Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

Abstract

There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of -6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85 degrees C.

Figure 1

Alignment of ORF56 from pRN1 with copy control proteins (CopG) from other rolling circle plasmids. ORFC from pWVO1 (accession no. JQ1198), CopA from ppsc22 (accession no. X95843), Cop protein from pSBO2 (accession no. AB021465), CopG (formerly RepA) from pLS1 (accession no. A25599), ORF52 from pRN2 (accession no. U93082), Cop-6 from pE194 (accession no. M59209) and ORF56 from pRN1 (accession no. U36383). DNA binding has been shown for CopG from pLS1 (26) and Cop-6 (27). The GOR IV secondary structure prediction (c, coil; e, β-strand; h, α-helix) is given below the alignment.

Figure 2

Purification of recombinant ORF56. Protein from various steps of the purification protocol was analyzed on a 16% SDS–Tris–Tricine polyacrylamide gel (12) stained with Coomassie Blue. Lane 1, whole cell extract of an uninduced control culture; lane 2, whole cell extract of an induced E.coli (pET28-orf56) culture; lane 3, crude extract (centrifuged whole cell extract) of an induced culture; lane 4, heat step supernatant; lanes 5–7, pooled peak fractions of purified ORF56; lanes M1 and M2, molecular mass markers.

Figure 3

UV CD spectra of ORF56. (A) CD spectra of 10 µM ORF56 free (continuous line) and complexed with 3.3 µM wt DNA (broken line) corrected for the background of DNA alone in standard buffer. (B) Decrease in the CD signal at 213 nm as a function of added wt DNA. The titration is stoichiometric (10 µM ORF56).

Figure 3

UV CD spectra of ORF56. (A) CD spectra of 10 µM ORF56 free (continuous line) and complexed with 3.3 µM wt DNA (broken line) corrected for the background of DNA alone in standard buffer. (B) Decrease in the CD signal at 213 nm as a function of added wt DNA. The titration is stoichiometric (10 µM ORF56).

Figure 4

Chemical crosslinking of ORF56 with DMS. ORF56 was incubated with and without crosslinker and analyzed on a denaturing peptide gel. Lane 1, no DMS; lane 2, 10 mM DMS added. The crosslinking reaction was repeated in the presence of binding site DNA (wt): lane 3, 10 mM DMS; lane 4, no DMS.

Figure 5

The promoter region of the orf56 gene. Thin horizontal arrows indicate primers used for amplifying the promoter DNA. The primers for amplifying the promoter region were 5′-TCTCTACTTTCCTAACTCTCTG and 5′-GTATGGTCTACCCATTTG. The sequence immediately upstream of the start codon is shown in detail. Thick arrows indicate the two imperfect inverted repeats within the 47 bp MseI fragment. The putative BRE, TATA box and box B of the promoter are also indicated. The boxes were assigned according to rules formulated by Reiter et al. (24), Qureshi and Jackson (34) and Bell et al. (25).

Figure 6

Specific binding of ORF56 analysed by EMSA. (A) A MseI restriction digest of the promoter PCR fragment (61, 59 and 47 bp) was probed with 3 µM ORF56. The 47 bp fragment is bound by ORF56 even in the presence of 40 ng/µl competitor DNA (right-most lane). (B) Binding of ORF56 (5 µM) to synthetic ³²P-labelled duplex DNA substrates. inv, short inverted repeat; ct, negative control DNA with no repeat structure; wt, longer inverted repeat. The wt DNA was also probed in the presence of 200-fold excess of poly[d(I·C)·d(I·C)] (right-most lane). See Table 1 and Figure 5 for the sequences and the positions of the synthetic DNA substrates.

Figure 7

Fluorescence anisotropy titration of fluorescently labelled wt DNA with ORF56. (A) Titration of 2 nM fluorescein-labelled wt DNA. The experimental points and a fit based on the A₄B model are given. The error bars are the standard errors of each titration point calculated from four to six measurements. In this fit the anisotropy of the unbound DNA is 0.099 and 0.159 for the bound DNA. The dissociation constant based on the tetramer concentration is 1.1 nM. (B) Competition experiment with 2 nM fluorescein-labelled wt DNA/20 nM ORF56 and increasing amounts of unlabelled wt DNA. The fit of the data yields a dissociation constant for the unlabelled DNA of 0.88 nM. (C) Stoichiometric titration of 50 nM fluorescein-labelled wt DNA with ORF56.

Figure 7

Fluorescence anisotropy titration of fluorescently labelled wt DNA with ORF56. (A) Titration of 2 nM fluorescein-labelled wt DNA. The experimental points and a fit based on the A₄B model are given. The error bars are the standard errors of each titration point calculated from four to six measurements. In this fit the anisotropy of the unbound DNA is 0.099 and 0.159 for the bound DNA. The dissociation constant based on the tetramer concentration is 1.1 nM. (B) Competition experiment with 2 nM fluorescein-labelled wt DNA/20 nM ORF56 and increasing amounts of unlabelled wt DNA. The fit of the data yields a dissociation constant for the unlabelled DNA of 0.88 nM. (C) Stoichiometric titration of 50 nM fluorescein-labelled wt DNA with ORF56.

Figure 7

Fluorescence anisotropy titration of fluorescently labelled wt DNA with ORF56. (A) Titration of 2 nM fluorescein-labelled wt DNA. The experimental points and a fit based on the A₄B model are given. The error bars are the standard errors of each titration point calculated from four to six measurements. In this fit the anisotropy of the unbound DNA is 0.099 and 0.159 for the bound DNA. The dissociation constant based on the tetramer concentration is 1.1 nM. (B) Competition experiment with 2 nM fluorescein-labelled wt DNA/20 nM ORF56 and increasing amounts of unlabelled wt DNA. The fit of the data yields a dissociation constant for the unlabelled DNA of 0.88 nM. (C) Stoichiometric titration of 50 nM fluorescein-labelled wt DNA with ORF56.

Figure 8

Specificity, salt and temperature dependance of the ORF56 DNA-binding activity. (A) Competition experiments with non-specific DNA. ORF56 (20 nM) was allowed to bind to 2 nM fluorescently labelled wt DNA. Afterwards increasing amounts of competitor DNA were added. Filled squares, competition with rep DNA (single repeat substrate); open circles, competition with calf thymus DNA at 25°C; filled circles, competition with calf thymus DNA at 53°C. The calf thymus DNA concentration refers to the concentration of base pairs, which equals the concentration of overlapping sites. (B) Salt dependence of the dissociation constant. wt DNA (2 nM) was titrated in 10 mM Tris–HCl, pH 7.5, 0.01% Tween 20 with 125–300 mM KCl. The dissociation constant K_d at each salt concentration was calculated and plotted as a function of added salt. The calculated slope of the plot is 6.8 ± 0.2. (C) Temperature dependence of DNA binding. The temperature dependence of the dissociation constant was assayed at eight temperatures in the range 17–57°C. Fluorescently labelled wt DNA (5 nM) was titrated in cacodylate buffer with 150 mM KCl. The dissociation constants K_d were calculated at each temperature and plotted as lnK_d versus 1/T (van’t Hoff plot). The data was fitted according to equation 4 (see Materials and Methods). ΔC_p = –6.2 kJ/mol, T_H = 39°C and T_S = 47°C.

Figure 8

Specificity, salt and temperature dependance of the ORF56 DNA-binding activity. (A) Competition experiments with non-specific DNA. ORF56 (20 nM) was allowed to bind to 2 nM fluorescently labelled wt DNA. Afterwards increasing amounts of competitor DNA were added. Filled squares, competition with rep DNA (single repeat substrate); open circles, competition with calf thymus DNA at 25°C; filled circles, competition with calf thymus DNA at 53°C. The calf thymus DNA concentration refers to the concentration of base pairs, which equals the concentration of overlapping sites. (B) Salt dependence of the dissociation constant. wt DNA (2 nM) was titrated in 10 mM Tris–HCl, pH 7.5, 0.01% Tween 20 with 125–300 mM KCl. The dissociation constant K_d at each salt concentration was calculated and plotted as a function of added salt. The calculated slope of the plot is 6.8 ± 0.2. (C) Temperature dependence of DNA binding. The temperature dependence of the dissociation constant was assayed at eight temperatures in the range 17–57°C. Fluorescently labelled wt DNA (5 nM) was titrated in cacodylate buffer with 150 mM KCl. The dissociation constants K_d were calculated at each temperature and plotted as lnK_d versus 1/T (van’t Hoff plot). The data was fitted according to equation 4 (see Materials and Methods). ΔC_p = –6.2 kJ/mol, T_H = 39°C and T_S = 47°C.

Figure 8

Specificity, salt and temperature dependance of the ORF56 DNA-binding activity. (A) Competition experiments with non-specific DNA. ORF56 (20 nM) was allowed to bind to 2 nM fluorescently labelled wt DNA. Afterwards increasing amounts of competitor DNA were added. Filled squares, competition with rep DNA (single repeat substrate); open circles, competition with calf thymus DNA at 25°C; filled circles, competition with calf thymus DNA at 53°C. The calf thymus DNA concentration refers to the concentration of base pairs, which equals the concentration of overlapping sites. (B) Salt dependence of the dissociation constant. wt DNA (2 nM) was titrated in 10 mM Tris–HCl, pH 7.5, 0.01% Tween 20 with 125–300 mM KCl. The dissociation constant K_d at each salt concentration was calculated and plotted as a function of added salt. The calculated slope of the plot is 6.8 ± 0.2. (C) Temperature dependence of DNA binding. The temperature dependence of the dissociation constant was assayed at eight temperatures in the range 17–57°C. Fluorescently labelled wt DNA (5 nM) was titrated in cacodylate buffer with 150 mM KCl. The dissociation constants K_d were calculated at each temperature and plotted as lnK_d versus 1/T (van’t Hoff plot). The data was fitted according to equation 4 (see Materials and Methods). ΔC_p = –6.2 kJ/mol, T_H = 39°C and T_S = 47°C.

Figure 9

Thermal denaturation of wt+CGC DNA in the absence and presence of ORF56. The temperature profiles of 1 µM wt+CGC DNA (open circles) and of 1 µM wt+CGC DNA and 4 µM ORF56 (closed circles) are shown. Every second data point is presented. The melting temperature T_m was obtained by fitting according to equation 5 (see Materials and Methods). T_m was 71 and 86°C, respectively.