Molecular Biology - Chapter 12

copy number

number of copies of a single type of plasmid that can be maintained within a cell. - Relative to the number of origins that can be present

What is the name of the region of F plasmid that is required for the initiation of transfer?

oriT

When an F plasmid is not integrated and is free, what is the name of its independent replication origin?

oriV

Partition complex

parS site + ParB + maybe IHF

How is conjugation similar to bacteriophage replication?

"Infects" recipient with a copy of the plasmid

Strand Displacement

- A viral DNA grows by displacing one strand of DNA, then replicating the other independently - Remaining single-stranded dna forms a duplex origin (circular structure, complementary pairing at the ends) which can then be used to initiate synthesis - Used by adenovirus and phi29 DNAs - uses other methods, such as a terminal protein, to provide a primer

What is purpose of strand displacement

- A way to get around the linear replicon problem

Immunity for plasmids?

- Ability of plasmid to prevent another plasmid of the same type to become established in a cell - Specific number of a certain type of plasmid must remain CONSTANT remember?

What role does parS play in the plasmid?

- Acts as a centromere - When ParB (and maybe IHF) binds to it, it creates a partition complex which allows larger complexes to form, and pairs 2 plasmids together - Par A is necessary to position plasmids

Plasmid

- Circular Dna. Not part of chromosome. - Independent from chromosome. Has the ability to independently replicate. - Normally in bacteria - Number of plasmids from generation to generation remains CONSTANT - can be transferred to other organisms

CounterTranscript regulatory system? Example?

- Complementary to RNA primer - Forms an antisense structure of loops and stuff that then affects ability of RNA to form a good hybrid/primer - Pairing between countertranscript is enhanced with a protein - RNA 1/ RNA1/ RNA I and the Rom protein

What are possible special mechanisms that are theorized to solve the issue of linear replicon replication?

- Converting linear to circular - terminus hairpin - variable end (# of copies at a short end varies (like eukaryotic telomerase)) - Terminal protein

What is the purpose of partition systems for plasmids?

- Ensure that duplicated plasmids are segregated correctly during cell division - Maintain single copy

Ri Plasmid

- Hairy root or crown gall disease

Hfr cell

- High frequency recombination - Contain an integrated F factor into their genome - Supports recombination/sexual reproduction because part of genome is conjugated, rather than just f factor - Higher rates of recombination than cells without integrated f factor

What are some impacts of the F plasmid presence?

- selective conjugation (+ and -) - any transfer of the full plasmid will convert a negative bacteria to a positive - if plasmid is integrated, conjugation might also transfer bacterial chromosome as well and may not include full f plasmid

Agropine plasmids

- tumors do not differentiate and die early - opines of agropine type

The free F plasmid is maintained at a level of _____ per bacterial chromosome.

1 plasmid

2 major partition systems? Which plasmids are they generally used for?

1.) ATPase acts as actin, and pushes the plasmids apart via polymerization - Used in R1 plasmid - Well understood 2.) ATPase uses nucleoid to position plasmids - Used in F plasmid and P1 - Not very well understood

Assignment of mitochondria to daughter cells is:

random/stochastic

Integration host factor (IHF)

host-encoded binding proteins

If a multimeric dna length is cleaved at a unit length, then the rolling circle is being used to:

replicate the phage replicon - generate monomers

Why can F positive bacteria only mate with f negative bacteria?

traS and traT -> surface exclusion proteins - cells with pili are unable to mate with other cells with pili

Heteroplasmy

mtDNAs with variations in the same cell (not identical)

Plasmid incompatibility is _____________ feedback control

negative (prevents more output)

When an integrated F plasmid initiates conjugation, orientation of transfer is directed ________ the transfer region.

away from - allows for recombination of bacterial dna into recipient, but may or may not include part of or the entire transfer region

What 3 loci in Agrobacterium chromosome are required for the bacteria to bind to the plant cell?

chvA, chvB, and pscA

What is rolling circle replication?

- In a circular dna, only one strand is replicated via strand displacement. Can go around the circle multiple, multiple times, due to circular structure. - Structure is a direct result of circular structure - Forms single-stranded multimer (complete sequence repeated multiple times, forming a long linear strand) - Cleavage (cut off) at a single unit length or of multimers

What type of nucleic acid genome can lytic phages have? Lysogenic phages? Plasmids/episomes?

- Lytic phages -> any type of genome. Infective particles - Lysogenic phages -> Double stranded genome - Plasmids/Episomes -> Double stranded genome

How are plasmid immunity and phage immunity similar?

- Make it impossible for another element of the same type to become established.

Multicopy plasmids

- More than one copy of a plasmid per chromosome origin - Segregation system is unnecessary

Describe phage phiX174 replication/structure?

- Normally single stranded (+) strand - (+) strand is used to synthesize complementary (-) strand and generates a duplex circle. - Replicated using rolling circle and "A" protein

Lysogeny?

- Phage can be integrated into a bacterial genome and become a STABLE component of genome. - Will enter lysogenic cycle (remember? Cell will not immediately die/enter lytic cycle)

What are negative consequences of F-Pili, and why are F positive cells at a disadvantage?

- Pili provide sites where phages can attach. - susceptible to infection

Crown Gall Disease

- Plant disease - Induced by agrobacterium tumefaciens - creates tumors/growths in plants - Ti Plasmid

Compatibility Group

- Plasmids that are in the same "family" - 2 different plasmids, but their origins are similar enough to be indistinguishable at an essential maintenance stage (regulated by a similar system) - Cannot coexist in the same cell if it's a single copy system - Results in incompatibility

Episomes?

- Plasmids that can integrate into a chromosome. - can also exist independently - All episomes are plasmids. Not all plasmids are episomes.

Segregation/Distribution of mtDNA to daughter mitochondria is ___________ .

- Random/Stochastic

How is conjugation of an independent F plasmid different from conjugation of bacterial genome with integrated F plasmid?

- Recipient cell may not include the full F plasmid - conjugated dna from a full genome may not include the entire F plasmid

TraI protein

- Relaxase (like phiX174 - nicks oriT and binds to 5' end along with TraY - Catalyzes unwinding - TraI bound DNA

Terminal Protein

- Serves as a primer for some linear viral replicon dna - Binds to 5' end, provides a cytidine nucleotide + 3'OH end - Associated with dna polymerase - Used in adenovirus replication - Essential for binding nuclear factor i

Octopine plasmids

- Similar to nopaline plasmids, but synthesized opine is different - opines of octopine type - Undifferentiated tumors

Addiction System

- Survival mechanism used by plasmids - If plasmid dies, cell does too - Plasmid will make both a killer and antidote - If plasmid dies, antidote will decay and the poison will kill the cell - " We hang together or we HANG together" LMAOOOO THAT'S METAL AS F***

Why are the ends of linear replicons a potential problem during DNA replication?

- Synthesis proceeds in a 5'-3' direction. - A strand that runs in a 3'-5' direction poses no problem, because the polymerase can run off of the end. A primer is only required for the beginning, not the end. (leading strand) - However, for the lagging strand, a primer would be required to replicate the very end, and removal of a primer would result in a shortening of the strand. - So in order to preserve the entire replicon, synthesis must start/include the very last base, and researchers do not know if this is possible.

"A" Protein

- encoded by phiX174 - Relaxase - Creates a nick in dna at a specific origin, binds to 5' end, and releases once replication is complete, allowing a new circle of dna to be produced. - Generates a new (+) strand - Involved in circularization of new dna

Plasmids F, P1, and R1 use the same partition system. Which system is this? Describe it?

- genes parA and parB with target site parS (not always called this, just call them par sites for now. ) - parA and par B are trans-acting loci, and produce the proteins ParA (partition ATPase) and ParB - ParA and ParB bind together, which allows them to bind to the parS site. - deletions of any of these genes will prevent proper partition -Partition systems depend on the properties of the ATPase

Nopaline plasmids

- genes that synthesize opine and nopaline in tumors - Shoots with abnormal structures (tumors) - differentiate into different embryonic structures - Aka teratomas

Single copy plasmids

- one replication per cell division, one copy per cell - Equal with bacterial chromosome - require partition/segregation systems

Plasmid R1 partition system? Describe names for structures and overall how it works

- parS = parC - ParA/ATPase = ParM - ParB = ParR - ParM acts as a cytoskeletal element. Resembles actin. With ATP, creates a filament like structure. - ParR and parC bind together, and connect 2 daughter plasmids. ParM binds and polymerizes and pushes R1 plasmids apart

Conjugation

2 cells come in contact and transfer genetic material - one bacteria to another - conjugation bridge

How many tra and trb genes are directly associated with conjugation?

4 traD, traI, traM, and traY

Replicon definition

A DNA/RNA that replicates from a SINGLE origin

What most often results from conjugation between Hfr and F-?

A recombinant F- cell

Rolling circle provides a means of ____________ the original replicon.

Amplifies - Can generate a strand of multimers of original sequence - Amplified material consists of a large # of repeating units

How can linear replicons appear in a cell?

As extrachromosomal units (independent) or on the telomeres of eukaryotic chromosomes.

Relaxosome

Bacterial complex required for conjugation In f plasmid, formed by TraI and TraY protein

Extrachromosomal Replicons

DNAs not part of a chromosome. Independently replicate. - 2 types: plasmids and bacteriophages

The probability of a specific region of bacteria being transferred in conjugation of Hfr cell depends on:

Distance from oriT

How is mtDNA replication unique?

Each strand is replicated using a different origin, which are intiated at different times (once intial replication exposes second origin) Produces a d-loop When second origin is exposes, it releases the displaced strands and replication of both strands continues

F positive bacteria are able to conjugate/mate with:

F negative bacteria only

Stochastic districution of mtDNA results in:

Heteroplasmy

F plasmid can integrate using a recombination event using sequences called:

IS sequences

Why is F plasmid known as fertility factor?

It allows the conjugation of DNA. (provides genes necessary for the process)

What is the T-DNA function?

It transforms the plant cell for agrobacterium use

The phiX174 dna can only be nicked by the A protein if it is:

Negatively supercoiled

Phages and plasmids are generally able to reside in a cell as an independent unit. Is this always the case? Why?

No, it is not always the case. - Certain types of phages/plasmids can be integrated into a (bacterial) genome, which can be inherited and passed along normally. - Known as lysogeny (phages) or episomal (plasmids)

Conjugation between a f+ cell and an f- will always turn the f- into a f+ cell. Is this the case when conjugation occurs between an Hfr and f-?

No. Conjugation of an integrated f plasmid will cut off randomly (because the chromosome is longer than an independent plasmid). And because conjugation is directed away from the transfer region, the conjugated material will most likely not include the entire f plasmid.

Different types of Ti/Agrobacteria plasmids?

Nopaline/teratomas plasmids, octopine plasmids, agropine plasmids, Ri plasmids

What structure do f positive bacteria possess that f negative bacteria do not?

Pili

What is the subunit protien that makes up Pili/pilus? What gene encodes for it?

Pilin traA

Functions involved in partition were first identified in ___________ .

Plasmids

F+ bacteria?

Possess F factor/plasmid

How does TraM help initiate transfer process?

Recognizes that a mating pair has formed

purpose of traJ gene in the transfer region?

Regulator gene

In the Ti plasmid, what does the vir region do?

Required to release and intiate transfer of T-DNA

phage phiX174 is replicated using what sort of mechanism?

Rolling circle

Multimer?

consisting of multiple monomers - Many, many repeating units

Transferred part of Ti plasmid is called:

T-DNA

TraI bound dna is transferred to _________ (secretion complex) by Tra___ .

T4SS, TraD

Structures of a replicon generated by replication depend on relationship between the __________ and the ____________________ .

Template, replication fork

What does incompatibility between plasmids suggest?

That the maintenance system is determined by the replicon/plasmid, not the cell. - Repressor is synthesized that measures concentration of origins

There are 2 major classes of partition systems. These depend of the properties of:

The ATPase (not always called ParA....)

What region of the f plasmid is required for conjugation?

The transfer region ( tra and trb loci)

Plasmids that exist in multiple copies can be distributed to daughter cells stochastically. What does this mean?

There are enough copies of plasmids overall to ensure that each daughter cell will receive plasmids. - Distributed RANDOMLY

Rate of plasmid loss in bacterial populations? Why?

Very low. Less than 10^-7. - Multiple methods exist to ensure the survival of a plasmid

Is the F plasmid an episome?

Yes can integrate into an e. coli genome

The ColE1 compatibility system is controlled by:

an RNA regulator - RNA primer forms hybrid with dna upstream of origin - This RNA can be regulated with 2 things :complementary RNA and a specific protein