Poster abstracts

Poster number 98 submitted by Monali NandyMazumdar

Mechanism of translation activation by the transcription factor RfaH

Monali NandyMazumdar (Microbiology, The Ohio State University), Irina Artsimovitch (Microbiology, The Ohio State University)

Abstract:
Escherichia coli RfaH, a paralogue of general transcription factor NusG, activates expression of long, poorly translated, horizontally transferred virulence operons which are potential targets of Rho-mediated polarity. RfaH consists of two domains connected by a flexible linker. The N-terminal domain is recruited to the RNA polymerase halted at a specific ops site and reduces transcription pausing and termination thereafter. The C-terminal domain interacts with the ribosomal protein S10. By physically linking the RNA polymerase to the translational machinery, RfaH may (i) enable ribosome recruitment during initiation and (ii) couple transcription and translation during elongation. The first activity may be particularly important because RfaH-controlled operons do not possess optimal Shine-Dalgarno (SD) elements. Indeed, deletion of the SD element from a model operon confers strong dependence on RfaH. Although many bacterial operons share the same feature, the mechanism of non-SD led translation initiation remains unclear, and deciphering the mechanism of RfaH could provide potential insights into this hitherto unknown cellular process.
Here, we investigate the contribution of leader region features to RfaH mediated translation activation using a reporter in which the ~200 nt rfb operon leader region with the ops site located in the middle is followed by the lux operon. First, we show that RfaH has roughly the same effect on lux expression when the spacing between the ops and the translation start is varied from 44 to 435 nts, suggesting that sequences around the ops and the translation initiation region are the most important. Second, we found that RfaH does not influence the start site selection by the ribosome. This may imply a start codon scanning mechanism by the 30S subunit captured by RfaH bound to the leading RNAP. We also show that the ribosomal protein S1, which acts as a translation enhancer in the absence of SD, is not required for RfaH-mediated activation. Thus RfaH appears to be the only component required for the potential coupling and translation of its target operons. In the future, we will dissect the fine details of the leader region and carry out footprinting experiments to map the site of the ribosome recruitment by RfaH.

Keywords: RfaH, Translation, Ribosome