Fine-structure deletion analysis of the transcriptional silencer of the proU operon of Salmonella typhimurium

Abstract

Transcriptional control of the osmotically regulated proU operon of Salmonella typhimurium is mediated in part by a transcriptional silencer downstream from the promoter (D. G. Overdier and L. N. Csonka, Proc. Natl. Acad. Sci. USA 89:3140-3144, 1992). We carried out a fine-structure deletion analysis to determine the structure and the position of the silencer, which demonstrated that this regulatory element is located between nucleotide positions ؉73 to ؉274 downstream from the transcription start site. The silencer appears to be made up of a number of components which have cumulative negative regulatory effects. Deletions or insertions of short nucleotide sequences (<40 bp) between the proU promoter and the silencer do not disrupt repression exerted by the silencer, but long insertions (>0.8 kbp) result in a high level of expression from the proU promoter, similar to that imparted by deletion of the entire silencer. The general DNA-binding protein H-NS is required for the full range of repression of the proU operon in media of low osmolality. Although in the presence of the silencer hns mutations increased basal expression from the proU promoter three-to sixfold, in the absence of the silencer they did not result in a substantial increase in basal expression from the proU promoter. Furthermore, deletion of the silencer in hns ؉ background was up to 10-fold more effective in increasing basal expression from the proU promoter than the hns mutations. These results indicate that osmotic control of the proU operon is dependent of some factor besides H-NS. We propose that the transcriptional regulation of this operon is effected in media of low osmolality by a protein which makes the promoter inaccessible to RNA polymerase by forming a complex containing the proU promoter and silencer.The proU operon, which specifies the three constituent proteins of a transport system for osmoprotectants such as glycinebetaine and proline (5), is induced Ͼ100-fold by high osmolality in Salmonella typhimurium and Escherichia coli. Transcriptional control of the proU operon is mediated at least in part by a cis-acting negative element, or silencer, that is located ϳ100 to 200 bp downstream of the promoter (7, 18). In S. typhimurium, deletion of this element resulted in up to a 40-fold increase in expression from the proU promoter in media of low osmolality (18).Thus far, attempts to isolate mutations in regulatory genes that are unique to the proU operon have not been successful. This impasse has led to the formulation of two models which propose that the transcriptional control mechanism of the proU operon is unusual in that it does not involve any specific regulatory proteins. Both models are based on the assumption that the proU promoter, which does not have a good match to the consensus optimal promoter (Fig. 1), is inherently weak and needs some activating factor for high-level expression in media of high osmolality. According to one model, potassium glutamate, which accumulates to high concentrations in media of high osmolality ...

DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression

“…Thus, the distance between the promoter and the DRE, and their relative orientations with respect to the face of the helix, can be varied with little effect on expression or regulation. This is consistent with other reports (26). Importantly, this result ensures that interpretation of the following in vivo experiments is not influenced by the precise position at which the curves were inserted.…”

“…These results with the control plasmid were as expected (13). It should be noted that, as reported previously, residual osmotic regulation was observed when the DRE was deleted showing that sequences other than the DRE play some role in regulation (13,26,28). Re-insertion of the DRE between the promoter and lacZ restored normal osmoregulation (pAF456; induction ratio of 71.0).…”

“…At the proU promoters of E. coli and S. typhimurium, H-NS represses transcription by binding to the DRE, located some 100 -200 bp downstream of the transcription start site (13,25,26). To determine whether H-NS binding per se is sufficient to maintain repression of the proU promoter, defined sequences were cloned between the proU promoter and a downstream reporter gene, replacing the DRE.…”

“…proU appears to be one of the simplest H-NS-responsive promoters as no trans-acting factor, other than H-NS itself, appears to be required for its regulation. proU expression is derepressed in hns mutants (6,7,24), although residual osmoregulation is retained (13,25,26). Repression of proU by H-NS requires an intrinsically curved region of DNA located around 200 bp downstream of the transcription start site, within the first structural gene of the proU operon (the downstream regulatory element, DRE) (12, 26 -28).…”

“…Repression of proU by H-NS requires an intrinsically curved region of DNA located around 200 bp downstream of the transcription start site, within the first structural gene of the proU operon (the downstream regulatory element, DRE) (12, 26 -28). However, the binding preference of H-NS for the DRE in vitro is, at most, only a few-fold greater than for generic DNA, and the precise distance between the DRE and the proU promoter does not appear to be critical (13,25,26). It is not known how interaction of H-NS with the DRE might influence transcription.…”