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LF206: Molecular Cell Biology > Lecture 1. Bacterial Genomes > Flashcards

Lecture 1. Bacterial Genomes Flashcards

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1
Q

What is our traditional view of prokaryotic genomes based on?

A

Everything we know about E. coli
Circular singular DNA molecule that is located in the nucleoid (doesn’t stain well as full of DNA)

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2
Q

Is there any difference between prokaryote DNA and eukaryote DNA?

A

DNA essentially the same (in the B form)
Two polynucleotide chains are in opposite orientation
Regular right-handed double helix
Diameter of 2 nm and making a complete turn every 3.4 nm.
There are ~10.5 base pairs per turn of the helix

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3
Q

What are the certain flexibilities within the basic B-form?

A

The number of base pairs per turn of the helix can be altered
The helix in the cell is not straight but coiled in 3D space
There are certain sequence features where bends occur

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4
Q

How does the DNA coil on top of itself?

A

Through supercoiling

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5
Q

When does supercoiling occur?

A

When additional turns are introduced into the DNA double helix (positive supercoiling) or if turns are removed (negative supercoiling) which makes the elements of the genome more accessible for proteins but tightens up the genome

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6
Q

What is supercoiling controlled by?

A

The cell, the cell manages their genome constantly reshaping it for their needs

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7
Q

What enzymes help rearrange the genome?

A

Topoisomerases

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8
Q

How is the torsional stress in the molecule accommodated?

A

Two ways
Formation of superhelices
Altering number of base pairs per turn of helix

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9
Q

What is the linking number (L)?

A

Total number of times that the two strands of the double helix of a closed molecule cross each other when constrained to lie in a plane

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10
Q

What is the role of type I topoisomerases?

A

Break one strand of DNA, pass the other complete strand through the gap and seal the break
Linking number changed by ±1
Topoisomerase I of E. coli, relaxes negatively supercoiled DNA
Make supercoiling more severe

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11
Q

What is the role of type II topoisomerases?

A

Break both strands of the DNA, pass another part of the helix through the gap and change the linking number by ±2

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12
Q

What enzyme creates negative supercoils in E. coli?

A

DNA gyrase

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13
Q

What do type I topoisomerases remove?

A

Reduces supercoiling by removing negative supercoils

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14
Q

What is the role of DNA gyrase?

A

DNA gyrase of E. coli is a heterotetramer of 2 subunits (A and B)
Creates negative supercoils (using ATP)
Opens up strands and is essential for DNA replication

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15
Q

How is DNA organised in E. coli?

A

The single circular DNA molecule is organised into a series of supercoiled loops (40-50) that radiate from a central protein core
Highly organised structure
Organse and control genome like eukaryotes that is controlled by the protein core

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16
Q

What does the protein component of the E. coli nucleoid include?

A

DNA gyrase and DNA topoisomerase I that maintain the supercoiled state of the DNA
At least four proteins, the most abundant being HU (heat unstable), that are involved in packaging the DNA (we now know there are many more than just 4)
HU forms a tetramers around which the DNA is wound – approximately in units of 60 bp, cover about/binds to 1/5 of the genome

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17
Q

How many HU protein are in a single E. coli cell?

A

~13,000 HU proteins in a single E. coli cell

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18
Q

What do archaea have instead of proteins related to HU (heat unstable)?

A

Proteins related to eukaryotic histones
One of the pieces of evidence used to suggest that eukaryotes descended form archaea

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19
Q

Why is the idea that prokaryotic genome based on E. coli isn’t entirely correct?

A

Some bacteria have a linear not circular genome (Borrelia burgdorferi (Lyme disease), Streptomyces coelicolor (antibiotic producer), Agrobacterium tumefaciens (plant tumours))
Others have multipartite genomes (genomes divided into two or more DNA molecules)

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20
Q

With multipartite genomes, what is it difficult to distinguish?

A

It can be difficult to distinguishing a genuine component of the “essential” genome from a plasmid

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21
Q

What are plasmids?

A

A plasmid is a (often small) DNA molecule that usually codes for non-essential genes
Some very large plasmids may also carry essential genes

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22
Q

What are essential parts for Vibrio cholerae?

A

Main chromosome (2.961 Mb - million bp)
Megaplasmid (1.072 Mb)
The megaplasmid in Vibrio cholerae is larger than chromosome 2 in Deinococcus radiodurans (nomenclature is confused)

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23
Q

What is the importance of circular plasmid cp32 in Borrelia burgdorferi?

A

Always 5 or 6 copies within the cell, implies important to the cell all of the time

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24
Q

What is it important to remember when talking about microbes in the real environment?

A

Not every microbe is E. coli
May be carrying multiple copies of nearly identical copies of genes

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25
Q

What is an example of a genome that is smaller than many megaplasmids?

A

Nasuia deltocephalinicola, a leafhopper symbiont, genome size 112kbp, 137 genes
Relies on the host as symbiote so can shorten genome

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26
Q

What are important components of the genome of bacteria?

A

Horizontal gene transfer (HGT)
Acquisition of all antimicrobial resistance genes spreads by horizontal gene transfer

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27
Q

Why is horizontal gene transfer (HGT) important?

A

Important in bacterial evolution
‘evolution in quantum leaps’

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28
Q

What are prophages?

A

Phage-like elements found inside bacterial genomes
Linked to pathogenesis and other important phenotypes

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29
Q

What are examples of toxins that are encoded by prophages?

A

Diptheria toxin (C. diptheriae)
Shiga toxin (E. coli)
Cholera toxin (V. cholerae)
Neurotoxin (presence of prophage is the only difference between C. botulinum and benign clostridium)
Leukocidin (s. aureus)

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30
Q

What are genomic islands (GIs)?

A

Horizontally acquired genomic regions
Often mutated so masking/destroying their transmission and integration modes
Can confer fitness to occupy a particular ecological niche
Some linked to pathogenesis (Pathogenicity Islands - PAIs)
Most carry function that allows the bacteria to survive under certain conditions

31
Q

What are transposable genetic elements?

A

Elements within all organisms that can jump between genomes
Some don’t carry anything but most carry function that allows the cell to survive better under certain conditions

32
Q

How does DNA replicate in all organisms?

A

Leading and lagging strands
DNA Pol III (in bacteria) does most work
DNA Pol I fills the gaps in the Okazaki fragments on the lagging strand
Polymerase I has a role in the removal of RNA primers from Okazaki fragments

33
Q

What is the replicon?

A

The basic unit of replication (multiple copies of genomes can make it hard to recognise)
A DNA molecule or sequence with a functional origin of replication
Each replicon MUST be replicated at least once per cell division cycle (to make sure there is always a copy that survives)

34
Q

What two basic features link replication to the cell cycle of all organisms?

A

Initiation of replication commits the cell to a subsequent division
Cell division cannot occur until the round of replication associated with a particular initiation has been completed
If it tries to replicate without proper replication, cell will be produced with incomplete genome

35
Q

What happens in the cell cycle and replication in E. coli (how do you replicate a circular molecule)?

A

Single origin of replication (oriC), therefore a single replicon
Bidirectional replication leading to a theta structure (looks like θ) as two sites of replication move

36
Q

How was E. coli oriC characterised?

A

DNA digested with a restriction enzyme and then ligated into a plasmid lacking an origin of replication

37
Q

What is the length of a region of oriC in E. coli and what does it contain?

A

245bp long and very highly structured, it contains:
14 copies of the sequence GATC
4/5 copies of a 9 bp sequence in the right hand 2/3 of oriC
3 copies of an AT-rich 13 bp sequence in the left hand 1/3 of oriC
These sequences serve a very specific role that allows DNA to be replicated

38
Q

How does initiation occur at the start of E. coli replication?

A

~20 monomers of DnaA (protein) to the 4/5 x 9bp repeats in the RH part of oriC causing the DNA to bend. This forms the closed complex, causing the 3 AT-rich 13bp repeats to melt and create the open complex allowing enzymes to attach to the DNA molecules

39
Q

What is the role of DnaB helicase?

A

DnaB helicase is loaded onto the melted DNA with DnaC (open complex)
ATP is hydrolysed and DnaC released (DnaB now on the different strands)
DnaB unwinds the DNA bidirectionally in a process that requires Single Strand Binding protein (SSB) and DNA gyrase (SSB protects single stranded DNA from defensive mechnaisms and DNA gyrase solves the supercoiling problems)
Primase synthesises a primer RNA molecule on both strands and replication commences

40
Q

What directly controls the initiation of replication in E. coli?

A

By the GATC motifs in the oriC
The Dam methylase methylates the adenine residues GATC motifs in oriC
Replication will only initiate if ALL 14 copies of GATC in oriC are methylated

41
Q

How does the cell know when it has a new copy of its genome and prevent further division?

A

Newly synthesised GATC sequences are unmethylated, recognises two genomes now present
Semi-conservative replication means each new copy contain one old (methylated) and one new (unmethylated) strand
New DNA molecules are hemi-methylated (“half”-methylated)
New DNA containing hemi-methylated oriC DNA is sequestered to the membrane so will not be available for replication
New round of replication will not start till cell divides/committed to division

42
Q

When does re-methylation occur in the cell cycle?

A

About 1/3 of the way into it

43
Q

How is replication terminated in E. coli?

A

Termination occurs when DNA replication is completed
The two replication forks which began at the origin (oriC), and moved in opposite directions away round the genome, approach one another
In the Termination phase they fuse in a region opposite to oriC, the terminus region
The terminus region is a ‘replication fork trap’
It has a series of DNA sites at which arrest (or pausing) of fork progression occurs
The replication fork will stop for a period of time, before carrying on if necessary

44
Q

How many DNA replication terminators are there in E. coli and how are they distributed?

A

10 terminators in E. coli facing in different directions (5 each way), but forks must meet at the terminating site (face towards terminating site)
They are distributed over 42.5% of the chromasome

45
Q

How many DNA replication terminators are there in B. subtilils and how are they distributed?

A

9 terminators in B. subtilis facing in different directions
Distributed over 9.9% of the chromasome

46
Q

Why are the E. coli DNA replication terminators distributed over a much longer distance in the chromosome when compared with B. subtilis?

A

E. coli doubles in 15-20 minutes vs B. subtilis doubling in 40-50 minutes
Therefore the DNA terminators in E. coli have to be spread out because there needs to be more control over replication, allows things to go slow on one side and fast on the other and still work

47
Q

What are DNA terminator (ter) sites?

A

Polar in their action
Arrest a fork from one direction but not the other

48
Q

What is the function of Tus protein?

A

A terminator protein, must be bound to ter site to halt the fork of replication

49
Q

If a clockwise fork passes the anticlockwise ter sites, what will halt the fork?

A

Clockwise terminal site (ter)+ Tus

50
Q

What happens when the anticlockwise fork meets the clockwise fork at the ter site?

A

Replication stops

51
Q

What happens if the anticlockwise fork doesn’t meets the clockwise fork at the ter site?

A

The clockwise fork continues to the next ter after a pause where it may terminate

52
Q

What is the last opportunity for the clockwise fork to terminate?

A

terJ

53
Q

What is homologues recombination essential for?

A

The generation of genetic diversity and DNA repair

54
Q

What is required for recombination to occur?

A

The two molecules must have homologous regions of the order of 100-500bp

55
Q

What is the Meselson-Radding model?

A

Explains how recombination starts (improvement on the Holliday model)

56
Q

How does recombination of DNA start?

A

Cleavage - one strand is cleaved by an endonuclease (cells nick DNA to allow for recombining molecules and fix things)
Chain displacement - DNA synthesis displaces a chain (chain hanging around, not doing anything)
Invasion – The single stranded chain invades a homologous dsDNA molecule - catalysed by RecA (attaches and breaks bond in another DNA molecule - single stranded DNA loop sticks out)
Chain removal – the displaced chain is digested (loop degrades but nick still present)
Ligation – produces a Holliday junction
Branch migration - increases heteroduplex, catalysed by RuvAB

57
Q

What happens in isomerisation in DNA recombination?

A

The strands of the Holliday junction spontaneously cross and uncross, does not require catalysis
Two molecules are the same, just twisted, completely natural and nothing else needed

58
Q

What happens in resolution in DNA recombination?

A

The crossed strands of the Holliday junction cleaved by RuvC. Products depend on configuration of junction at cleavage
Unlike Holliday model outcome is be asymmetric (asymmetrically horizontal or vertical resolution)

59
Q

What enzyme is involved in ~99% of all recombination in E. coli?

A

RecBCD (conglomeration of 3 different proteins)

60
Q

What are the five roles of RecBCD?

A

ssDNA exonuclease (5’ → 3’ and 3’ → 5’) - eats bits of DNA from one end of the strand to the other
ssDNA endonuclease - makes nicks in DNA
dsDNA exonuclease - eat DNA completely
DNA-dependent ATPase - ATPase only works to give energy when attached to DNA
DNA helicase (prefers blunt dsDNA ends) - separates bits of DNA out so enzymes can work on them

61
Q

How does RecBCD function on the DNA?

A

RecBCD binds tightly to the end of a dsDNA substrate and unwinds it using its helicase activity
As RecBCD unwinds the helix it degrades both ssDNA strands using its dual 5’ → 3’ and 3’ → 5’ exonuclease activities
RecBCD moves in steps and each step is 23 bp long in a unique mechanism of action
Called the “quantum inchworm”

62
Q

What are χ sites (5’GCTGGTGG3’)?

A

Sites of recombination (1009 χ sites in E. coli genome)

63
Q

What happens when RecBCD encounters a χ site?

A

RecBCD’s enzymatic activities are dramatically altered:
3’ → 5’ exonuclease activity inhibited
5’ → 3’ exonuclease activity stimulated helicase activity unaffected
The outcome is that RecBCD produces a ssDNA tail with a 3’ end and RecA binds to the ssDNA tail, which invades

64
Q

What drives strand invasion?

A

RecA coated ssDNA

65
Q

What is transposition and what are inversion sequences?

A

Some DNA sequences which do not transpose, but can alter their orientation with a DNA molecule - inversion sequences (DNA faces the other way)
Inversions sequences control gene expression in some organisms

66
Q

What is the main example of transposition?

A

Phase variation in Salmonella spp.

67
Q

What is phase variation in Salmonella spp.?

A

Most Salmonella spp. can produce two different types of flagellum (phase 1 and phase 2 H antigens - both act as antigens)
Growing phase 1 cells will produce some phase 2 cells and vice versa
Phase is determined by the orientation of the hin (H-inversion) region

68
Q

What is the hin region of Salmonella spp.?

A

H inverting region
Is 995 bp long and bounded by two 14 bp inverted repeats

69
Q

What is the structure of the H1 and H2 genes in Salmonella spp.?

A

The H1 gene has it own promoter and operator and is physically separated from the hin region
The H2 gene is in an operon with the rep (repressor) gene that encodes a repressor for the H1 gene
The promoter for the H2-rep operon lies within the hin region

70
Q

What happens in phase 2 of phase variation?

A

When hin region inverts the H2 promoter is now in correct orientation
Phase 2 - H2 and Rep are expressed
Rep represses H1 expression
Does this completely randomly

71
Q

What happens in phase 1 of phase variation?

A

H1 is expressed as the H2 promoter is in the wrong orientation (H1 phenotype expressed)

72
Q

What are examples of phase variable genes and what do they come from?

A

Capsular polysaccharide - H. influenzae
Fimbriae - E. coli
S-layer expression - C. fetus
LPS antigenicity - H. influenzae
Haemoglobin ultilisation - N. gonorrhoeae
Glagella - S. typhimurium
Opc - N. meningitidis

73
Q

What do most phase-variable genes code for?

A

Surface components

LF206: Molecular Cell Biology (18 decks)

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