Revision as of 14:43, 31 October 2017

Rop protein - global copy number inhibitor (SynORI framework)

Rop (also known as a repressor of primer) is a small protein responsible for keeping the copy number of ColE1 and related bacterial plasmids low in E. coli. Inhibits plasmid replication of all SynORI framework plasmid groups non-specifically, also other ColE1 replicon plasmids.

In SynORI multi-plasmid framework, Rop acts as a global copy number regulator, by reducing the copy number of all the plasmid groups at the same time, by acting non-specifically to each plasmid group's RNA II and RNA I sequences, but rather binding to kissing-loop complex that mentioned RNA molecules form.

Sequence and Features

Figure 1. Main principles of ColE1 plasmid family replication. Rop protein interaction region marked in red square. (Citation needed)

Introduction

Biology

Repressor of primer (ROP) is a small dimeric protein that participates in ColE1 plasmid family copy number control, by increasing affinity between two complementary RNAs - RNA I (Replication inhibitor) and RNA II (Replication activator) (Fig. 1). ^[1] By increasing affinity of the two RNA molecules Rop decreases the rate of of plasmid replication initiation events.

Figure 2. Structure of the ColE1 Rop protein, at 1.7 angstroms resolution.^[2]

Rop dimer is a bundle of four tightly packed alpha helices that are held by hydrophobic interactions (Fig. 2).

Usage with SynORI (Framework for multi-plasmid systems)

About SynORI

SynORI is a framework for multi-plasmid systems created by Vilnius-Lithuania 2017 which enables quick and easy workflow with multiple plasmids, while also allowing to freely pick and modulate copy number for every unique plasmid group! Read more about [http://2017.igem.org/Team:Vilnius-Lithuania SynORI here]!

Rop protein in SynORI

Rop protein does not recognise specific sequences of RNA I and RNA II molecules, but instead recognises the RNA I - RNA II kissing loop complex secondary structures. That means it can act as a global copy number modulator, which bypasses the selective control of each plasmid group.

For example: You have a two-plasmid system, with specific RNA I concentrations set so that
first plasmid group has an average copy number of 100, and another group at 50 copies. Rop 
protein can be used to globally lower the copy number of each group  - from 100 to 50 and 
from 50 to 25 copies respectively. The degree of copy number reduction depends
on Rop concentration in a cell.

Characterization of Rop protein (Vilnius-Lithuania 2017)

Constitutive Rop protein effect on plasmid copy number

To be updated!

Inducible Rop protein effect on plasmid copy number

To be updated!

References

1. ↑ Castagnoli L, Scarpa M, Kokkinidis M, Banner DW, Tsernoglou D, Cesareni G. Genetic and structural analysis of the ColE1 Rop (Rom) protein. The EMBO Journal. 1989;8(2):621-629.
2. ↑ Banner DW, Kokkinidis M, Tsernoglou D. Structure of the ColE1 Rop protein at 1.7 Å resolution. J Mol Biol. 1987 m.;196(3):657–75.