LF206 lecture 1 - bacterial genomes

the traditional view of prokaryotic genome (based on ______) was a single, c______ DNA molecule localised within the _________.

E.coli

circular

nucleoid

a majority of ___ in living cells occurs in the B form.

what is it?

DNA

• 2 polynucleotide chains in opposite orientation

• regular right-handed double helix

• diameter of 2nm and makes a complete turn every 3.4 nm

• ~10.5 base pairs per turn of the helix

there are certain flexibilities within the basic B-form

• the number of base pairs ___ ____ of the helix can be altered

• the helix in the cell is not _______ but curled in __ space

per turn

straight

3D

DNA in the ______ genome of E. coli is supercoiled

_______ supercoiling occurs when additional turns are introduced into the DNA double helix when twisted in a ____-handed fashion.

_______ supercoiling occurs when turns are _______ and are twisted in a left-handed fashion

circular

positive, right

negative, removed

define torsional stress

when a twisting force (torque) is applied causing internal stress to resist the twisting

when DNA undergoes torsional stress how is it accommodated? [2]

1. formation of superhelices (a helix coiling itself into a helix)

2. altering the number of base pairs per turn of helix

define linking number

total number of times that both strands of the double helix (of a closed molecule) cross each other when constrained to lie in a plane

Type I Topoisomerases break ___ strand of DNA and create a covalent DNA-____________ bond.

then pass the _ ‘strand under the other strand and ______ the _____.

the ______ number is changed by +or- 1.

Type _ topoisomerase of E.coli _______ly supercoils DNA

one

phosphotyrosine

3

seal

break

linking

I

negatively

Type __ topoisomerases break both strands of ___ and pass _______ ___ of the helix through the gap. the linking number changes by + or - ___.

DNA ______ of E.coli is a hetero________ of 2 subunits (which are?) creates negative supercoils using ___

II

DNA

another part

2

gyrase

heterotetramer

A and B

ATP

topoisomerases are _______ that alter the ______ (shape) of DNA .

they often relieve _______ stress by creating transient (define this) breaks in DNA = ______ supercoiling.

but can also introduce positive supercoiling

enzymes

topology

torsional

transient = temporary

negative

how is DNA organised in bacteria (E.coli)?

• single circular DNA molecule in a series of supercoiled loops (40-50) radiating from a central protein core

the protein component of the E.coli nucleoid includes

• DNA ______ and DNA topoisomerase _ to maintain the ______ state

• at least 4 proteins involved in packaging DNA - the most abundant is ______ (HU)

• HU forms ______ around what DNA is wound

• approximately 1 HU for every __ bp, cover ___ of the genome

• ______ don’t have protein related to HU, instead they have proteins related to eukaryotic _______

• HU is also known as _______-_______ ______ (NAP)

gyrase, I, supercoiled

heat unstable

60 base pairs, 1/5

tetramers

Archaea, histones

nucleoid-associated protein

some bacteria have a linear genome (give 1 example)

some have multipartite genomes (define this)

define plasmid

1. Borrelia burgdoferi (Lyme disease), Streptomyces coelicolor (antibiotic producer), Agrobacterium tumefaciens (plant tumours)

2. genomes divided into 2 or more DNA molecules

3. (often small) DNA molecule that usually codes for non-essential genes - large plasmids can carry essential genes

what is a prophage?

genome of a bacteriophage (a virus that attack bacteria)

DNA replication in E. coli

there is a leading and ______ strand

DNA pol I

DNA pol III

1. long dayyyyyy

what is a replicon?

each replicon must be _________ at least once per cell ______ cycle

the basic unit of replication with a functional origin of replication

replicated

division

many important genes in bacteria have been acquired by __________ ___ ______ (HGT).

examples of HGT

horizontal gene transfer

• prophages

• genomic islands

• transposable genetic elements

genomic islands

acquired by HGT genomic regions often mutated so masking/destroying their modes of transmission (cannot leave the cell) and confer fitness to occupy a niche.

transposable genetic elements can ____ from one location in the genome to another

‘jump’

Cell cycle in E.coli

1. ________ of replication commits the cell to a subsequent _______.

2. Cell division cannot occur until the round of ______ associated with a particular initiation has been _________.

3. E.coli has a single ______ __ _________ (oriC) therefore has a single _____.

4. E.coli replicates __-directionally leading to a ___ structure

5. initiation commences with the binding of ~__ monomers of Dna_ to 4 out of the _ 9 base pair repeats in the _______ ______ (RH) part of ____

6. this forms a _______ complex

7. an ____ complex forms when the 3 __-rich 13-bp repeats ___

8. DnaB _______ is loaded onto the melted DNA with _____

9. ATP is __________ and DnaC is _______

10. DnaB unwinds DNA ________lly in a process that requires ______ _______ _______ (SSB) and DNA gyrase

11. ______ synthesises a primer RNA molecule on both strands and replication commences

1. initiation, division

2. replication, completed

3. origin of replication, replicon

4. bi, theta

5. 20, DnaA, 5, ???????, oriC

6. closed

7. open, AT, melt

8. helicase, DnaC

9. hydrolysed, released

10. bidirectionally, Single Strand Binding protein

11. primase

what controls whether a round of replication is initiated?

replication only continues if all 14 copied of ____ in oriC are __________.

____ _________, methylates adenine residues GATC motifs in oriC.

after replication, GATC copies on the _____ ___________ strand are not methylated.

DNA molecules are ____-methylated as the old strand is methylated and the new strand is ___________. this is governed by semi-_________ replication.

hemi-methylated oriC DNA is sequestered at a ________site and is not available for replication

re-methylation occurs ~_____ of the way into the cell cycle

GATC

methylated

Dam methylase

newly synthesised

hemi-methylated

unmethylated

semi-conservative

membrane

1/3

how is replication terminated?

both replication forks (which began at oriC) approach one another and fuse at the terminus region.

terminus region is a replication fork trap

has a series of DNA sites that arrest fork progression

DNA ___________ (ter) sites are polar in their action - meaning what?

___, a terminator protein, must be bound to the ter site to ____ ___ _____.

In E.coli the ter sites are distributed over ____% of the chromosome but in B. subtilis they are spread much ________er (what percentage?)

terminator

they will arrest a fork approaching from one direction but not the other

Tus

halt the fork

42.5%

narrower

9.9%

this E.coli DNA,

what sites does the clockwise fork pass?

what sites does the clockwise fork halt? - if it doesn’t initially halt there, where else will it?

terH, i, E,D and A

terC + Tus

should halt when anticlockwise fork arrives but will continue to terB

if anticlockwise doesn’t arrive, clockwise continues after a pause and terJ is the last opportunity to terminate.

why is a fork arrested from only 1 direction?

Tus protein can only inhibit helicase (at the apex of the fork) in one direction.

inhibiting helicase prevents DNA from unwinding and therefore replicating.

_____________ recombination is essential for the generation of _______ diversity and ___ repair

for recombination to occur the 2 molecules must have ________ ______ of the order (approx.) of 100-500bp

homologous

genetic

DNA

homologous regions

Meselson-Radding model

1. one DNA strand is cleaved by an endonuclease

2. DNA synthesis displaces a chain

3. the single stranded chain invades a homologous double-stranded DNA molecule - catalysed by RecA

4. the displaced chain is digested

5. remaining single strand gaps are ligated producing a Holliday junction

6. branch migration catalysed by RuvAB increases DNA heteroduplex

7. isomerization: strands of the Holliday junction spontaneously cross and uncross - no catalysis required.

8. resolution: crossed strands of Holliday junction are cleaved by RuvC

   1. unlike Holliday model, products can be asymmetrical

DOUBLE STRAND HOMOLOGOUS RECOMBINATION

1. RecBCD binds to the end of a ______ substrate and unwinds using its ______ activity

2. as RecBCD unwinds the helix, it ______ both ______ strands using its dual _’→_’ and _’→_’ ___nuclease activities

3. RecBCD moves in _____ and each ____ is __ bp long. this is called the ________ _______

4. when RecBCD encounters a χ (what symbol is this and what is it?) site its enzymatic activities are altered

   1. _’→_’ exonuclease activity is inhibited

   2. 5’ → 3’ exonuclease activity is _______

   3. helicase activity is _______

5. RecBCD produces a ssDNA___ with a _’ end and RecA binds to the ssDNA tail which ______

1. dsDNA, helicase

2. degrades, ssDNA, 3’→5’, 5’→3’, exonuclease

3. steps, step, 23, quantum inchworm

4. chi, sites of recombination 5’ GCTGGTGG 3’ (there are 1009 chi sites on the E.coli genome)

   1. 3’→5’

   2. stimulated

   3. unaffected

5. tail, 3’, invades

DNA sequences which do not transpose alter their orientation within a ___ molecule - __________ sequences.

most Salmonella spp. can produce two different types of ________

• phase _ and _ _ antigens

phase is determined by the orientation of ___ (H-inversion) region

this region is ___ bp long and bounded by two ___ bp _______ repeats

this region encodes for _______ which catalyses inversion

differences between H1 and H2 genes

DNA

inversion

flagellum

phase 1 and 2 H antigens

hin

992

14

inverted

invertase

H1 gene has its own promoter and operator & is physically separated from the hin region

H2 gene is in an operon with the rep gene that encodes a repressor for H1 gene

H2-rep promoter lies within the hin region