Prokaryotic Genetic Systems Flashcards
What occurs during rapid growth of bacterial cells?
No B period and replication cycles overlap (not possible in eukaryotes)
What is DAPI and what does it do?
What did DAPI staining reveal?
Fluorescent due that stains AT rich regions of the genome
DAPI staining revealed the bacterial nucleoid occupies 50-75% of the cell volume
How big is the E.coli chromosome?
What is the problem with this?
Why do many bacterial cells often contain more DNA than this?
4.7Mb chromosome in a circle of 1.5mm (minimum amount of DNA required in E.coli cell)
Needs to fit into a cell of 1 by 0.5 um
The have accessory DNA and rapidly growing cells undergo multiple rounds of replication simultaneously = more than one chromosome
To what size would condensation by thermal motion (Brownian motion randomly coiling the DNA) reduce the DNA?
10um; greater degree of condensation required
How can nucleoids be isolated? What are the differences in the two structures produced?
With membrane associated or membrane free (MA or MF)
MA has much more protein and also has phospholipid and four stable DNA binding proteins
HNS, HU, FIS, And IHF
How can nucleoid be disrupted?
What does this show?
Heat, RNase, DNase, proteinase
Held together by weak interactions and DNA RNA and protein each have role in maintaining structures
What are the prokaryotic analogues of histones?
How do they structure DNA?
How is it similar to eukaryotic?
What is special about these loops?
How was this found originally?
DNA binding proteins like HU and H-NS (non sequence specific)
At low concentrations HU bends DNA compacting the nucleoid, at high concentrations HU dimers interact with DNA to form extended filaments
H-NS (histone like nucleoid structure in protein) has two DNA binding domains that it uses to bridge between distant sites on DNA
Dimers plus bringing creates scaffold on which nucleoid can be organised, like eukaryotic DNA, the chromosomes is organised into loop domains
They are topologically independent of one another; a nick in one loop will not relax neighbouring loops
Electron microscopy
What do the evidence for loop domains in prokaryotic nucleoid (also evidence for size of loop domains)?
What size were they approximated to be?
What size does supercooling reduce the nucleoid to?
Originally Electron microscopy:
Later cells irradiated with x Rays and it was found that 160 nicks (one dose of X-ray assumed to equate to one Nick and 160 doses were required) relaxed >95% of super helical tension
Suggests approx 120 loops or chromosome as loops are topologically independent of one another; a nick in one loop will relax that loop but not neighbours
50kb like in eukaryotes (50-100kb)
1um
The size loops were estimated to be from this experiment were found to be incorrect with later experiment with SwaI
How are loop bases thought to be anchored?
Not quite sure so look into further
Scaffold associated regions SAR using BIME and REP
REP has 1-12 tandem repeats of 40bp palindromic sequences which bind gyrase (a enzyme like eukaryotic topoisomerase II)
How was it concluded that DNA loops are variable in size and position and nucleoid is fluid?
Restriction endonuclease SwaI was expressed in living cells (got gene into cells by plasmid uptake). Specific 6bp cleavage sequence of enzyme known so knew where the 117 cleavage sites of the enzyme were in DNA. Cleavage relaxed loop the site was in but not neighbouring loops (topologically independent) Knew of 300 genes in E.coli whose expression varied with supercoiling so used these as reporters when DNA microarray with RNA harvested form cells (shows which genes were transcribed) used to assess effects of cleavage on expression (by seeing which RNA transcripts were absent they could tell which genes had been switched off) Genes showed little response to cleavage if cleavage site more than 10kb away from gene - the size of loops is 10kb
Then introduced new cut sites by genetic engineering for SwaI at varying distances from supercoiling sensitive genes. If domain boundaries fixed to genes should see discrete switches between expression, did not see that so boundaries fluid
This experiment corrected the earlier x-Ray experiment which estimated loops to be 50kb
How have fluorescence and atomic force microscopy shown nucleoid structure to be dynamic?
They have facilitated observance of nucleoid through the different growth stages and revealed that it changes, in particular nucleoid is condensed in non-growing bacteria and more open whilst in log phase
Fluidity in macroscopic structure as well as microscopic
How is eubacteria DNA coiled?
How is negative supercoiling introduced?
How is supercoiling maintained?
What does negative supercoiling mean for the expression of the DNA?
Negatively supercoiled or under wound
DNA gyrase (type II topoisomerase) requiring ATP which makes cuts in both strands of the DNA
Topoisomerase I removed supercoils without ATP by cutting one strand and allowing it to wind around the other to release stress
Antagonistic action of the isomerases a maintains constant level of supercoiling
Negative supercoiling Is slightly under wound and makes DNA more open and accessible for transcription
How is DNA supercoiled and linked (geometry)
What is Lk when DNA is not supercoiled?
What is it when it is supercoiled?
What causes writhe?
The two strands are wrapped around one another in a double helix with negative supercoiling
Two strands in helix are linked by H bonds and base pair surface interactions but also linked geometrically by intertwining of DNA backbones into T twists
One inter-link for each twist of double helix
Lk = linkage between the strands when the helix is not supercoiled = the twist
When the DNA is supercoiled, Lk = W + T
W = writhe = number of times backbone crosses itself
Writhe results from a slight undoing (4%) of the helix turns
antagonistic action of DNA gyrase (supercoils) and topo I which releases stress, (relaxation of helix turns introduces writhe)
What is the eukaryotic cell cycle?
What is the prokaryotic cell cycle?
What period also features in each cell cycle?
G1 phase - growth is mass
S phase - replication of DNA
G2 phase - growth in mass
Mitosis - separation of sister chromatids
Cytokinesis - separation of daughter cells
B period - growth in mass
C period - replication of DNA
(No second growth phase)
D period - separation of daughter chromosomes and daughter cells
G0 in eukaryotic cells and persister formation in prokaryotes, used to think only cells in limiting conditions would enter dormancy phase but many bacterial cells exist in this state even when nutrients are available, makes them very stress resistant (problem in disease)
What two experiments we’re used to estimate the size of prokaryotic DNA loops?
X-Ray (incorrectly estimated to be 50kb)
SwaI estimated to be 10kb
Why can translation and traction ion be coupled in prokaryotes?
What does this allow?
Lack of nuclear membrane
Regulation of transcription by attenuation
What is attenuation?
What does it rely on?
Mechanism of control in bacterial operons which results in premature termination of transcription
The fact that transcription and translation occur simultaneously in prokaryotes
