Abstract
It has been shown that Fis activates transcription of the ribosomal promoter rrnB P1; however, the mechanism by which Fis activates rrnB P1 transcription is not fully understood. Paradoxically, although Fis activates transcription of rrnB P1 in vitro, transcription from the promoter containing Fis sites (as measured from rrnB P1-lacZ fusions) is not reduced in a fis null mutant strain. In this study, we further investigated the mechanism by which Fis activates transcription of the rrnB P1 promoter and the role of Fis in rRNA synthesis and cell growth in Escherichia coli. Like all other stringent promoters investigated so far, open complex of rrnB P1 has been shown to be intrinsically unstable, making open complex stability a potential regulatory step in transcription of this class of promoters. Our results show that Fis acts at this regulatory step by stabilizing the interaction between RNA polymerase and rrnB P1 in the absence of NTPs. Mutational analysis of the Fis protein demonstrates that there is a complete correlation between Fis-mediated transcriptional activation of rrnB P1 and Fis-mediated stabilization of preinitiation complexes of the promoter. Thus, our study indicates that Fis-mediated stabilization of RNA polymerase-rrnB P1 preinitiation complexes, presumably at the open complex step, contributes prominently to transcriptional activation. Furthermore, our in vivo results show that rRNA synthesis from the P1 promoters of several rRNA operons are reduced 2-fold in a fis null mutant compared with the wild type strain, indicating that Fis plays an important role in the establishment of robust rRNA synthesis when E. coli cells are emerging from a growth-arrested phase to a rapid growth phase. Thus, our results resolve an apparent paradox of the role of Fis in vitro and in vivo in the field.
Highlights
In nutrient-rich media, most RNA polymerase (RNAP)1 molecules inside rapidly dividing Escherichia coli cells are engaged in transcription of a small set of genes in the genome, whose products, such as rRNA and tRNA, are primarily involved in translation [1, 2]
We investigated the mechanism underlying the Fis-mediated transcriptional activation of the rrnB P1 promoter and the role of fis in rRNA synthesis as a function of cell growth in E. coli
Our results showed that Fis stabilizes the interaction between RNAP and rrnB P1, a regulatory step in transcription of the stringent promoter, leading to transcriptional activation of the promoter
Summary
In nutrient-rich media, most RNA polymerase (RNAP)1 molecules inside rapidly dividing Escherichia coli cells are engaged in transcription of a small set of genes in the genome, whose products, such as rRNA and tRNA, are primarily involved in translation [1, 2]. The fact that Fis activated transcription from rrnB P1 by the stringent RNAPs to a greater extent than by wild type RNAP under the conditions used indicates that Fis manifests a greater effect on the relatively unstable initiation complexes.
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