Prokaryotic Chromatin

Question 1

Describe prokaryotic cells and their genome.

Answer 1

Prokaryotic cells are generally small, simple capsules which constrain and crowd the biomolecules that they contain. Their genome must function within a very small physical volume.

Question 2

How are prokaryotic chromosomes organised?

Answer 2

The basic, text-book, view of the prokaryotic nuclei suggests a condensed, looped structure not dissimilar to the eukaryotic metaphase chromosome. (Dense scaffold and extended DNA loops).

Question 3

Do prokaryotes compartmentalise their genome into a nucleus?

Answer 3

No. Everything is mixed together. Chromosomes in the cytoplasm form a general DNA:protein complex called the nucleoid.

Question 4

Do prokaryotes compartmentalise their DNA metabolism?

Answer 4

No. Transcription, translation, DNA replication and cell division often all occur simultaneously.

Question 5

What is a replichore?

Answer 5

Prokaryote chromosomes usually have a single oriC sending replicase complexes in opposite directions around the circular chromosome. Each domain of replication is termed a replichore.

Question 6

What is the terminus (ter)?

Answer 6

In bacteria, replication forks meet and resolve at a region called terminus which is opposite oriC.

Question 7

What is the Tus protein?

Answer 7

In E.Coli ter motifs bind a protein called Tus, which prevents replication forks moving in from the opposite replichore.

Question 8

How are the two chromosomes separated?

Answer 8

A site-specific recombinase separates the two chromosomes.

Question 9

What does the movement of RNA polymerases and DNA polymerases do to the DNA ahead of it and behind it?

Answer 9

The movement of both RNA polymerases and DNA polymerases drive positive supercoils into DNA ahead of the enzyme and leave a wake of negative supercoils behind. Supercoiling affects the topology and compaction of DNA domains.

Question 10

How is supercoiling of regions of prokaryotic DNA modulated?

Answer 10

By a wide variety of topoisomerase enzymes which relax or increase supercoiled states and Nucleoid Associated Proteins (NAPs) including condensin-like SMC factors.

Question 11

Describe prokaryotic SMCs.

Answer 11

The prokaryotic SMCs are classed as condensins but appear to play both condensin and cohesion -like roles in the nucleoid. Hi-C experiments reveal that SMC proteins organise prokaryotic nucleoids into indicate and dynamic structure. Prokaryotic SMC proteins create a scaffold and subdivide the nucleoid into loops like eukaryotic M-phase DNA.

Question 12

What are prokaryotic nucleoids subdivided into?

Answer 12

TAD like structures called macro domains and smaller micro domains/CIDS (chromosomal interacting domains)

Question 13

What is the structure of the prokaryotic nucleoid described as being similar too?

Answer 13

The condensed eukaryotic M-phase chromosome but with full transcriptional activity

Question 14

What does the pairing of chromosome arms in bacteria require?

Answer 14

SMC factors. SMC actively zippers-up the arms of the chromosome from the ori macrodomain.

Question 15

What are the typical arrangements of prokaryote chromosomes?

Answer 15

Prokaryote chromosomes (at least in rod-shaped bacteria) appear to be broadly maintained in their circular forms, but folded either longitudinally or transversely with respect to OriC and ter by SMC action. This arrangement is species-specific.

Question 16

What is the parB protein and where does it binds?

Answer 16

Bacterial SMC proteins gain access to DNA via a “loading protein called parB which binds to multiple parS sites found close to oriC.

Question 17

How do SMCs actively move DNA through the loops that they create (DNA loop extrusion)?

Answer 17

They use ATP hydrolysis.

Question 18

What is thought to actively drive the separation of daughter chromosomes during bacterial cell division?

Answer 18

SMC zippering (prokaryotes do not have a spindle and don’t do proper cell division, they do this passively by replicating their DNA then their SMC proteins come along and squeeze them apart).

Question 19

What is though the help drive formation of TADs?

Answer 19

Loop extrusion by cohesion. Cohesin extrudes loops of chromatin until it meets a pair of CTCF bound insulators. This aids the phase transition process and allows logical demarcation of TAD boundaries.

Question 20

The SMC factors clearly play a major role in defining prokaryotic chromosome structure. What are the other NAPs doing (at the structural and functional level)?

Answer 20

Non-SMC NAPs have some sequence specificity (i.e. they often prefer short regions of A/T-rich DNA) however they do not bind highly-specific motifs like eukaryotic TFs. At a structural level they bind DNA to BEND, BRIDGE, WRAP and STIFFEN DNA sequences.

At a functional level they often have two roles depending on how and where they bind to DNA in the genome:

1) Architectural proteins with help trap supercoils
2) Context-dependent DNA-binding transcription factors (TFs).

Question 21

Describe the structure of non-SMC NAPs.

Answer 21

Non-SMC NAPs are often small DNA-binding proteins with short alpha-helical regions separated by loops. This organisation is reminiscent of histone proteins, although there is no sequence similarity. Most of them form dimers and/or multimers. Although there is a vague similarity to histone structure most prokaryotic non-SMC NAPs interact relatively sparsely with DNA. Most prokaryotic nucleotides have a mass ratio of protein:DNA 0.02 whereas the eukaryote histone octamer:DNA ratio is much higher, 1.0 (50/50 protein to DNA).

Question 22

Where are TF binding motifs located in prokaryotes?

Answer 22

In prokaryotes TF binding motifs are closely associated with promoter elements and can activate or repress transcription helping or hindering RNA pol access to the promoter.

Question 23

What is Fis?

Answer 23

An abundant dimeric NAP expressed in early bacterial growth stages. When bound close to a promoter it can act as a conventional TF (activator or repressor depending on the gene). Fis is produced as bacterial cell cultures begin growth, the amount rapidly decreases as cells enter exponential growth. Fis also bridges and stabilises negatively supercoiled DNA domains (Fis is therefore sometimes a bridger).

Question 24

What is HU?

Answer 24

HU is an abundant NAP with roles in normal nucleoid structure, transcription, RNA binding and recombination. Alpha and beta subunit levels fluctuate according to growth phase and signals; binds and bends 36bp regions of DNA but no specific binding sequence. HU isolated dimers cause flexible bends in DNA, but aggregates are also found which form wrapped, helical structures with are stiff. (HU is therefore a bender, wrapper and a stiffener).

Question 25

HU: what do flexible bends at promoters allow?

Answer 25

Flexible bends at promoters allow gene-regulatory DNA to form loops allowing communication between other TFs

Question 26

What does HU act as at terminators?

Answer 26

A recruitment site for topoisomerase which process transcription-induced supercoils.

Question 27

What is mycoplasma gentalium and what is the role of HU in mycoplasma gentalium?

Answer 27

The simplest/smallest prokaryotic genome. HU is so versatile that it is M.genitaliums only DNA binding factor/TF/NAP.

Question 28

What is alba?

Answer 28

Alba and proteins containing alba-domains occur in most archaea phyla. Alba function has diversified hugely throughout archaeal evolution. Depending on the concentration of Alba (this can be species of cell-condition specific) it can bind to the archaeal nuclei in many different ways. Can form homodimers and heterodimers and also has little tails. Can bind as isolated dimers or can form bridged complexes or huge aggregates.

Question 29

What function does Alba have in Sulfolobus (a crenarcheaote)?

Answer 29

Alba acts as a reversibly-acetylated regulator of transcription. (These archaea cells don’t have histones but use Alba very much like a histone and acetylate and deacetylate it as a PTM that regulates their gene expression).

Question 30

What is Pat?

Answer 30

Pat = Protein Acetyl Transferase. Pat acetylates the K16 residue in Alba which has an activating effect on transcription similar to histone acetylation in eukaryotes.

Question 31

What is Sir2?

Answer 31

Sir2 shows structural similarities to eukaryotic HDAC co-repressors and de-acetylates the K16 residues in Alba having a repressive effect on transcription, similar to histone deacetylation in eukaryotes.

Question 32

Do any prokaryotes have histones?

Answer 32

It was originally believed that NAPs and histones were characteristic of, and exclusive to, the prokaryotes and eukaryotes respectively however recent genomics and biochemistry has discovered that “proper” histones (DNA wrappers) exist within certain archaeal subphyla including that from which the eukaryotes evolved. Eukaryotic and archaeal histone fold sequences are homologous: around 25% amino acid sequence identity and archaeal histone dimer X-ray crustal structures reveal identical histone fold structure to eukaryotes (the histone fold is therefore likely >2 billion years old. Archaea species usually only have one or two core histone types.

Question 33

Describe the structure of archaeal histones compared to eukaryotic histones.

Answer 33

Histone polypeptide chain organisation and the presence of histone tails differs between the archaea and eukarya. Our histones are almost always heterodimers and have long tails that can be modified whereas the archaea histones don’t have long tails, these don’t get modified and they can be either homodimers or heterodimers (much simpler)

Question 34

What is Haloferax volvanii and describe the structure and positioning of its nucleosomes.

Answer 34

Haloferax volvanii was isolated from the hyper-saline dead sea and requires 4M NaCl in its growth medium to survive. H Volcanii makes histone tetramer nucleosomes (each consisting of 3 histone dimers and 60bp DNA) often in specific positions with active gene-regulatory DNA configured into chromatin free regions.

Question 35

What is Termococcus Kodakarensis?

Answer 35

Thermococcus Kodakarensis was isolated from a volcanic fumarole on Kodakara island in the South Japan Sea. T Kodakarensis makes multi-histone dimer super-nucleosomes in specific positions with active gene-regulatory DNA configured into chromatin free regions.

Question 36

What are T. kodakarensis super-nucleosomes made from?

Answer 36

Histone dimers with unrestricted interaction capacity which allows them to wrap variable lengths of DNA.

Question 37

How is the position (and size/number of histone dimers in T. Kodakarensis) determined?

Answer 37

It is encoded/hard-wired into underlying DNA sequence. (This is a difference between us and them).

Question 38

Where did histone originate?

Answer 38

Histones have their origin in the archaea and have since evolved to form at least three different types of nucleosomes.

Question 39

Is there any over-arching principle to chromatin and chromosome structure across all domains of life - why do all cells use proteins to bend, bridge, stiffen or wrap DNA?

Answer 39

Maybe, everything from histones to NAPs is always about managing supercoiling? Prokaryotic NAPs trap plectonemic supercoils and nucleosomal DNA is a supercoil.