Analysis of the Fis-Dependent and Fis-Independent Transcription Activation Mechanisms of the Escherichia coli Ribosomal RNA P1 Promoter

Martin Zacharias, Hans Ulrich Göringer, Rolf Wagner

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

Abstract

The role of the curved DNA sequence upstream to the Escherichia coli ribosomal RNA PI promoter in transcription activation was studied. This sequence region had been shown to activate transcription from PI in vivo and in vitro and to harbor binding sites for the trans-activating protein Fis. We have constructed a series of linker scanning mutants spanning the region -104 to -47, relative to the transcription start site. DNA fragments carrying the mutations show altered gel electrophoretic mobilities, consistent with reduced DNA bending angles compared to the wild-type sequence. Using gel retardation assays, qualitative as well as quantitative differences in the binding of the trans-activating protein Fis to the mutant DNA fragments could be observed. The effects of the mutations on rrnB PI promoter activation were studied in vivo in fis+and fis-backgrounds. A reduction in the promoter strength for some of the linker mutants correlates with altered Fis binding to two of the known Fis binding sites. Shifting the Fis binding region by half a helical turn, relative to the promoter core sequence, abolishes Fis-mediated activation almost totally, whereas activation is partly restored by a shift of a complete helical turn. For one mutant, which does not show alterations in Fis binding, a decrease in the promoter strength was observed in a fis-strain. From the results, we conclude that two upstream activating mechanisms, one Fis-dependent and one Fis-independent, influence the rrnB PI promoter strength. Sequence determinants for the Fis-independent mechanism are closer to the promoter core region than the Fis binding sites. In addition, the study demonstrates that both the helical geometry and the absolute distance of the UAS region relative to the promoter are crucial for transcription activation.

Original languageEnglish
Pages (from-to)2621-2628
Number of pages8
JournalBiochemistry
Volume31
Issue number9
DOIs
StatePublished - 1 Feb 1992
Externally publishedYes

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