Fagan L1-3

Question 1

Why do bacteria prioritise gene regulation?

Answer 1

Evolution has selected for efficiency and flexibility above all else in order to pass genes on.

Question 2

What is the structure of generic bacterial DNA?

Answer 2

Most bacteria have singular circular structure. Chromosomes are densely coding so little junk DNA. Few introns with no spliceosome required whilst some have self splicing. Operons are common where group of genes have similar function. Transcriptional units tend to be orientated in the same direction as replication to avoid DNAP crashes.

Question 3

What is the structure of an E.coli DNA?

Answer 3

4.6MB genome with circle of DNA 1.6mm long inside a 2μm cell. Genome is highly organised and constrained into domains but still needs to be accessible for polymerases and so highly dynamic. It is constrained in multiple domains which are all independently controlled.

Question 4

How do you introduce a positive supercoil?

Answer 4

Over twisting the DNA in the same direction of the helix.

Question 5

How do you introduce negative supercoiling?

Answer 5

Under twisting the DNA in the opposite direction of the helix.

Question 6

What are the consequences of supercoiling?

Answer 6

It compacts DNA but can also add or remove energy that can be used for transcription.

Question 7

How does E.coli introduce supercoiling?

Answer 7

It has topoisomerase 1-4. Topo2 or gyrase is the only one that can introduce negative supercoiling. Topo1 introduces a single stranded break, holds both ends and passes the intact strand through and then relegates to relax a negative supercoil or introduce positive supercoiling.

Question 8

How does Topo2/Gyrase work?

Answer 8

GyrB/GyrA complex allows GyrB to bind DNA and GyrA to make a double stranded break, whilst remaining covalently bound to each other. GyrA (ATPase) hydrolyses ATP to allow a conformational change that passes the intact strand through the break. GyrB relegates the break.

Question 9

What do bacterial promoters consist of?

Answer 9

A -35 (TGTTGACA) and -10 (TATAAT) sequence. In perfect promoters, there is 17bp between them so one RNAP is required as different amount of base pairs causes rotation so sequences are out of phase.

Question 10

What is the Shine Dalgarno sequence?

Answer 10

(AGGAGG) and is the ribosome binding site that can initiate the ATG start codon.

Question 11

What does bacterial RNAP consist of?

Answer 11

A core polymerase - 2 alpha subunits, 1 beta, 1 beta’ and 1 omega. Requires a sigma factor creating a holoenzyme allowing promoter recognition.

Question 12

How is bacterial transcription initiated?

Answer 12

Polymerase binds non specifically and scans a sigma70 subunit recognises a promoter. This is closed complex. DNA is unwinded forming open complex. Sigma factor is kicked out and transcription starts.

Question 13

What are the two mechanisms for transcription termination in bacteria?

Answer 13

Rho dependent and Rho independent.

Question 14

What is Rho dependent transcription termination in bacteria?

Answer 14

Rho binds to Gc-rich sequence after ORF which wraps around the Rho hexamer. Rho ATPase activity drives spooling which makes contact with polymerase and stops transcription.

Question 15

What is Rho independent transcription termination in bacteria?

Answer 15

The terminator is a stable GC-rich terminator stem loop. Once transcribed, contact between the stem loop and the polymerase stops transcription.

Question 16

What do sigma factors do?

Answer 16

Using alternative sigma factors directs transcription of different sets of genes. When the conditions are optimum for the bacteria sigma70 is the dominant sigma factor. When environmental conditions change, the sigma factors are required to change in order to become more versatile and survive.

Question 17

How are sigma factors regulated?

Answer 17

Through transcript of secondary structures, proteolysis, anti-anti-sigma factors etc.

Question 18

How many sigma factors does E.coli have and what are they?

Answer 18

E.coli has 6 alternative sigma factors:
* σ70 – housekeeping σ-factor(σA)
* σ38 – general stress response σ-factor (σS)
* σ32 – heat shock/cytoplasmic stress σ-factor (σH)
* σ28 – flagellar and motility gene σ-factor (σF; FliA)
* σ24 – extracytoplasmic stress σ-factor (σE)
* σ19 – ferric citrate transport σ-factor (σI)
* σ54 – nitrogen-related σ-factor (σN)

Question 19

What is sigma70?

Answer 19

It is the housekeeping sigma factor. Promoters have -10 (TATAAT) and -35 (TGGACA). It is the most abundant sigma factor during exponential growth.

Question 20

What are sigma70s anti factors?

Answer 20

Rsd and HscC. Rsd binds to region four, the activator contact and -35 recognition, and blocks binding to RNAP core. This allows the lower affinity sigma38 to bind to RNAP. HscC acts like a DnaK chaperone and binds to sequestering sigma70. HscC is a lot less specific and so can bind anywhere on sigma70, stopping its action.

Question 21

What is sigma38?

Answer 21

It is the general stress factor. Sigma38 promoters are similar to sigma70. Its abundance is about 1/3 of sigma70 with a lower affinity for RNAP core than sigma70.

Question 22

What regulates sigma32?

Answer 22

Temperature. Lower temps causes degradation under 42 degrees due to sensitive secondary structures.

Question 23

What is sigma28?

Answer 23

Activates expression of motility and flagella synthesis. Promoters have an extended -10 element (GCCGATAA) with the GC rich region being important. Only few promoters so does not control many genes. Half as abundant as sigma70 but decreases under heat shock.

Question 24

What links flagella regulon expression to assembly?

Answer 24

A cascade. Class 2 operons are most important and controlled by sigma70 with the proteins encoded forming the base. One of the operons – fliAZY makes sigma28 which can go on to make class3 operons.

Question 25

What is sigma54?

Answer 25

Controls expression of nitrogen related genes. Promoters have consensus -12 (TTGCA) and -24 (CTGGNA) elements and upstream enhancer binding sites. About 1/10th as abundant as sigma70. Dependent on the hydrolysis of ATP by AAA+ transcription factors bound upstream of enhancer regions. This often requires IHF, a nucleoid associated protein, to bend DNA. There are also no recognised anti-sigma54 factors.