Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery

Abstract

The small regulatory protein Crl binds to σS, the RNA polymerase stationary phase σ factor. Crl facilitates the formation of the σS-associated holoenzyme (EσS) and thereby activates σS-dependent genes. Using a real time surface plasmon resonance biosensor, we characterized in greater detail the specificity and mode of action of Crl. Crl specifically forms a 1:1 complex with σS, which results in an increase of the association rate of σS to coreRNApolymerase without any effect on the dissociation rate of EσS. Crl is also able to associate with preformed EσS with a higher affinity than with σS alone. Furthermore, even at saturating σS concentrations, Crl significantly increases EσS association with the katN promoter and the productive isomerization of the EσS-katN complex, supporting a direct role of Crl in transcription initiation. Finally, we show that Crl does not bind to σ70 itself but is able at high concentrations to form a weak and transient 1:1 complex with both core RNA polymerase and the σ70-associated holoenzyme, leaving open the possibility that Crl might also exert a side regulatory role in the transcriptional activity of additional non-σS holoenzymes. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.