Difference between revisions of "Part:BBa K2259000"
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Interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]]. | Interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]]. | ||
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Rop dimer is a bundle of four tightly packed alpha helices that are held by hydrophobic interactions (Fig. 2). | Rop dimer is a bundle of four tightly packed alpha helices that are held by hydrophobic interactions (Fig. 2). | ||
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===Regulative RNA II molecule in SynORI=== | ===Regulative RNA II molecule in SynORI=== | ||
| − | + | RNA II gene is foundational and central biobrick of SynORI system. | |
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=Characterization of RNA II (Vilnius-Lithuania 2017)= | =Characterization of RNA II (Vilnius-Lithuania 2017)= | ||
Revision as of 20:24, 23 October 2017
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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, 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 II molecule in SynORI
RNA II gene is foundational and central biobrick of SynORI system.
Characterization of RNA II (Vilnius-Lithuania 2017)
Constitutive Rop protein effect on plasmid copy number
To be updated!