This Website is using style sheets. Your browser does not support style sheets.

The RNA Chaperone Activity Website

MFPL website
Uni Wien

RCA assays

Many different assays have been used to monitor RNA chaperone activity. These assays help to define different classes. Some proteins, for example the E. coli protein Hfq (host factor for phage Q replication), have RNA annealing activity, but no RNA unfolding or RNA strand displacement actitivity. In contrast, ribosomal protein S1 has RNA strand displacement but no RNA annealing activity. The E. coli histone-like protein StpA has both, RNA annealing and strand displacement activities.

 
(A) To monitor basic RNA-RNA interactions such as annealing and strand displacement in vitro, fluorophore-labelled RNAs can be used. Hybridisation of CyDye-labelled short ribooligonucleotides results in fluorescence resonance energy transfer (FRET), and this reaction is accelerated in the presence of RNA chaperones. For strand displacement, a double-labelled RNA duplex is incubated with an excess of non-labelled competitor strand. RNA chaperone activity is required to facilitate strand dissociation and formation of single-labelled, FRET-inactive RNA duplexes. In contrast to radioactively labelled RNAs, which have to be gel-separated at distinct time points prior quantification, fluorescence signals can be measured in real time in solution.
(B) Pre-RNAs containing the thymidylate synthase group I introns have to fold correctly to undergo splicing. For the cis-splicing assay, the purified transcript is folded by heat-renaturing, and the reaction is initiated by addition of a guanosine cofactor. Proteins with RNA chaperone activity significantly increase the population of molecules with a catalytically active structure that are splicing competent. (C) To further strengthen the requirement for RNA-RNA interactions and correct folding, the pre-RNA can be transcribed in two parts, which are then hybridised. Trans-splicing is started by an exogenous guanosine cofactor that is ligated 5' to the 5'-intron part in the first transesterification step. The second reaction results in ligated exons and intron release. Proteins with RNA chaperone activity facilitate this reaction, especially at low temperatures.
(D) The hammerhead ribozyme assay monitors the cleavage of a substrate RNA that has to anneal to the ribozyme. Moreover, by changing the ribozyme to substrate ratio, multiple turnover conditions can be investigated that require cleavage product release and hence, strand dissociation. Thereby, two different activities of an RNA chaperone - annealing and strand displacement - can be assessed.
(E) For the folding trap assay, a stop codon mutant of the thymidylate synthase pre-RNA is expressed in vivo. Without unwinding by the prematurely stopped ribosome, 3'-terminal intron sequences preferentially pair with exon 1 sequences, thereby stabilising a misfolded, splicing-incompetent precursor. Co-expression of RNA chaperones partly alleviates this splicing deficiency by removing the misfolded structure. The RNA then folds correctly and the ribozyme splices. (F) Transcription termination can be caused by the formation of a stem-loop in the nascent transcript, followed by a poly(U)-stretch. This prevents the RNA polymerase from reaching a downstream reporter gene (chloramphenicol acetyl transferase, CAT), making the cells chloramphenicol sensitive. With RNA chaperones over-expressed in the cell, the terminator stem is 'melted', CAT is transcribed and the cells become chloramphenicol resistant (CmR).
 
sponsored by
 
FWF website
 
FWF website, SFB
 
Spezialforschungsbereich (SFB)