Part:BBa_K2259000

SynORI framework RNA II - Replication Initiator (Group A)

RNAII acts as a pre-primer and begins the synthesis of plasmid DNA leader strand. The transcript folds into a secondary structure which stabilises the interaction between the nascent RNA and the origin's DNA. This hybrid is attacked by RNase H, which cleaves the RNA strand, exposing a 3' hydroxyl group. This allows the extension of the leading strand by DNA Polymerase I. Lagging strand synthesis is later initiated by a primase encoded by the host cell.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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

Introduction

Biology

ColE1 plasmid replication overview

ColE1-type plasmid replication begins with synthesis of plasmid encoded RNA II (also called primer transcript) by RNA polymerase which initiates transcription at a site 555bp upstream of origin of replication. The RNA transcript forms a RNA - DNA hybrid with template DNA near the origin of replication. Hybridized RNA is then cleaved at the replication origin by RNAse H and serves as a primer for DNA synthesis by DNA polymerase I (Figure 1. A).

Initiation of replication can be inhibited by plasmid encoded small RNA, called RNA I . Synthesis of RNA I starts 445 bp upstream of the replication origin and proceeds in the direction opposite to that of RNA II synthesis, and terminates near the RNA II transcription initiation site. RNA I binds to RNA II and thereby prevents formation of a secondary structure of RNA II that is necessary for hybridization of RNA II to the template DNA (Figure 1. B).

For RNA I to inhibit primer formation, it must bind before the nascent RNA II transcript extends to the replication origin. Consequently, the concentration of RNA I and the rate of binding of RNA I to RNA II is critical for regulation of primer formation and thus for plasmid replication.

Interaction between RNA I and RNA II can be amplified by Rop protein BBa_K2259010

Test test

testest

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

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]!

Regulative RNA II molecule 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 RNA II (Vilnius-Lithuania 2017)

Constitutive Rop protein effect on plasmid copy number

To be updated!