MBB 331 Lecture 10

What happens to the target site after transposition

The target sequence becomes duplicated

What enzyme performs transposition

Transposase

What type of cuts are made in the target DNA?

Staggered cuts

Why do staggered cuts create target-site duplications

Gap filling after insertion copies the target sequence on both sides of the transposon

original target sequence now appears where

on both sides of the transposon

What gene is essential in most transposons

Transposase

What happens if transposase is missing

The transposon cannot move

What additional genes are often found in composite transposons

Antibiotic resistance genes

What is a composite transposon

has transposons on either end of DNA sequence

Why are composite transposons important medically

They spread antibiotic resistance genes between bacteria

Why can Ac transpose independently

Ac contains a functional transposase gene

Why can't Ds transpose independently

Ds lacks a functional transposase and depends on Ac

What is the simplest bacterial transposon?

Insertion sequence (IS)

What does an IS contain

Transposase gene and inverted repeats

If IS was single stranded, what would happen

base pairs with itself

What are inverted repeats (IRs)

Short DNA sequences at opposite ends that are reverse complements

What is a composite transposon

Two IS elements flanking additional genes

What genes are commonly found between the IS elements

Antibiotic resistance genes

What is LTR retrotransposon

transposons containing long terminal repeats

What are Non-LTR retrotransposon

no repeats

What is site-specific recombination

DNA exchange occurring between specific recombination sites

Does transposition require extensive homology

No

What enzyme performs site-specific recombination

Recombinase

What genomic feature is required

Matching recombination sites

Where do we want to insert phage recombiation site

into the bacterial recombination sites

How do we know recombination is occuring

through crossover (X) between the two sites

What makes up the recombination site

two inverted repeats (two blue arrows facing each other) and a core (yellow arrow moving 5-3 facing right)

How do the inverted repeats look like

same sequence on either side different directionally

When does inversion occur

when the inverted sequences (FRT) face each other (different directions)

When does insertion/deletion occur

When FRT are facing the same way

When the pathway goes straight througha molecule it cause

deletion

recombination by tyrosine recombinase steps

1. cleavage

2. rejoining

3. isomerization

4. cleavage

5. rejoining

Is cut-and-paste transposition replicative

No

What happens to the donor site

The transposon leaves, creating a break

What enzyme performs the reaction

Transposase

Cut and paste transposition Step One

cut donor site on either side of transposon and expose 3' OH

Cut and paste transposition Step Two

3' OH attacks opposite strand, generating hairpin loop

Cut and paste transposition Step Three

Transposase cleaves hairpin generating 3'OH

Cut and paste transposition Step Four

OH attacks and creates strand transfer complex

Cut and paste transposition Step 5

Replication and ligation to fill and seal gaps

What happens with the donor

remains unchanged and is copied into recipent

end result of replicative and RNA intermediate transposition: Donor and Recipient have what

the same copy of transposon

replicative transposition Step One

transposon ends are nicked exposing 3'

DNA target site also nicked

replicative transposition Step Two

3OH’ Ends of transposon attack target DNA and join, creating a STRAND TRANSFER COMPLEX

replicative transposition step Three

3' ends of strand transfer complex are used as primer and form a cointegrate

Which protein forms the cointegrate

TnpA

Which protein resolves the cointegrate

TnpR (resolvase)

replicative transposition Step Four

cointegrate is resolved by homologous recombination (site specific recombination), catalyzed by resolvase

What key enzyme is required

Reverse transcriptase

retrotransposition Step one

transposon transcribed into RNA

retrotransposition Step Two

Reverse transcribe into dsCDNA

retrotransposition Step Three

Cleavage exposing 3' OH

retrotransposition Step Four

3’ OH of transposon attacks target site, inserts

filling and ligation of gap

What is the difference bt retrotransposition and regular transposition?

extra step creating 2nd strand using cDNA and we can then remove RNA and create cDNA ds

What does EP stand for

Extra chromosomally primed (LTR) retrotransposition

What primes reverse transcription

tRNA and anneals to transposon to create a dsDNA

Which enzyme inserts the new copy

Integrase will insert DNA transposon copy into DNA target site

What are LTR retrotransposons closely related to

Retroviruses

What does TP stand for

Target-primed retrotransposition

What happens first

Target DNA is nicked

What primes reverse transcription

The exposed 3' end of target DNA to convert to dsDNA

Examples of TP non LTR retrotransposons

LINEs and SINEs

EP (LTR) retrotransposition Step One

transposon transcribed into RNA

EP (LTR) retrotransposition Step Two

a tRNA or other RNA acts as a primer to

reverse transcribe into cDNA

EP (LTR) retrotransposition Step Three

Integrase catalyzes insertion of transposon copy into DNA target site

What does Ac stand for

Activator.

What does Ds stand for

Dissociation

Is Ds autonomous

No

Why can Ac move by itself

It encodes transposase

Why does Ds require Ac

Ds lacks functional transposase

What was the purpose of the CsCl gradient experiment

To detect insertion sequences

Why did λgal⁻ DNA have a different density than λgal⁺ DNA

λgal⁻ contained extra DNA from an insertion sequence

What conclusion was drawn from the CsCl experiment

Some mutations were caused by insertion of extra DNA rather than point mutations

In eukaryotes what's the three main transposition strategies

Ac/Ds = DNA transposons

LINEs/SINEs = non LTRretrotransposons

LTR retrotransposons = retrovirus-like elements

Two types of Non- LTR

Lines and Sines