Flashcards in Prokaryotic Chromatin Deck (39)
Describe prokaryotic cells and their genome.
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.
How are prokaryotic chromosomes organised?
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).
Do prokaryotes compartmentalise their genome into a nucleus?
No. Everything is mixed together. Chromosomes in the cytoplasm form a general DNA:protein complex called the nucleoid.
Do prokaryotes compartmentalise their DNA metabolism?
No. Transcription, translation, DNA replication and cell division often all occur simultaneously.
What is a replichore?
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.
What is the terminus (ter)?
In bacteria, replication forks meet and resolve at a region called terminus which is opposite oriC.
What is the Tus protein?
In E.Coli ter motifs bind a protein called Tus, which prevents replication forks moving in from the opposite replichore.
How are the two chromosomes separated?
A site-specific recombinase separates the two chromosomes.
What does the movement of RNA polymerases and DNA polymerases do to the DNA ahead of it and behind it?
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.
How is supercoiling of regions of prokaryotic DNA modulated?
By a wide variety of topoisomerase enzymes which relax or increase supercoiled states and Nucleoid Associated Proteins (NAPs) including condensin-like SMC factors.
Describe prokaryotic SMCs.
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.
What are prokaryotic nucleoids subdivided into?
TAD like structures called macro domains and smaller micro domains/CIDS (chromosomal interacting domains)
What is the structure of the prokaryotic nucleoid described as being similar too?
The condensed eukaryotic M-phase chromosome but with full transcriptional activity
What does the pairing of chromosome arms in bacteria require?
SMC factors. SMC actively zippers-up the arms of the chromosome from the ori macrodomain.
What are the typical arrangements of prokaryote chromosomes?
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.
What is the parB protein and where does it binds?
Bacterial SMC proteins gain access to DNA via a "loading protein called parB which binds to multiple parS sites found close to oriC.
How do SMCs actively move DNA through the loops that they create (DNA loop extrusion)?
They use ATP hydrolysis.
What is thought to actively drive the separation of daughter chromosomes during bacterial cell division?
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).
What is though the help drive formation of TADs?
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.
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)?
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).
Describe the structure of non-SMC NAPs.
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).
Where are TF binding motifs located in prokaryotes?
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.
What is Fis?
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).
What is HU?
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).
HU: what do flexible bends at promoters allow?
Flexible bends at promoters allow gene-regulatory DNA to form loops allowing communication between other TFs
What does HU act as at terminators?
A recruitment site for topoisomerase which process transcription-induced supercoils.
What is mycoplasma gentalium and what is the role of HU in mycoplasma gentalium?
The simplest/smallest prokaryotic genome. HU is so versatile that it is M.genitaliums only DNA binding factor/TF/NAP.
What is alba?
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.
What function does Alba have in Sulfolobus (a crenarcheaote)?
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).