Bacterial Gene Regulation Flashcards
Why has bacterial gene regulation evolved so greatly?
Due to their competitive and ever quickly environments bacteria they have to react efficiently to stimuli (must synthesise proteins).
What does it mean that the bacterial genome is a) efficient b) flexible
a) Quick protein production
b) Transcription can be altered.
What are 4 common structures of the bacterial genome?
1) DNA stored as a single circular chromosome
2) Densely coding (few introns)
3) Operons found commonly
4) Transcriptional units tend to be orientated in the same direction as chromosome replication.
What is meant by describing bacterial DNA as densely coding?
There is little space between genes encoding for proteins (short intergenic distances), so are few introns and repeating junk DNA meaning no spliceosome present.
What is the average intergenic distance (DNA between genes) in bacteria?
60-70bp
What is an operon?
Multiple genes which encode for different RNA molecules are encoded from one structural unit, so these RNA molecules make a protein.
What are 3 ways bacterial DNA can be similar to eukaryotic DNA?
1) Can be linear
2) Can have one or more chromosomes
3) Genes can be transcribed as monocistronic transcripts.
How are the few introns present in some bacterial DNA removed?
As they are self splicing, so no spliceosomes are still present.
How is clashes between the RNA polymerase on each strand of DNA avoided?
> Transcriptional units tend to be orientated in the same direction as chromosome replication (a bit like anti-parallel but just on a circle instead of linear)
> Right branch genes in anticlockwise direction
> Left branch genes in a clockwise direction.
What is the average size of an E.coli bacterium and the average length of its genome, why is this an issue if ignored?
> E.coli is a 2um cell
E.coli genome = 4.6 Mb (mega bases) / 1.6mm long
> Not only is the genome so much longer than the cell, as bacteria are usually ready for division they normally have 2-8 copies of their genome present; which is too big for the cell.
How is the bacterial genome able to be a) Organised/ compact but still b) flexible/ accessible?
a) DNA is constrained into multiple domains held together by NAPs for organisation.
>These domains are further coiled for compactness.
b) Coiling is independent between loops (domains) so each can be further compressed or relaxed for transcription.
What does NAPs stand for?
Nucleoid associated proteins
What are NAPs (Nucleoid associated proteins) similar to in eukaryotic cells and why?
Similar to histones as they bind to DNA and help organise it in a compact way.
How many NAPs (Nucleoid associated proteins) does E.coli have?
6
What are the names of the 6 NAPs (Nucleoid associated proteins) E.coli has and what is their function?
- H-NS proteins can bridge between adjacent segments of DNA
- Fis and IHF proteins induce severe bends
3/4. HU (most conserved NAP in bacteria species) can condense DNA into a fibre by wrapping around DNA.
5/6. Dps and CbpA (expressed in stationary phase/ when E.coli are stressed) condense DNA to protect from damage
How many base pairs does Unsupercoiled (relaxed) DNA have per turn in the helix?
Approx 10 base pairs per turn.
What are the 2 ways DNA can be supercoiled and what occurs in both?
1) Positive supercoil
>Over-twist the helix (twist clockwise, same direction as helix), the DNA gets more coiled and loops form on the left side of the DNA.
2) Negative supercoil
>Under-twist the helix (anti-clockwise/ opposite direction of helix), and the DNA gets unwinds and forms a loop on the right side of the DNA
What is the difference and similarity between positive and negative supercoiling?
> Positive supercoiling coils the DNA more by a single stranded break by Topoisomerase I,II, or IV, causing the supercoiled loop to form on the left side. While negative supercoiling un-coils the DNA by a double stranded break by DNA Gyrase (Topoisomerase II) causing the supercoiled loop to form on the right side.
> Both cause loops of DNA to form which condenses the DNA molecule length, but as DNA is usually in negatively coiled state then positively supercoiling leads to relaxing DNA/ un-condencing.
As well as keeping DNA compact, what other mechanisms is supercoiling useful for?
The energy introduced into DNA from supercoiling can be used to make transcription more efficient as energy can be added or removed making the transition from DNA in a closed to open complex easier.
How does supercoiling store and release energy?
The cell expends chemical energy which is held in the knot/loop as it’s under tension. If the loop is loosened energy is released, if it is tightened energy is stored (energy is stored when DNA is negatively supercoiled, then when positive supercoiling relaxes the strand it releases energy).
What are the 3 enzymes regulate positive supercoils and what 1 enzyme regulates negative supercoils in E.coli?
1) Topoisomerase I, III, IV induce positive supercoils which overwinds DNA.
2) Topoisomerase II (Gyrase) negatively supercoils DNA (underwinds)
Is the majority of bacterial DNA negatively or positively supercoiled?
Most bacterial DNA is negatively supercoiled (more as it makes transcription more efficient)
What is DNA Gyrase (Topoisomerases II) composed of?
2 protein subunits, GyrB + GyrA
How does negative supercoiling occur in E.coli in 3 steps?
1) GyrB/GyrA complex (Gyrase) – GyrB binds DNA and GyrA makes a double-strand break, remaining covalently bound to each end of the break.
2) GyrA (an ATPase) hydrolyses ATP causing a conformational change that passes the intact double strand of DNA through the break, reducing unwinds the helix causing a supercoiled loop to form on the right
3)GyrB re-ligates the break, leaving the DNA negatively supercoiled (unwinding causes new supercoiled loop to form on the right)