Revision as of 09:43, 31 October 2017

SynORI framework RNA I - Plasmid replication Inhibitor (Wildtype)

RNA I acts as a plasmid replication inhibitor. RNA I gene transcript's secondary structures, which consists of three stem loops, binds to RNA II (replication initiator) secondary structures and - if succesful - inhibits plasmid replication initiation. Every SynORI RNA I part has it's wildtype promoter removed, and consequently, RNA I can be placed under any promoter signal pattern desired (Part:BBa_K2259086 is an exception, it is a fully wild type RNA I gene combined with wild type promoter).

• Caution! RNA I (Group A) indicates that this plasmid only interacts with the replication iniator RNA II (Group A) <LINK TO RNA II A> from SynORI (framework for multiplasmid systems) collection and is stable when placed with other SynORI plasmid groups. RNA II A will not be regulated with RNA I from another group!

See how this part fits into the whole SynORI framework by pressing here!

Sequence and Features

Introduction

Biology

ColE1 plasmid replication overview

Figure 1. Main principles of ColE1 plasmid family replication. (Citation needed)

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, see part:BBa_K2259010.

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 I molecule in SynORI

The main goal of RNA I in the framework is group-specific control of copy number. Different plasmid copy numbers are achieved by changing RNA I concentration in the cell.

Specific RNA II versions in multi-plasmid systems

RNA II interacts with inhibitory RNA I with three secondary RNA stem loops. In order to create plasmid groups with independent copy number control, one group's RNA II molecule must interact only with the same group's RNA I molecule.

For example if there are two plasmid groups in a cell - A and B - RNA II of A group
would only interact with RNA I A, and not RNA I B.

Figure 1. RNA I AND II group interaction example

Origin of RNA I biobrick

In order to flexibly control the synthesis of RNA I, the RNA I gene first needed to be inactivated in ColE1 origin of replication. That, however, was not a trivial task, as ColE1 ORI is an antisense system, which means that by changing RNA I promoter sequence, one also changes the RNA II secondary structure, which is crucial for plasmid replication initiation (Find how this problem was solved at http://2017.igem.org/Team:Vilnius-Lithuania team Vilnius-Lithuania wiki). This is the main reason why, in SynORI framework, the wildtype ColE1 ORI is split into two different parts - RNR I and RNA II .

<Picture of how RNA I promoter mutations might destroy RNA II secondary structure.>

Characterization of RNA I (Vilnius-Lithuania 2017)

Constitutive Rop protein effect on plasmid copy number

To be updated!

References