BCH 503: Prokaryotes (E coli)

Nucleiod

nucleus like, irregularly shaped region that contains all or most of the genetic material

genomic structure of prokaryotes

circular chromosome

one chromosome

less than 5Mbp

nucleoid

how to access the template

initiation

where to start

initiation

how to read and how to write

elongation

where and how to stop

termination

goal of initiation

to load and activate the DNA helicase (origin licensing)

Where does DNA replication start in prokaryotes?

one specific site called origin of replication or Ori

in prokaryotes what direction from ori does dna replication moves

both

Prokaryotes Have one______ ______

Bidirectional Origin

DNA Unwinding Element (DUE) in what region

AT rich region

Initiator binding element in what region

(DnaA binding sites)

Initiation of DNA Replication in E. coli

1. DnaA-ATP is bound to strong sites at oriC

2. Newly synthesized DnaA binds ATP (DnaA-ATP)

3. Threshold [DnaA-ATP] for filament formation at oriC

4. DnaA-ATP opens AT-rich region of oriC

5. Helicase (DnaB) is loaded onto

ssDNA by DnaC at AT-rich region

6. Primase (DnaG) associates

with DnaB, releasing DnaC

Primer synthesis and DNA Synthesis (i.e., elongation) occurs in the ____ direction

5’ to 3’ direction

To initiate DNA replication, E. coli must have

a high enough concentration of the ATP-bound initiator protein (DnaA-ATP

DnaA-ATP allows e. coli to

bind the Initiator Binding Element found in the Origin of Replication.

binding the Initiator Binding Element causes

the WNA unwinding element to unwind

once the DNA is unwound…

the loading element (DnaC) will loas the DNA helicase (DnaB) onto the Dna unwinding element

DnaG (primase) will then bind DNA reulting in

the release of DnaC and activation of DnaB

Regulation of DNA Replication in E. coli

concentration of DNa ATP ( must be high enough)

Initiation of DNA replication is regulated by

the concentration of DnaA-ATP.

After initiation, hemi-methylated promoter is bound by a protein

called

SeqA

SeqA prevents

DnaA-ATP binding

(SeqA dissociates when the promoter is methylated, allowing DnaA to bind the promoter.)

goal of elongation

to replicate dna

E. coli has five DNA polymerases:

DNA polymerase I

DNA polymerase III

Dna Polymerase II, IV and V (are TLS polymerase)

DNA Polymerase I

okazaki fragment maturation

DNA polymerase III

Main replicative polymerase in E. coli

Processive

DNA Polymerase II, IV, and V are TLS Polymerases

Specialized polymerases that can read through DNA damage

DNA Polymerase III subnuits

α-subunit

ε-subunit

β-subunit (or β-clamp)

γ-complex

α-subunit

• Polymerase activity

ε-subunit

Stimulated by θ-subunit

• Proofreading

β-subunit (or β-clamp

sliding clamp protein

γ-complex

γ- and τ-subunits

• Clamp-loading complex (or clamp loader)

• Also associated with helicase

The main replicative DNA polymerase in E. coli is called

DNA polymerase III

what replicates most of both strands of DNA in E. coli

dna ploymerase III

the core enzyme consists of ____ activity and ___ activity

polymerase activity (alpha subunit) and proofreading activity (ε-subunit)

The holoenzyme consists of multiple subunits, including the

sliding clamp ( beta subunit) and the clamp loader (γ-complex)

The clamp loader is also associated with

helicase DNAB

Leading Strand:

extended continuously

lagging strand

semi discontinuous (synthesized in parts)

Okazaki Fragments

transient components of DNA during lagging strand synthesis

okazaki fragment maturation

connection of Okazaki fragment to lagging strand

okazaki fragment maturation requires

removing primer from previous okazaki fragment

& DNA polymerase I

& DNA ligase

exonuclease (removing primer) direction

5’ to 3’

polymerase (extends fragment) direction

5’ to 3’

exonuclease (proofreading ) direction

3’ to 5’ direction

Synthesis of both strands is

simultaneous

topoisomerase

technically not part of replisome t

topoisomerase is required for

elongation ( it relaxes positive supercoils)

DNA gyrase

Type II topoisomerase

main replicative topoisomerase in Ecoli

main replicative topoisomerase in Ecoli

DNA gyrase

Dna gyrase function

relieves positive supercoils in front of replication fork

Leading strand is synthesized continuously or in parts?

as one continuous strand

the lagging strand is synthesized continuously or in parts ?

in parts

~1000nt fragments called

okazaki fragments

lagging strands are synthesized

at the same time

5’→3’ exonuclease activity of DNA polymerase I

removes primers

DNA polymerase I will also fill in

the removed nucleotides using its polymerase function (extends the Okazaki fragment).

NA ligase is required to

seal the dna strands

goal of termination

to stop dna replication and separate newly synthesized chromosomes

Does E. coli want one replisome doing more work than the other?

no

it halts the replisome using

TUS proteins

TUS means

terminus utilization substance

tus is bound by

a dna element called Ter

Ter means

termunus binding site

how many tus/ter complexes are there

10

e coli has a ___ chromosomes and a ____ fork

Circular chromosome + Bidirectional Fork

Termination occurs when

the replication machinery collide on the DNA

DNA elements (ter) are bound by

proteins (tus)

tus will stop the replication machine in a ___ manner

asymmetrical manner stalling its process

the second replication machinery will eventually collide with the first resulting in

in both replisomes falling off the dna template

Circular chromosomes are interlinked, and must be separated (decatenated)

using

topoisomerase IV