Transposon ORF2p

Transposon L1 Insertion

               

Target-Primed Reverse Transcription (TPRT) and DNA integration.

Target DNA
L1 RNA
L1 cDNA
Proteins

A Initial Target Binding & Cleavage

ORF2p binds to the target DNA. The Endonuclease (EN) domain makes the first cut, exposing a 3'-OH group. The L1 RNA acts as a template, hybridizing its poly(A) tail to the target site.

ORF2p Complex Upstream 5' Downstream EN Cut L1 RNA AAAAAA 3'

B TPRT Macromolecular Complex

A structural abstraction of the components during first strand synthesis. PCNA clamps the DNA, ORF2p facilitates Reverse Transcription, and RNase H2 is recruited.

O2

ORF2p (Endonuclease / RT)

Bends target DNA; houses active sites for cutting and reverse transcription.

PCNA (Ring Clamp)

Binds to the PIP box on ORF2p; encircles DNA and recruits repair factors like RNase H2.

RH

RNase H2

Recruited by PCNA to degrade the RNA template after cDNA synthesis is underway.

Host Factors Network

TOP1 PARP1 p53 BRCA1

C Revised Insertion Model (Process Timeline)

The chronological progression inferred from biochemical and genetic evidence. This minimalist model strips away protein bulk to isolate the behavior of the nucleic acid strands.

1

First EN Cut

2

TPRT: Priming

3

First Strand Synthesis

4

Non-templated Addition (NTA)

5

Template Jumping

6

Second (Staggered) EN Cut

7

Strand Transfer

8

Second Strand Synthesis

RNase H* Degradation
9

Ligation and Repair

New L1 (~6kb) TSD TSD
ⓘ The final product (Step 9) results in a complete ~6,000 bp insertion flanked by Target Site Duplications (TSDs) of ~12–18 bp created by the staggered cuts.
A Kinetic Mechanism

Target Cleavage & RNA Priming

Adenine
Thymine
Guanine
Cytosine
ORF2p Endonuclease
3'
C
A
A
A
A
G
C
5'
5'
G
T
T
T
T
O
H
3'
C
G
3'
3'
A
A
A
A
L1 Transcript
5'

Animation Controls

Step 0 of 4

Target Sequence Localization

B Kinetic Mechanism

cDNA Synthesis & Template Jump

New cDNA
NTA Bases
RT EN
3'
C
A
A
A
A
T
G
C
C
A
5'
5'
G
T
T
T
T
3'
A
A
A
A
U
G
C
5'
A
C
G
G
T

Animation Controls

Step 0 of 4

RT Complex Initiation

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