Prokaryotic Chromosome Structure and Function

Question 1

What are the 3 domains of life?

Answer 1

Bacteria
Archaea
Eukarya

Question 2

How does the volume of prokaryotic and eukaryotic genomes differ?

Answer 2

Prokaryotic genomes must function iwth a very small physical volume, they are packed into a much, much smaller space than eukaryotic genomes.
Prokaryotic cells are generally small, simple capsules which physically constrain and crowd the biomolecules that they contain.

Question 3

What is the basic textbook view of the prokaryotic nucleoid?

Answer 3

The basic textbook view suggests the prokaryotic nucleoid is a condensed looped structure not dissimilar to the eukaryotic metaphase chromosome.
There is a dense scaffold in the middle and lots of loops of DNA approx 10bp long coming off this scaffold.
This textbook view is actually correct!

Question 4

Compare and contrast the structure of the eukaryotic M phase chromosome and the prokaryotic nucleoid?

Answer 4

They are similar in structure, both have a dense scaffold in the middle with lots of loops of DNA coming off this scaffold. However, in prokaryotes, these loops are approx. 10kb long and in eukaryotes they are somewhat longer, approx. 100kb.
Supercoiling of DNA is essential in the condensation of the metaphase chromosome, topoisomerases and condensins both create supercoils and trap them in a scaffold to generate the condensed M phase chromosome in eukaryotes.
The same general use of topoisomerases and condensin-like proteins is also used by prokaryotes which are really dynamic in terms of supercoiling due to constant interplay of transcription and replication in prokaryotic nucleoids. The movement of both RNA and DNA polymerases in prokaryotes drives positive supercoils ahead of the polymerase and negative supercoils behind generating a similar structure as seen in the eukaryotic M phase chromosome.

Question 5

Describe the compartmentalisation of the prokaryotic genome?

Answer 5

Prokaryotes do not compartmentalise their genome into a nucleus unlike eukaryotes.
This lack of compartmentalisation extends to everything that the DNA and chromosome does, they don’t compartmentalise their metabolism, they do not have separate cell cycles, they don’t stop their transcription to replicate their DNA and then package up the EDNA and divide it etc.
Everything happens simulatenously, transcription, translation, DNA replication, cell division, it is all happening simultaneously, nothing is compartmentalised.

Question 6

How many protein coding genes are in the E. coli genome?

Answer 6

4288

Question 7

How many operons are in the E. coli genome?

Answer 7

~2500

Question 8

How many rDNA operons are in the E. coli genome?

Answer 8

7

Question 9

How many tRNA genes are in the E. coli genome?

Answer 9

86

Question 10

What is the size of the E. coli genome?

Answer 10

4.5Mbp

Question 11

What is the average size of archael genomes?

Answer 11

2Mbp

Question 12

Describe the organisation of prokaryotic genomes?

Answer 12

Prokaryotic genomes are circular compared to linear eukaryotic genomes.

Question 13

How many oriCs are in a prokaryotic genome?

Answer 13

Prokaryotic chromosomes usually have a single oriC sending replicase complexes in opposite directions around the circular chromosome.

Question 14

What is the name given to each domain of replication initiated from the single OriC in prokaryotic genomes?

Answer 14

Replichore.

Question 15

In bacteria where to the replichores meet and resolve?

Answer 15

At a region called ter (terminus).

In E. coli, ter motifs bind a protein called Tus which prevent replication forms moving in from the opposite replichore.

Question 16

What is the protein that binds to ter motifs in E. coli and what is its function?

Answer 16

A protein called Tus binds a specific sequence at the ends of chromosomes that deal with termination of DNA replication, there are called ter and binding of Tus to ter prevents replication forms moving in from the opposite replichore.

Question 17

How does the movement of DNA and RNA polymerases alter the structure of DNA in prokaryotes?

Answer 17

RNA polymerases and DNA polymerases are often moving simultaneously in prokaryotic genomes because remember there isn’t compartmentalisation physically or biochemically in prokaryotic genomes.
All the time, these polymerases are driving the formation of positive supercoils ahead of them and leaving behind negative supercoils, a dynamic collection of supercoiling events.

Question 18

Besides topoisomerases, what are the other key players in the supercoiling of prokaryotic DNA?

Answer 18

Supercoiling of prokaryotic DNA is also manipulated by nucleoid-associated proteins which include condensin-like structural maintenance of chromosome factors.

Question 19

What is the role of prokaryotic SMCs?

Answer 19

Prokaryotic SMCs are classed as condensins but appear to play both condensin and cohesin-like roles in the nucleoid - they are condensing things and then holding them together.
The SMC proteins are at the base of the loops that come from the scaffold, these SMC proteins are holding stuff together (exactly like in euakryotes).
These SMCs also play a dynamic role in organising the structure of the nucleoid within a prokaryotic cell - they are driving longitudinal organisation of the chromosome such that instead of being circular, the chromosome is being folded into an elongated/sausage shape.

Question 20

What are the different roles of structural maintenance of chromosome factors in eukaryote chromosome structure and function?

Answer 20

In eukaryotes, cohesins hold loops together in the interphase nucleus, hold insulators and gene loos together whereas condensins bring the metaphase chromosome together and help it condense by trapping supercoils created by condensin I and topoisomerase II alpha.

Question 21

When we compare amino acid sequence of SMCs in prokaryotes what do they resemble most?

Answer 21

They resemble condensins more than cohesins but are probably doing both sorts of roles, condensing things and holding them together.

Question 22

What is the name given to the prokaryotic equivalent of a TAD?

Answer 22

Macrodomain

Question 23

Do prokaryotes have TADs?

Answer 23

There are ‘blobby’ domains which are TAD-like and essentially are equivalent to TADs, these are termed macrodomains and are equivalent sizes to eukaryotic TADs - approx 1Mbp.
The prokaryotic nucleoid is subdivided into TAD-like structures called macrodomains and even smaller microdomains (~50-250Kbp)/chromosomal interacting domains (CIDs).

Question 24

How many macrodomains make up the E. coli genome?

Answer 24

4

Question 25

What is the approximate size of E. coli macrodomains?

Answer 25

1Mbp

Question 26

What is the best model to think of the prokaryotic nucleoid?

Answer 26

The best way to think of the prokaryotic nucleoid is as being exactly the same as the human condensed metaphase chromosome, held together by a scaffold with loops (albeit smaller loops) highly supercoiled yet completely transcriptionally active (unlike the metaphase chromosome in eukaryotes).

Question 27

What is the key difference between the prokaryotic nucleoid and the eukaryotic M phase chromosome?

Answer 27

The prokaryotic nucleoid is exactly the same structurally as the M phase chromosome yet is entirely transcriptionally active even in this highly supercoiled, semi-compacted state.

Question 28

What do 3C experiments reveal about interactions between the oriC and ter sequences?

Answer 28

With 3C contact probability maps we see an extra signal running perpendicular to the diagonal.
The inference of this is that the chromosome must be folded in on itself and therefore that the OriC and terminator are interacting with one another.

Question 29

What is meant by longitudinal organisation of the prokaryotic chromosome?

Answer 29

The scaffold is at the centre of each bit of chromosome and the circular chromosome folds into an elongated shape - longitudinal organisation.
This is driven by the SMC proteins as shown upon SMC depletion we lose this feature.

Question 30

In what organisation do prokaryotes maintain their chromosomes?

Answer 30

Broadly speaking, prokaryotic chromosomes (at least in rod-shaped bacteria) appear to be broadly maintained in their circular forms but are then folded eitehr longitudinally or transversely with respect to oriC and ter by SMC proteins.

Question 31

How are SMC proteins loaded onto the DNA in prokaryotes?

Answer 31

In E. coli and in Bacillus, there exists a set of proteins that load SMC rings onto DNA, this protein is called parB which binds a motif called ParS and this is the loading factor that helps SMC proteins load onto the chromosome and this normally occurs at the OriC.

Question 32

What are parB proteins?

Answer 32

parB proteins are crucial in the loading of SMC factors onto DNA and do this by binding to a parS motif which normally occurs at the OriC.

Question 33

Briefly describe an experiment that shows that SMC proteins are actively loading on DNA and are dynamic in their activity?

Answer 33

By engineering a single parS site into a chromosome and providing a parB from an inducible promoter, it can be shown that SMC acitvley zippers up the arms of the chromosome from the ori macrodomain.
The implication is that SMC proteins are dynanmic, actively loading onto DNA and moving along DNA relative to the underlying sequence.
This is an active biochemcial process, SMCs use ATP hydrolysis to actively move through the loops f DNA they create - this is ATP-dependent DNA loop extrusion.

Question 34

How do SMCs move thorugh the DNA loops that they create?

Answer 34

SMCs use ATP hydrolysis to activley move through the loops that they create - this is ATP-dependent DNA loop extrusion.

Question 35

What other function do SMC proteins have when zippering up chromosomes and condensing them up?

Answer 35

SMC zippering is now thought to actively drive separation of daughter chromosomes during bacterial cell division.
In this zippering up process in prokaryotes, it is thought this actually divides the chromosomes when they do DNA replication.
Prokaryotes don’t have a proper spindle and don’t complete cell division in the same way as eukaryotes, instead this process appears to be completely passive, they replicate their DNA and then the SMC proteins come along and squeeze them apart - a completely different cell division process to eukaryotes.

Question 36

How are SMCs associated with TAD formation in eukaryotes?

Answer 36

A current model exists suggesting cohesin extrudes loops of chromatin until it meets a pair of CTCF bound insulators. Additionally, DNA loop extrusion as the SMC proteins actively move through the DNA loops they generate is involved in driving TAD formation.
This aids phase transition process and allows demarcation of TAD boundaries.

Question 37

Describe the structure of non-SMC NAPs?

Answer 37

Non-SMC NAPs are often small (70-200 amino acids) DNA-binding proteins with short alpha-helical regions separated by loops.
This organisation is reminscent of histone proteins although there is no sequence similarity.
Most non-SMC NAPs form dimers/multimers.

Question 38

What is the approximate size of non-SMC NAPs?

Answer 38

70-200 amino acids.

Question 39

How do non-SMC NAPs differ from histones?

Answer 39

non-SMC NAPs are reminscent of histones despite having no sequence similarity, there are some key differences….
Most prokaryotic non-SMC NAPs interact relatively sparsely with DNA, in prokaryotes, protein:DNA ratio is low, there is only the odd protein, most DNA compared to human cells where the ratio is 50:50, therefore, in prokaryotes, these non-SMC NAPs are much more sparsely dotted around DNA.

Question 40

Compare the mass ratio of protein:DNA in prokaryotic nucleoids vs. eukaryotic chromosomes?

Answer 40

Most prokaryotic nucleoids have a mass ratio of protein:DNA of ~ 0.2.
Compared to a much higher mass ratio of histone octamer:DNA of 1 in eukaryotes.

Question 41

Do non-SMC NAPs have any sequence specificity?

Answer 41

Non-SMC NAPs have some sequence specificity, they often prefer short regions of A/T rich DNA, however unlike eukaryotic transcription factors, they don’t bind highly specific motifs.

Question 42

What are non-SMC NAPs doing at the structural level?

Answer 42

At the structural level, they bind DNA to bend, bridge, wrap and stiffen DNA sequences.

Question 43

What are different categories of structural functions performed by these non-SMC NAPs?

Answer 43

Benders - able to put extreme bends in DNA
Bridgers - able to bridge and link bits of DNA and will sometimes form filaments.
Wrappers
Stiffeners

Question 44

At the functional level, what are the two roles of non-SMC NAPs?

Answer 44

Depending on how and where they bind DNA in the genome, non-SMC NAPs have two very distinct roles…

1. Architectural proteins which help trap supercoils.
2. Context-dependent DNA-binding transcription factors - even though they don’t have absolute binding specificity, they do act as true proper transcription factors.

Question 45

What is Fis and what is its function?

Answer 45

Fis is an abundant dimeric NAP expressed in early bacterial growth stages.
When bound close to a promoter, Fis can act as a conventional transcription factor, some genes have sites that Fis can bind and depending on the type of gene, Fis will either be an activator or a repressor.
Elsewhere in the genome, Fis is nothing to do with expression and can bind DNA and bridge supercoiled loops and here Fis is a purely architectural factor trapping supercoils and preventing them from unwinding.
Therefore, Fis is sometimes a bridger and bridges and stabilises negatively supercoiled DNA domains.

Question 46

How does Fis production change with bacterial cell culture?

Answer 46

Fis is produced as bacterial cell cultures begin to grow, but the amount of Fis produced rapidly decreases as cells enter the exponential growth.

Question 47

Which bacterium has only a single DNA binding factor and what is it?

Answer 47

Mycoplasma genitalium has the smallest prokaryotic genome and has a single DNA-binding factor called HU which is so versatile it’s the only one required.
HU is the only architectural protein and transcription factor that Mycoplasma uses to regulate all its genes.

Question 48

What is HU and what is its function?

Answer 48

HU is an abundant nucleoid-associated protein with roles in normale nucleoid structure, transcription, RNA binding and recombination.
HU is a bit histone-like, it comes in different forms, it can be a homodimer or heterodimer etc.
Isolated dimers cause flexible bends in DNA, but aggregates are also found which form wrapped helical structures which are stiff.
Therefore, HU is a bender, wrapper and a stiffener.
At terminators, HU acts as a recruitment site for topoisomerases which process transcription-induced supercoils.
At promoters, HU induces flexible bends to allow gene-regulatory DNA to form loops allowing communication between other transcription factors.

Question 49

What are the two examples of non-SMC bacterial NAPs to learn?

Answer 49

HU and Fis.

Question 50

In what phyla would you find Alba and proteins containing Alba-domains?

Answer 50

Archaea.

Question 51

What is the function of Alba?

Answer 51

Alba is an archael nucleoid-associated protein that can form dimers, homodimers, heterodimers but also has waggly tails coming out of it - like histones but not all related (convergent evolution).
Alba can form bridged complexes, it can form huge aggregates and there are some archaeal cells which if stressed will coat their entire chromosome in Alba and turn the chromosome into a tiny blob to protect it from extreme environments.

Question 52

How do cells use Alba concentrations to cope with different environments?

Answer 52

Depending on the concentration of Alba it can bind to the archaeal nucleoid in different ways.
If cells get stressed, they can completely coat their entire chromosome in Alba which condenses the chromosome into a tiny blob to protect it from an extreme environment.

Question 53

What is the significance of the waggly tails on the Alba protein?

Answer 53

The waggly tails on the Alba protein are acetylated by a protein acetyltransferase (PAT) which acetylates the K16 residues on the tails of Alba and turns Alba into a transcriptional activator - this acetylation has an activating effect on transcription similar to histone acetylation.
Sir2 shows structural similarities to eukaryotic HDAC co-repressors and deacetylates the K16 residues on Alba having a repressive effect on transcription - similar to histone deacetylation in eukaryotes.

Question 54

What is responsible for acetylation of K16 residue on Alba?

Answer 54

PAT (protein acetylase)

Question 55

What is responsible for deacetylation of K16 on Alba?

Answer 55

Sir2 shows structural similarities to eukaryotic HDAC co-repressors and deacetylates the K16 residue on Alba.

Question 56

Which group of archaea have Alba?

Answer 56

Crenarchaeote

Question 57

Are NAPs essential for bacterial nucleoid structure?

Answer 57

No one NAP is absolutely required for bacterial nucleoid structure - instead they work together and dynamically to define the overall pattern of intra-chromosomal interaction.
No one knockout of Fis, SMC or HU for example completely abolishes intra-chromosomal domain formation.

Question 58

Are NAPs exclusive to prokaryotes/histones specific to eukaryotes?

Answer 58

It was originally believed NAPs were exclusive to prokaryotes and histones exclusive to eukaryotes.
However, in recent years proper histones have been identified within certain archael subphyal - interestingly, these are the archaeal subphyla that we as humans/eukaryotes have evolved from.

Question 59

How do the histones identified in certain archaeal subphyla compare to eukaryotic histones?

Answer 59

Eukaryotic and archaeal histone fold sequences are homologous yet only have have approximately 25% amino acid sequence.
These archaeal histones are true histone with the 3 alpha helices with loops.
This is clearly an ancient protein fold that evolved very early in evolution of life.
Eukaryotes have 4 core histones as well as a range of variant histones, whereas archaeal species usually have only 1 or 2 core histone types.
Eukaryotic histone core is modular build from assemblign dimers, archaeal histones aren’t quite as complex.
Eukaryotic histones have long tails that can be modified whereas archaeal histones don’t have long tails and don’t always get modified.
Archaeal histones either have one or two histone folds per polypeptide no/minimal tails and no post-translational modification and form homo or heterodimers.
Whereas eukaryotic histones have either one histone fold per polypeptide, they are assembled in heterodimers and have long tails for post-translational modifications.

Question 60

How old is the histone fold?

Answer 60

The histone fold is an ancient protein fold that evolved early in the evolution of life.
The histone fold is likely to be more than 2 billion years old.

Question 61

What was discovered when chromatin-seq was applied to archaeal histones?

Answer 61

It was discovered there are 2 completely new forms of beads-on-a-string chromatin.

Question 62

Describe the nucleosomes of Haloferax volcanii?

Answer 62

Haloferax volcanii was isolated from hyper-saline dead sea and requires 4M NaCl in its growth medium to survive and it makes half-sized nucleosomes.
These nucleosomes are made of a histone tetramer (consisting of 2 histone dimers with 60bp of DNA wrapped around) compared to histone octamers found in higher eukaryotes.
The nucleosomes of H. vocanii are specifically positioned with active gene regulatory DNA configured in chromatin free regions.

Question 63

Describe the nucleosomes of Thermococcus kodakarensis?

Answer 63

Thermococcus kodakarensis was isolated from a volcanic fumerole in the South Japan sea.
T. kodakarensis makes multi-histone dimer super nucleosomes again in specific positiosn iwth active gene regulatory DNA configured into chromatin free regions.
These super nucleosomes are multimers of different histone dimers of various different sizes with chromatin-free regions surrounding their promoters.
These histone dimers that make up these super-nucleosomes have unrestricted interaction capacity and therefore, super-nucleosomes can wrpa variable lengths of DNA.

Question 64

Which archaeal species makes half-sized nucleosomes?

Answer 64

Haloferax volcanii isolated from the hyper-saline dead sea makes half-sized nucleosomes consisting of 2 histone dimers and only wraps 60bp of DNA.

Question 65

Which archaeal species makes super-nucleosomes?

Answer 65

Thermococcus kodakarensis isolated from a volcanic fumerole in South Japan Sea makes multi-histone dimer super-nucleosomes.

Question 66

How is nucleosome position in Haloferax volcanii and Thermococcus kodakarensis controlled?

Answer 66

The position of archaeal nucleosomes is encoded and therefore hard-wired into underlying DNA sequence.
In these two speices, they don’t have chromatin remodellign enzymes and so it is the DNA sequence and only the DNA sequence that tells them where to put a particular nucleosome and how big it should be.
This is one of the key differences between eukaryotes and archaea.