lecture 4- Transposons: mobile genetic elements

Transposons

-most important thing to genetic mutation and study of bacterial genetics

-mobile-genetic element that kills genes if inserted into open reading frame (mutagenesis)

-revert at low frequency

-pull mutation in E. coli

insertion mutagenesis

-transposons insert in open reading frame and fill gene

-if in lac z, lac Y and lac A aren’t made in E. coli (normally in a point mutation if one isn’t made the others are still made)

Universal genetic elements of transposons

-inverted DNA sequences at their ends

ex.) 3’-GAC———-5’

5’—————-CAG3’

-contains transposase gene

transposase gene

-enzyme that catalyzes transposition and has multiple enzyme activities:

1.) recognizes the inverted repeat sequences and cuts next to them to released transposon: ends are blunt

2.) cuts DNA where the transposon inserts: ends are staggered

3.) has single-stranded DNA ligase activity

-transposons can be moved anywhere in the genome

General structure of transposons

-inverted repeats make transposons

-cuts where 5’ base overlaps

-if target site is 3 + 3 base pairs there’s a higher chance of being in the ORF

-new base pairs typically ligate out of reading frame

-DNA polymerase fills gaps

insertion sequences

-don’t have transposons (not many in E.coli)

-can’t jump around without interruption easily, very dense DNA sequence

-not like humans with 90% noncoding DNA

more about transposons

-make blunt-ended cuts in donor DNA

-transposase ligates genome into 5’ single-stranded ends of target DNA

-cellular DNA polymerase extends 3’ cut ends and ligase joins extended 3’ ends to other 5’ end

can tell where a transposon is because…

-they cause direct repeats

-flanks of DNA on ends of repeats

Composite transposons (early bacterial genetics)

-to move a piece of chromosomal DNA, transposons must be the same on either side

-transposase cuts outside of both inverted repeats rather than inside so whole thing moves and not just the chromosomal DNA

-moves to a new position in genome → leads to genetic changes

-can be inserted flipped around

-new genome now contains transposons and host DNA

-can be important to medicine because you can insert an antibiotic resistance gene

-figure of process on slide 7 of lecture 4

R-factor plasmid

-if selectable then becomes stable in population

-multi-drug resistant plasmids originated from composite transposons

-can jump onto a plasmid not just a chromosome

There is no replication cycle so how is there copies of transposons?

-transposon is already in replication fork

-if jumps to itself before the replication fork, transposon is now doubled

-makes more info during replication only

Methlyation

-prevents transposase binging to DNA

-prevents transposase synthesis (also inhibited by base pairing)

-prevents direct repeats

-effective inhibitor of transposase to control transposase levels

Another inhibitor of transposase

-2 promotors, one that makes transposase and one that travels out

-causes overlap of base pairs

-cells hate this and transposase is inhibited

Regulation of Tn10 transposition

-all previous methods linked to methylation

-DNA is undermethylated when its methylated because the daughter strand isn’t methylated

-undermethylation makes transposase better

-transposons can jump a lot of places, even phages