Part:BBa_K2259000

SynORI framework RNA II - Replication Initiator (Group A)

RNA II acts as a plasmid replication initiator. The transcript folds into a secondary structure which stabilises the interaction between the nascent RNA and the plasmids DNA. This RNA-DNA 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 and consequently, the start of plasmid replication.

• Caution! RNA II (Group A) indicates that this plasmid only interacts with regulatory RNA I (Group A) part:BBa_K2259024 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

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]

Introduction

Biology

ColE1 plasmid replication overview

Figure 1. Main principles of ColE1 plasmid family replication

ColE1-type plasmid replication begins with the 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 the 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.

The interaction between RNA I and RNA II can be amplified by Rop protein, see part:BBa_K2259010.

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

RNA II gene is foundational and central biobrick of SynORI system and by far the only one that is mandatory for the framework to run. The two main functions of RNA II is as follows:

1. Initiating plasmid replication
2. Interacting with RNA I of specific plasmid group (See below)

RNA II alone, as only part of SynORI system, produces highest plasmid copy number a specific group can offer. For further modification it can be constructed together with inhibitor of replication - RNA I or global plasmid copy number modulator - ROP protein.

Inactivation of RNA I promoter

It immediately becomes clear that in order to control the copy number of a plasmid one could simply change RNA I promoter. But, as RNA I and RNA II are two antisense molecules, changes made to the sequence will affect both of them. Location of RNA I promoter coincides with the RNA II secondary structures, which are crucial to replication primer formation. Even if one could somehow manage to change the RNA I promoter to another one without disabling replication initiation, it would still not be a convenient because picking another promoter would require a large pool of resources every time.

There are two main aspects that needs to be taken in mind and balanced out when inactivating RNA I promoter as they are both interconnected:

1. Changes made to RNA II secondary structure needs to be minimal, otherwise Ori may become inactive or plasmid copy number drop too low
2. Promoter inactivation has to be done while leaving it with minimal activity (or ideally inactivating it completely)

These aspects are combined by compiling a simulative algorithm which compares every possible combination of -10, -35 mutations and then compares them to predicted RNA II secondary structures made by CoFold.

Once RNA I promoter is disabled in the ColE1 origin of replication, it can be moved to another plasmid location and used as a separate unit. Also, RNA I promoter can now be changed without damaging the replication initiation.

RNA II and RNA I in the engineering of unique plasmid groups for multi-plasmid system

As RNA I and RNA II interact mainly with the three stem loops that form kissing complexes, we have decided to use this fact to our advantage in order to engineer different plasmid groups by adding unique, group-specific sequences to RNA I and RNA II stem loops.

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.

The inactivation and transfer of RNA I gene away from RNA II allow us to use different sequences for RNA I and RNA II molecules that are not necessarily ideal complements of each other.

Since there are three stem loops responsible for RNA I – RNA II interaction for each of the plasmid group we have decided to:

• Use two different unique sequences in the first two stem loops, in order to maximize same group specificity.
• For the third loop, we have decided to keep RNA II unchanged, and add either G/C mutations (GC type RNA I) or make RNA I completely non-complement to RNA II (NC type RNA I).

We did not want to introduce new specific sequences into the third loop of RNA II sequence. That is because according to literature RNA II secondary structure at third loop are very sensitive to any mutations and has a high chance of ruining the replication initiation. Just because we chose not to interfere with the third loop of RNA II, we could not leave RNA I gene unchanged. If every group would have the fully compatible third loop, the background cross-group inhibition would be too large.

So now we have 5 different RNA II genes corresponding to groups A B C D and E.

Also, we have 10 different RNA I alternatives: A, B, C, D, E with each having a version of either G/C or NC mutations.

So for example, if we have a part named RNA I (B-NC), it means: This RNA will only selectively regulate RNA II molecule by having specific B group sequences in first two stem loops. Also, in the third stem loop every nucleotide is not complementary to RNA II third loop.

These different plasmid groups (A-E) can then be co-maintained in a cell with a specific, pre-selected copy number. Copy number control principle is the same for every group, but each group is only specific to its own group.

Figure 1. RNA I AND II group interaction example

Origin of RNA II biobrick

If RNA II and RNA I are naturally an antisense system, why are there two separate constructs in SynORI system?

In order to flexibly control the synthesis of RNA I, the RNA I gene first needed to be inactivated in the ColE1 origin of replication. That, however, was not a trivial task, because by changing RNA I promoter sequence, one also changes the RNA II secondary structure, which is crucial for plasmid replication initiation. This is the main reason why, in the 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.>