lec 1

Comprehensive vocabulary flashcards covering the biology of bacterial plasmids, mechanisms of origin firing, host range constraints, and copy number control across R1, ColE1, and pSC101 plasmid models.

Plasmid

A typically circular, covalently closed circular (CCC) double-stranded DNA molecule that replicates independently of chromosomal replication and generally carries non-essential accessory genes, such as those for antibiotic resistance and environmental adaptation.

Is plasmid replication independant from chromsome replication?

YES! but it can require the hosts replication machinery

• eg. DNA polyermase, primases

Theta replication

A mechanism of plasmid replication resembling the replication of most bacterial chromosomes, characterized by the formation of an intermediate structure resembling the Greek letter theta ($\theta$).

Rolling circle replication

A unidirectional nucleic acid replication mechanism used by specific plasmids and viruses to generate multiple copies of circular DNA.

Origin of replication ($\text{ori}$ or $\text{oriV}$)

The specific DNA sequence on a plasmid where replication initiates; it dictates:

• the host range of the plasmid

• its copy number.

Origin of transfer ($\text{oriT}$)

The distinct site on a conjugative plasmid where single-stranded DNA cleavage and transfer initiate during bacterial conjugation.

Host range

The spectrum or collection of bacterial genera and species in which a particular plasmid is capable of establishing and replicating.

Narrow host range plasmid

A plasmid that can replicate only within a specific bacterium (such as Escherichia coli) and its closely related species, relying primarily on host replication and initiation machinery.

Broad host range plasmid

A plasmid capable of replicating across diverse bacterial genera by initiating replication independently of the host and utilizing flexible interactions between plasmid-encoded and host initiation factors.

Plasmid copy number

The regulated, characteristic number of plasmid molecules maintained per host cell, controlled by plasmid-encoded negative regulatory systems.

R1 plasmid (DNA binding protein)

A low-copy plasmid model whose replication initiation is under negative regulation by an antisense RNA (CopA) and secondary transcriptional repression by a protein (CopB).

RepA (R1 plasmid)

The essential plasmid-encoded replication initiation protein that exerts a positive effect on R1 replication by initiating synthesis at $\text{oriV}$.

Tap (R1 plasmid)

A small leader peptide whose translation is translationally coupled to RepA, meaning its translation is required for RepA translation to take place.

CopA

A rapidly turned-over antisense RNA in the R1 plasmid that base-pairs with the $P_1$ transcript to inhibit Tap translation, thereby sequestering the RepA ribosome binding site (RBS) and preventing RepA translation.

CopB

A plasmid-encoded transcription factor in R1 that represses the $P_2$ promoter once sufficient plasmid copies enter the cell, leaving transcription of repA solely dependent on $P_1$.

Translational coupling

A regulatory phenomenon wherein the efficient translation of a downstream open reading frame (repA) strictly depends on the active translation of an immediately preceding reading frame (tap).

ColE1 plasmid

A plasmid that serves as the basis for pUC and pET cloning vectors, whose replication control relies on an unstable antisense RNA (RNA I) aided by the Rop protein.

pUC vector -ColE1

An all-purpose high-copy cloning vector derived from the ColE1 plasmid origin of replication.

pET vector - ColE1

A specialized recombinant plasmid expression vector used for protein overexpression, derived from the ColE1 family.

RNA II (ColE1 plasmid)

A pre-primer transcript of approximately $550\, \text{nt}$ that forms an RNA-DNA hybrid at $\text{oriV}$ and is cleaved by RNase H to expose a free $3'\text{-OH}$ group for DNA Polymerase I to prime DNA synthesis.

RNA I (ColE1 plasmid)

A short-lived, rapidly degraded antisense RNA transcript of approximately $110\, \text{nt}$ that base-pairs with RNA II to inhibit replication initiation when plasmid density reaches a critical threshold.

Rop protein

A ColE1 plasmid-encoded homodimeric RNA-binding protein that stabilizes the RNA I–RNA II duplex to promote negative regulation of replication.

Kissing complex

The initial loop-loop RNA-RNA interaction intermediate formed between the stem-loops of RNA I and RNA II before full duplex formation.

RNase H

A host ribonuclease that cleaves RNA strands annealed within RNA-DNA hybrids; in ColE1, it cuts RNA II to generate the critical $3'\text{-OH}$ end required for replication priming.

pSC101 plasmid

A plasmid that regulates its copy number via iteron control, combining negative autoregulation of RepA with plasmid 'handcuffing'.

Iterons

Directly repeated DNA sequences of approximately $17\text{--}22\, \text{nt}$ located within the origin of replication ($\text{oriV}$) that serve as binding sites for plasmid replication initiator proteins.

Negative auto-regulation (RepA)

A mechanism in iteron plasmids such as pSC101 where high levels of the RepA protein bind to the repA promoter region to shut down its own transcription.

Handcuffing (Plasmid coupling)

A steric negative regulatory mechanism occurring at elevated plasmid concentrations, where RepA initiator proteins bridge and link together iteron regions of two separate plasmid molecules, preventing replication initiation.