Lecture 4: (finishing Bacterial gene expression) and Plasmids

Polycistronic mRNA

• Multiple proteins made form this mRNA

• Often operons

Proteins must be translated and properly ___ to be stable and active. Some can fold automatically but others need help from ___

Folded, chaperones

Chaperones; examples?

• Proteins that help other proteins fold into their final conformation

• Heat shock proteins (Hsp, Dnak), trigger factor

Do all proteins exist in the cytoplasm?

No, some are secreted or transported out of the cytoplasm. They can be fully secreted or membrane anchored

Sec system

transports unfolded peptides across the membrane that can then be folded

Tat system

Transports folded proteins

Signal sequences

Sequences on the N terminus of a peptide that can target a protein for transport out of the cell or for anchoring to the cytoplasmic membrane

Reporter gene

gene whose product is stable and easy to assay, conveniently detectable and quantifiable

Plasmids

• DNA molecules that exist within bacteria separately from chromosome

• Important for bacterial adaptation + evolution

• Useful tool in molecular biology

T or F: plasmids are often circular but can be linear

True

T or F: number of copies of plasmid in a bacteria can vary and bacteria can have more than one type

True

Are plasmids partitioned?

Yes, they encode RNA and proteins. They replicate as the cell grows, and are passed down to daughter cells

T or F: plasmids generally encode essential functions for bacterial growth

False: they usually do NOT encode essential functions

T or F: plasmids have the same origin of replication as chromosomes

False

How were plasmids HISTORICALLY named?

Were named based on phenotype they provide to bacteria carrying them.

• R-factor plasmids = resistance to several antibiotics

how are plasmids named now?

• Now plasmids are given number and letter names, much like strains of bacteria

• A small “p” for plasmid goes before capital letters that describe the plasmid or person who isolated or constructed it

• Numbers usually go after the letters to identify the construct

• Ex: JAT101, pJAT102 (modified), pJAT103 (different modification)

What is the benefit of harboring plasmids? Examples?

Plasmids give bacteria a selective advantage under certain conditions

• enzymes for certain carbon source utilization

• Resistance to substances (antibiotics)

• Synthesis of antibiotics

• Synthesis of toxins + proteins that aid in infection of higher organisms

5 reasons to not just have plasmid genes as part of the chromosome

1. Decreases competitiveness

2. Allows for smaller chromosomes

3. By distributing different plasmids among species members, ensures some members will survive

4. Allow bacteria to occupy larger ecological niches

5. Contributes to the success of the plasmid itself]

Basically, it is energetically costly and allows for some heterogeneity in population

Plasmids are generally circular with no free ends that are ___ closed

Covalently

In plasmids, having no free ends to rotate leads to ___

Supercoiling

Supercoiling makes the plasmid run ___ on an agarose gel

Faster

T or F: plasmids have replicons that allow them to replicate independently

True

Requirements for replicons

• must have at least 1 origin of replication (ori site)

• Must code for the proteins required for replication to initiate (the rest of the replication machinery can be borrowed from the host like ligases, primates, helicases etc)

What are the 2 types of mechanisms that plasmids can replicate by

• Theta replication (most common)

• Rolling circle replication

Steps of Theta Replication

1. Opening of the 2 strands of DNA at the ori region creates a structure that looks like the Greek letter theta

2. RNA primer begins replication

3. Replication fork can proceed in 1 or both directions around the DNA

Steps of Rolling Circle Replication

1. Rep protein recognizes a palindromic sequence (double strand origin, DSO) in the DNA and makes a nick

2. Free 3’ OH primer for DNA pol III to make new strand using other strand as template, displacing the nicked single strand

3. The displaced single stranded DNA is replicated using only host-encoded proteins

   • RNA polymerase makes a primer at a different single strand origin (SSO) and DNA pol III synthesizes the other strand starting at the primer using the single strand as a template

4. Rep is used once for each round of replication and is then destroyed —> the level of Rep controls the plasmid number

Genes required for replication of plasmids are located very close to the ___

Ori region

Functions of the ori region often determine plasmid properties like:

• host range

• Copy number

• Incompatibility

What is a host range for a plasmid

All types of bacteria in which the plasmid can replicate (broad vs species specific)

Copy number of a plasmid

The average number of that plasmid per cell

Incompatibility of a plasmid

The ability of 2 plasmids to co-exist in the same cell

Narrow vs wide host range

Narrow = can only replicate in a few species (ex: exist only in E.coli and closely related species — pET and pUC protein expression vectors)

Wide = can replicate in many types of bacteria, both Gram + and Gram -

Broad host range plasmids must encode their own ___

Proteins required for initiation of replication

__ and __ for replication genes of broad-host-range plasmids have evolved so they can be recognized by a wide variety of bacteria

Promoters and ribosome binding sites

How to determine the host range of a plasmid

1. Largely done by trial + error

2. Requires a way of introducing plasmid to other bacteria (transformation system, electroporation, or some are self-transmissible or mobilizable via conjugation)

3. Requires a way to select for bacteria which have taken up the plasmid (ex: antibiotic resistance)

3 ways of introducing plasmid to other bacteria

1. Transformation system

2. Electroporation

3. Conjugation (self-transmissible/mobilizable)

T or F: bacteria can possess more than 1 type of plasmid at a time

True

T or F: all plasmids can co-exist inside the same bacteria

False, some cannot coexist inside the same bacteria

If 2 plasmids can NOT coexist stably, they are said to be member of ____

The same incompatibility (Inc) group

If 2 plasmids can coexist stably, they are said to be of ___

Different incompatibility (Inc) groups

Plasmids can be incompatible for a number of reasons like:

• they can regulate each other’s replication

• They can share the same partitioning (par) functions

Why does copy number of a plasmid need to be regulated?

• to prevent overproduction of the plasmid

• To prevent too few copies being made

Relaxed plasmids are ___ copy number plasmids

High

Stringent plasmids are

Low copy number plasmids

Relaxed plasmids need only have a mechanism that inhibits replication when ____

Plasmid concentration reaches a certain level

Stringent plasmids replicate only once or very few times during each cell cycle and have tighter ___

Mechanisms to regulate replication

Cloning vectors

Replicons that DNA can be added into

Advantages of plasmids as cloning vectors

• don’t kill host cells

• Relatively easy to purify

• Small size since few genes are required for replication

Requirements for creating a cloning vector

• plasmid origin (oriV)

• Selectable markers (like antibiotic resistance genes)

• Multiple cloning site (downstream of promoters and TIRs)

Multiple cloning site

• designated site for addition of DNA with several unique restriction enzyme sites

• Downstream of promoters and TIRs

T or F: plasmids are ideally small and relatively high copy number

True