Binding of the Unorthodox Transcription Activator, Crl, to the Components of the Transcription Machinery

Patrick England,Lars F Westblade,Gouzel Karimova,Véronique Robbe-Saule,Françoise Norel,Annie Kolb

doi:10.1074/jbc.m807380200

Abstract

The small regulatory protein Crl binds to sigmaS, the RNA polymerase stationary phase sigma factor. Crl facilitates the formation of the sigmaS-associated holoenzyme (EsigmaS) and thereby activates sigmaS-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 sigmaS, which results in an increase of the association rate of sigmaS to core RNA polymerase without any effect on the dissociation rate of EsigmaS. Crl is also able to associate with preformed EsigmaS with a higher affinity than with sigmaS alone. Furthermore, even at saturating sigmaS concentrations, Crl significantly increases EsigmaS association with the katN promoter and the productive isomerization of the EsigmaS-katN complex, supporting a direct role of Crl in transcription initiation. Finally, we show that Crl does not bind to sigma70 itself but is able at high concentrations to form a weak and transient 1:1 complex with both core RNA polymerase and the sigma70-associated holoenzyme, leaving open the possibility that Crl might also exert a side regulatory role in the transcriptional activity of additional non-sigmaS holoenzymes.

Highlights

Fitness in environments containing nutrients at low concentrations [6]
Variants of ␴S with the N-terminal portion of the protein deleted are responsive to Crl [24]; to immobilize ␴S and ␴70 to the solid surface, we used a noncovalent orientated strategy, relying on the stable interaction between the His12 moiety appended to the N termini of both ␴S and ␴70, and an anti-His5 monoclonal antibody attached to the dextran surface of the sensor chip
This study aimed to elucidate the mechanism by which Crl promotes E-␴ association and involved the two principal ␴ factors of Enterobacteria: ␴70, the ␴ factor required for vegetative growth, and ␴S, the ␴ factor required for stationary phase survival

Summary

Introduction

Fitness in environments containing nutrients at low concentrations [6]. ␴S abundance and activity are tightly controlled by the interplay of a complex set of regulators that affect transcription, translation, and the stability of the protein. ␴S concentration is not the sole parameter controlling the expression of rpoS-dependent genes Another important checkpoint is the formation of E␴S, which in Escherichia coli is restricted by the competition of ␴S with six other ␴ factors for binding to a limited amount of E (9 –11). High resolution structural studies of thermophilic bacterial E␴ reveal that ␴ is spread out along one face of E, forming an extensive protein-protein interface with the ␤ and ␤Ј subunits (36 –38) Both free ␴70 and ␴S are unable to recognize promoter DNA [39, 40],4 most likely a result of the intramolecular interactions between ␴2 and ␴4 [34]. We find that Crl is able to bind directly to E, forming a transient complex that might be related to the reported Crl-dependent activation of transcription by other E␴ species [30]

Objectives

Results

Conclusion