Abstract
The putative overlapping consensus sequences (-129 to -105) for binding of fumarate nitrate reductase regulator- and integration host factor (IHF)-like proteins to puc operon upstream DNA of Rhodobacter sphaeroides was protected from DNase I digestion by purified Escherichia coli IHF. The binding of E. coli IHF to the purported IHF-binding site in the puc upstream DNA is highly sequence-specific. The recorded binding affinity was significantly lower than that of E. coli IHF to the lambda attP site. Employing site-directed changes in the DNA sequence within the -129 to -105 region, a loss in IHF binding, as monitored through gel retardation analysis, was correlated with alterations in puc operon expression monitored through the use of puc::lacZ transcriptional fusions. These results suggest that the IHF-binding site is involved in repression of puc operon transcription by oxygen as well as modulation of puc operon transcription levels by incident light intensity. Mutations specific to the upstream half of the putative fumarate nitrate reductase regulator-binding site of the puc upstream DNA did not show any physiological effects under the experimental conditions employed. Taken together, these studies reveal that the DNA sequence between -129 to -105 may involve facilitation of the interaction between upstream and downstream cis-acting regulatory sequences involved in puc operon expression.
Highlights
From the Vepartment of Microbiology and Molecular Genetics, Universityof Texas Health Science Centerat Houston, Houston
The putative overlapping consensus sequences (-129 of the protein found in the intracytoplasmic membrane [5,6,7,8,9]. to -105) for bindingof fumarate nitrate reductase regu- Following the removal or lowering of the partial pressureof 0 2 lator- and integration host factor (1HF)-like proteitnos below 2.5%, induction of the intracytoplasmic membrane takes puc operon upstream DNA of Rhodobacter sphaeroides place in theform of a series of invaginations arising from the was protectedfrom DNaseI digestion by purifiedEsch- cytoplasmic membrane [10].At the transcriptionallevel thepuc erichia coli IHF
The binding of E. coli IHF to the pur- operon gives rise totwo (0.5 and 2.3 kilobase pairs) transcripts portedIHF-binding site in the puc upstream DNA is which in addition to being subject to control by oxygen are highly sequence-specific.The recorded binding affinity highly regulated by light intensity [1, 11]
Summary
The binding of E. coli IHF to the pur- operon gives rise totwo (0.5 and 2.3 kilobase pairs) transcripts portedIHF-binding site in the puc upstream DNA is which in addition to being subject to control by oxygen are highly sequence-specific.The recorded binding affinity highly regulated by light intensity [1, 11]. Transcriptional response of the pucoperon to oxygen and light Taken together, these studies reveal that the DNA se- Aputative overlapping bindingsite (-129 to -105) consisting of quence between -129to -105 may involve facilitation of consensus DNA sequences recognized by the fumarate nitrate the interaction between upstream and downstreamcis- reductase regulator (FNR) and integration host factor (IHF). The abbreviations used are: bp, base pair(s); URS, upstream reguology and Molecular Genetics, University of Texas Health ScienceCen- latorysequence;DRS, downstream regulatory sequence; FNR,fumater at Houston,P.0.Box 20708, Houston,TX 77225. Strain or plasmid Strains E. coli S17-1 TG1 R. sphaeroides 2.4.1 Plasmids PBS pPX19 pSUP202 pLV106 pRS415 pCF200(-629) pCF606/C pCF607/G pCF608/C/G pCF609/hlG pCF610/A13 pCF611/"GGT pCF612f"/GT pCF613/GT pucEOxpreersosnion in R. sphaeroides
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