Abstract
The nahR gene from the NAH7 naphthalene degradation plasmid encodes a LysR-type transcriptional activator of the nah and sal promoters (Pnah and Psal, respectively) that responds to the inducer salicylate. In vivo methylation protection experiments with dimethyl sulfate showed that in the absence of inducer, NahR interacts in a similar manner with its target sites at Psal and Pnah. Both target sites also have very similar sequences comprised of a 4-base pair interrupted dyad containing two symmetrical guanines (-73 and -64 of Pnah; -71 and -62 of Psal), each located in adjacent major grooves on the same helical face, and both strongly protected by NahR. When inducer was present, several additional guanines of Pnah (-35, -45, and -58) and Psal (-42 and -40) became protected from methylation, while a guanine at -52 of Pnah became markedly enhanced for methylation, indicating that inducer and NahR-dependent interactions with these downstream sites of each promoter are quite different. Deletion of Psal sequences downstream of -30 did not affect its methylation patterns suggesting that NahR alone is responsible for the altered reactivities of these nucleotides. Similar in vivo methylation analyses with inducer-insensitive or inducer-independent NahR mutants also suggested that all alterations in methylation sensitivity are directly caused by NahR. It is more probable that the salicylate-induced reactivity changes result from direct NahR-guanine contacts which are required for, but not sufficient for transcription activation; however, they could also result from NahR-induced DNA contortions caused by upstream protein-DNA contacts.
Highlights
The nahR gene from the NAH7 naphthalene degra- hydrocarbons (e.g. ClcR, CatR, TfdS, and NahR), aminoacid dation plasmidencodes a LysR-typetranscriptional biosynthesis (e.g. IlvY, TrpI, and LysR), oxidative-stress reactivator of the nah and sal promoters (Pnah and Psal, sponse (OxyR), and synthesiosf tetrasaccharide signalmolerespectively) that responds to the inducer salicylate. cules involved in host-specific initiation of nitrogen fixation
Markedly enhanced for methylation, indicating that terminal regions contain a DNA-bindingdomainpartially inducer and NahR-dependent interactions with these comprised of a helix-turn-helix motif [11, 15].the downstream sitesof each promoterare quite different
Construction of Plasmids-Plasmids used in this study are diagrammed in Fig. l and were constructed as follows. pMS1313:a 1.1kh PstI-EcoRV fragment from pSC3 was purified by electroelution, ligated with PstI-SmaI digested pMS13 ( l ), transformed into E. coli JM107, and ampicillin-resistant transformants with pMS1313 were isolated. pMH9HindIII-digested pMS1313 DNA wasreligated under dilute conditions, transformed into E. coli JM83, and ampicillinresistant transformants with pMH9 were isolated. pSM10 an800-bp HindIII-EcoRI fragment from pSRl [2] was partially digested with SspI; the140-bp fragment containing Pealwas isolated by electroelution, ligated into SmaI-Hind111 digested pTZ18U, and transformed into E. coli JM107
Summary
Cells were harvested at inducer to anyof these preparationsof activator proteins has 10,000 X g for 5 min at 25 "C, resuspended in 2 ml of37 "C L broth not been demonstrated, in spite of the fact that concentrations and 10 p1 of dimethyl sulfate mixed in; after 1min at 37 "C methylas low as 0.2 PM are effective inducers i n vivo.' This may be ation was stopped by pouring the cells into centrifuge tubes containing due to substantial inactivation or alterationof the activators during purification, or absence of specific conditions required for biological activity (e.g. supercoiling of target DNA) To circumvent such problems we have utilized i n vivo methylation protection analysis ( i n vivo footprinting) to explore the 5 g of ice and 20mlof 0.25 M EDTA, pH 8, 0.15 M NaC1.
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