CopA and CopT: The Perfect RNA Couple

Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs.In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using in vivo reporter gene fusion expression and different in vitro methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays.Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn…

Contents

INTRODUCTION
What is antisense RNA?
Antisense RNA involved in regulation of plasmid copy number
The case of Plasmid R1: regulation through inhibition of translatio
Other antisense RNA regulated plasmid copy number systems
ColIb-P9: inhibition of pseudoknot formation
ColE1-relatives: inhibition of primer formation
Plasmid pIP501 and pT181: transcription attenuation
Antisense RNA systems encoded by the bacterial chromosome
MicF
OxyS
DsrA
RprA
The role of proteins in antisense/target RNA binding
hfq
Kinetics of binding of two hybridizing nucleic acids
Non-structured RNAs
Folded RNAs
Important structures for fast kinetics
Hairpin loops
U-turn
Bulges
Single stranded regions
Helical junctions
Methods for studying antisense-target RNA in real time
Surface plasmon resonance
Fluorescence resonance energy transfer
THE PRESENT INVESTIGATION
Scope of this investigation
Extended complex needed for rapid pairing (paper I)
Preference of directionality (paper I)

Author: Slagter-J├Ąger, Jacoba G.

Source: Uppsala University Library

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