s Transfer of an F factor is initiated when rolling circle replication begins at oriT. The free 5 end initiates transfer into the recipient bacterium. TraI bound to 5' end. The transferred DNA is converted into double-stranded form in the recipient bacterium. s s Direction Direction of transfer of transfer away from away from F plasmid. F plasmid. 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. Transfer continues until the process is interrupted by (random) breakage of the contact between donor and recipient bacteria. F plasmid F plasmid left behind. left behind. Conjugation
1. Tip of the F-pilus makes contact with recipient cell. a. pilus is composed of pilin subunits which form a hollow cylinder of 8 nm with 2 nm inner diameter. note: DNA does NOT transfer through this "tube". b. If potential recipient is F-positive, no connection is formed due to surface exclusion proteins coded by traS and traT of F-plasmid. 2. Pilus retracts bringing recipient closer for transfer. Conjugation
3. DNA transferred through channel formed the T4SS system. TraD coupling protein which directs the 5' end of the DNA to the channel. TraN and TraG may also participate in this process. 4. Transfer begins from oriT which is nicked by TraY/TraI complex at a nic site. (TraI actually nicks.) 5. TraY/TraI multimeric complex migrates around circle and unwinds DNA from 5' end at ca. 1,200 bp/sec. (Note: TraY recognizes the oriT sequence and binds first.) 6. Only one unit length is transferred. T4SS channel Alvarez-Martinez and Christie MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Dec. 2009, p. 775808 Overview of Conjugation
1. (Conjugative) Plasmids are either self-transmissible or mobilizable. A self-transmissible plasmid contains a tra region. A mobilizable plasmid only contains an oriT (mob site or bom site). 2. Tra systems are linked to their incompatibility group. Ftype IncF, RP4 plasmids IncP 3. Plasmids that have transfer systems that allow transfer of DNA to unrelated species are known as promiscuous plasmids. IncW plasmids, IncP plasmids, & IncN. Self-transmissible transfer
1. A site on the plasmid, known as the origin of transfer (oriT) is nicked by a specific endonuclease (TraI; TraY is also a part of the complex). F+ F- 2. A pore is formed between the two cells and only ONE strand of DNA is passed through to the other cell (5' end first). Free 5' 5' end 3. The single strand in each cell undergoes replication to form double stranded DNA. Only aasingle unit Only single unit length of F factor is length of F factor is transferred transferred F+ F+ Plasmid mobilization
1. The mob plasmid cannot transfer without another plasmid 2. The other plasmid (helper plasmid) may or may not be a self-transmissible plasmid but MUST contain tra functions (cell contact, nicking). 3. If the helper plasmid is selftransmissible it may also transfer. tra helper helper
Mob site Mob site tra tra tra tra Major Functions During Transfer (>40 genes)
1. TraY binds near oriT and recruits TraI (relaxase), 2. TraI has nuclease & helicase (ATP) activity. Function enhanced by TraY & IHF (integration host factor); 3. TraI is a transferase covalent attachment of the 5' end of the DNA to the protein (serves as the pilot protein) 4. TraD active transport, binds DNA, ATP/GTP binding sites, inner membrane protein, necessary for DNA transfer, directs 5' end to the T4SS channel.
Note: Protein A has the combined Note: Protein A has the combined activities of TraY and TraI. It activities of TraY and TraI. It recognizes the ori like TraY and does recognizes the ori like TraY and does the nicking like TraI. the nicking like TraI. Surface Exclusion
Reduces conjugation among cells carrying closely related plasmids (5 exclusion groups identified to date). Part of the Immunity System. 1. TraT outer membrane protein that blocks mating-pair formation 2. TraS blocks DNA transfer, inner membrane How does the F plasmid integrate into the host chromosome? When the F plasmid is integrated, how does it transfer host chromosome sequences to the recipient cell? There are two mechanisms of integration: 1. Homologous recombination 2. Transposition Depending on the site of F-plasmid integration, there are different Hfr strains. Transposition requires IS elements.
E. coli chromosome F-plasmid IS elements Hfr cell contains an Hfr cell contains an F++cell contains F cell contains integrated F plasmid. integrated F plasmid. episomal F plasmid episomal F plasmid integrated F-plasmid oriV After integration, F-plasmid After integration, F-plasmid replicates as part of host replicon. replicates as part of host replicon. oriV is suppressed. oriV is suppressed. oriC Chromosome Transfer: Hfr Recipient cell
Hfr cells 3. Free 5' end Chromosome Transfer: Hfr Recipient cell
(Part 2) 3.
oriT region only (~30 nt) 4. 5. Chromosome transfer
1. The transfer process uses the rolling circle method of replication. The complement to the transferred strand is synthesized in the recipient. It takes 100 min to transfer entire chromosome of E. coli. 2. The double-stranded transferred DNA is integrated into the recipient chromosome by double recombination. 3. F positive (+) strains support high levels of recombination and are described as Hfr strains (high frequency of recombination). Chromosome transfer
4. The transfer of the host chromosome is away from the tra region and F-plasmid, except for a small part around oriT. 5. Typically only relatively short stretches of DNA are transferred & are integrated into the recipient. Chromosome transfer
6. Host Chromosome transfer usually does not result in conversion of recipient cell to F +. 7. In host chromosome transfer, donor DNA integrates into the host genome by recombination or transposition
-In simple plasmid transfer, this does not occur. Chromosome transfer
8. Bacterial contact usually broken before DNA transfer complete; there is a gradient of transfer frequencies (most transfers near the site of insertion). 9. E. coli chromosome as a map divided into 100 minutes; the starting point for the gradient of transfer is different for each Hfr strain; determined by the F factor integration site.
0 &100 E. coli 25 75 50 Formation of Prime Factor Plasmids 1. Plasmids that "leave" the genome carrying chromosomal DNA are known as prime factors (F' plasmid). 2. They "leave" the chromosome by homologous recombination, resulting in a deletion in the chromosome. recombination The Bacterial Ti Plasmid Causes Crown Gall Disease in Plants
s Infection with the bacterium A. tumefaciens can transform plant cells into tumors. The infectious agent is a plasmid carried by the bacterium. s Agrobacterium/plant interactions
Acetosyringone is produced by wounded plant cells (phenolic compound). Agrobacterium in tumor at wound site transfers TDNA to plant cell. opines Agrobacterium infection succeeds only on wounded plants Agrobacterium in soil use opines as nutrients. Components of the Ti Plasmid
1- T-DNA: 23 kb region that is transferred to the plant (only the T-strand actually transferred) a-opine synthesis b-plant hormone synthesis 2-Virulence region (Vir): encodes all of the functions required for the transfer of the T-strand to the plant. T-DNA 23 kb vir genes
Encodes genes necessary for transfer to the plant pTi ~200 kb oriV Components of the Ti Plasmid
3-Opine catabolism: allows the bacterium to use octopine and nopaline nutrient (most other soil bacteria lack these genes) 4-Bacterial conjugation: The Tra region is similar to Tra in other bacteria. This is for transfer of the entire pTi between different Agrobacterium. T-DNA 23 kb
tra bacterial conjugation vir genes
Encodes genes necessary for transfer to the plant pTi ~200 kb opine catabolism
For opine metabolism by the bacteria oriV s The plasmid also carries genes for synthesizing and metabolizing opines (arginine derivatives) They are used by the tumor cell. Hormone Hormone synthesis synthesis Opine Opine synthesis synthesis T-DNA Carries Genes Required for Infection s s Part of the DNA of the Ti plasmid is transferred to the plant cell nucleus. The T-DNA is the transferred region that integrates into the plant chromosome. The vir genes of the Ti plasmid are:
-located outside the transferred region -required for the transfer process The vir genes are induced by phenolic compounds released by plants in response to wounding. acetosyringone The vir region is responsible for the transfer of T-DNA to the plant wounded plant cell. virA is the sensor. activated virG membrane constitutive virA
receptor for aceto-syringone virG -constitutive/inducible
positive regulator for other vir genes Note: activated virG causes its own promoter to have a new start point with increased activity. s 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. s s Transfer of T-DNA Resembles Bacterial Conjugation
T-DNA is generated when a nick at the right boundary creates a primer for synthesis of a new DNA strand.
VirD1/VirD2 VirD1/VirD2 VirE2 VirE2 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.
VirD2 nicks and VirD2 nicks and binds to 5' end. binds to 5' end. Transfer of T-DNA Resembles Bacterial Conjugation
s 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 integrated into the plant genome s s The mechanism of integration is not known. T-DNA can be used to transfer genes into a plant nucleus. Transfer of T-DNA Resembles Bacterial Conjugation
The Vir proteins that are transported to the nucleus include:
s s s VirD2 (contains 2 NLS sequences, nicks at border sequences) VirD1 (as a complex with VirD2) VirE2 (contains an NLS). The End of Chapter 12 ...
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This note was uploaded on 01/31/2012 for the course PCB 4533 taught by Professor G during the Spring '11 term at University of Florida.
- Spring '11