Characterization of the Detachable Rho-Dependent Transcription Terminator of the fimE Gene in Escherichia coli K-12

Abstract

The fim genetic switch in the chromosome of Escherichia coli K-12 is an invertible DNA element that harbors the promoter for transcription of the downstream fim structural genes and a transcription terminator that acts on the upstream fimE regulatory gene. Switches oriented appropriately for structural gene transcription also allow fimE mRNA to read through, whereas those in the opposite orientation terminate the fimE message. We show here that termination is Rho dependent and is suppressed in a rho mutant or by bicyclomycin treatment when fimE mRNA is expressed by the fimE gene, either from a multicopy recombinant plasmid or in its native chromosomal location. Two cis-acting elements within the central portion of the 314-bp invertible DNA switch were identified as contributors to Rho-dependent termination and dissected. These fim sequence elements show similarities to well-characterized Rho utilization (rut) sites and consist of a boxA motif and a C-rich and G-poor region of approximately 40 bp. Deletion of the boxA motif alone had only a subtle negative effect on Rho function. However, when this element was deleted in combination with the C-rich, G-poor region, Rho function was considerably decreased. Altering the C-to-G ratio in favor of G in this portion of the switch also strongly attenuated transcription termination. The implications of the existence of a fimE-specific Rho-dependent terminator within the invertible switch are discussed in the context of the fim regulatory circuit.Escherichia coli and other bacteria end the process of transcript elongation by employing one of the following two types of transcription terminator: intrinsic terminators and factor-dependent terminators (36,40,43,58). Intrinsic terminators typically consist of an approximately 20-bp stretch of DNA that is composed of a GϩC region of hyphenated dyad symmetry followed by a run of T residues. This specifies in the RNA transcript a stable stem-loop structure that is followed by up to eight U residues (8, 31, 50). These U residues base pair weakly with the corresponding A residues in the template DNA strand, and in combination with the formation of the RNA stem-loop structure, lead to destabilization of the RNA polymerase-template-transcript elongation complex and the consequent termination event (43, 61).Factor-dependent terminators utilize the hexameric ringshaped Rho protein to facilitate transcript release from regions of the template that are not characterized by intrinsic RNA-DNA hybrid helix instability (5,7,37,40,47). Rho-dependent terminators lack the obvious structural features that are associated with intrinsic terminators (7). In general, they consist of a bipartite structure that extends for up to 150 bp of DNA and have a high proportion of C relative to G residues (2,14,20,33,39,44,60,62). The Rho utilization (rut) site of a Rho-dependent terminator is a site on the RNA transcript which generally has little secondary structure and is recognized by the RNAbinding Rho factor, while the transcription stop point (tsp) is th...

Inactivation of ibeA and ibeT Results in Decreased Expression of Type 1 Fimbriae in Extraintestinal Pathogenic Escherichia coli Strain BEN2908

“…Whether the decreased expression of fimB and fimE is sufficient to explain the observed phenotype of BEN2908 ⌬ibeA will have to be investigated. It has been shown that when the IE is in the off orientation, the RNA encoding fimE is unstable due to early rho-dependent termination (22,27). It is therefore possible that the reduced level of fimE RNA that we observed was only a consequence, and not the cause, of the larger proportion of IE in the off orientation.…”

Statistical physics of a model binary genetic switch with linear feedback

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