Equilibrium DNA-binding of AraC protein. Compensation for displaced ions

Experiments on the AraC regulatory protein of Escherichia coli suggest a mechanism that DNA-binding proteins can use to reduce potentially drastic alterations in their affinity for DNA resulting from changes in salt concentration. Measurement of the net number of ions apparently displaced as AraC protein binds DNA and of fluorescence changes in the protein lead to the following picture. About 14 ions are displaced from the DNA as the protein binds the araI site. As the protein binds the DNA, however, it undergoes a conformational change and binds about ten ions. Consequently, the net order of the reaction is reduced from 15th to about fourth order in salt concentration.     

Altered DNA contacts made by a mutant AraC protein.

Mutant AraC proteins were selected for their ability to induce but not to repress, or their ability to repress but not to induce the araBAD operon. One such unusual mutant is able to bind to the araI site with an affinity only two to three-fold weaker than the wild type AraC protein, but the mutant protein was shown, both in crude extracts and when purified, to contact only two of the three major groove regions of the DNA that are contacted by the wild type protein.     

The missing nucleoside experiment: a new technique to study recognition of DNA by protein

We report a new technique for quickly determining which nucleosides in a DNA molecule are contacted by a sequence-specific DNA-binding protein. Our method is related to the recently reported "missing contact" experiment [Brunelle, A., & Schleif, R. F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6673-6679]. We treat the DNA molecule with the hydroxyl radical to randomly remove nucleosides. The ability of protein to bind to gapped DNA is assayed by gel mobility shift. Nucleosides important to protein binding are identified by sequencing gel electrophoresis. The missing nucleoside experiment can be used to scan a DNA molecule at single-nucleotide resolution in one experiment. The bacteriophage lambda repressor-OR1 and cro-OR1 complexes were analyzed to evaluate the method. For both proteins, the most important contacts are located in the protein monomer that binds to the consensus half of the operator. These contacts correspond well to those found by mutational studies, and in the cocrystal structure of the lambda repressor-operator. The missing nucleoside data show that the amino-terminal arms of lambda repressor make energetically important contacts with positions 7 and 8 and the central dyad base pair of the operator. The amino-terminal arm that makes the most extensive contacts to DNA appears to be the one that emanates from the repressor monomer that binds to the consensus half of the operator, in agreement with the cocrystal structure. The lambda cro protein does not have an amino-terminal arm, and the missing nucleoside experiment clearly shows a lack of contacts to DNA in the central region of the operator in this complex.     

Repression of the araBAD promoter from araO1.

DNA looping between the araO2 and araI sites holds the uninduced or basal level of expression of the araBAD genes from the pBAD promoter at a low level. Despite the presence of another and closer site potentially capable of mediating looping to araI, no repression from this site, called araO1, is observed. Here we show, using both in vivo and in vitro experiments, that the araO1 site is not normally occupied by AraC protein under repressing conditions, but that if AraC protein is overproduced and the araO2 site is absent, araO1 is then occupied and repression of pBAD can be observed.     

Contacts between Tet repressor and tet operator revealed by new recognition specificities of single amino acid replacement mutants.

We have analyzed the DNA binding properties of Tet-repressor mutants with single amino acid residue replacements at eight positions within the alpha-helix-turn-alpha-helix DNA-binding motif. A saturation mutagenesis of Gln38, Pro39, Thr40, Tyr42, Trp43 and His44 in the second alpha-helix was performed; in addition, several substitutions of Thr27 and Arg28 in the first alpha-helix were constructed. The abilities of these mutant repressors to bind a set of 16 operator variants were determined and revealed 23 new binding specificities. All repressor mutants with DNA-binding activity were inducible by tetracycline, while mutants lacking binding activity were trans-dominant over the wild-type. All mutant proteins were present at the same intracellular steady-state concentrations as the wild-type. These results suggest the structural integrity of the mutant repressors. On the basis of the new recognition specificities, five contacts between a repressor monomer and each operator half-site and the chemical nature of these repressor-operator interactions are proposed. We suggest that Arg28 contacts guanine of the G.C base-pair at operator position 2 with two H-bonds, Gln38 binds adenine of the A.T base-pair at position 3 with two H-bonds, and the methyl group of Thr40 participates in a van der Waals' contact with cytosine of the G.C base-pair at position 6 of tet operator. A previously unrecognized type of interaction is proposed for Pro39, which inserts its side-chain between the methyl groups of the thymines of T.A and A.T base-pairs at positions 4 and 5. Computer modeling of these proposed contacts reveals that they are possible using the canonical structures of the helix-turn-helix motif and B-DNA. These contacts suggest an inverse orientation of the Tet repressor helix-turn-helix with respect to the operator center as compared with non-inducible repressor-operator complexes, and are supported by similar contacts of other repressor-operator complexes.     

Mutations in the araC regulatory gene of Escherichia coli B/r that affect repressor and activator functions of AraC protein.

Mutations in the araC gene of Escherichia coli B/r were isolated which alter both activation of the araBAD operon expression and autoregulation. The mutations were isolated on an araC-containing plasmid by hydroxylamine mutagenesis of plasmid DNA. The mutant phenotype selected was the inability to autoregulate. The DNA sequence of 16 mutants was determined and found to consist of seven different missense mutations located within the distal third of the araC gene. Enzyme activities revealed that each araC mutation had altered both autoregulatory and activator functions of AraC protein. The mutational analysis presented in this paper suggests that both autoregulatory and activator functions are localized to the same determinants of the AraC protein and that the amino acid sequence within the carboxy-terminal region of AraC protein is important for site-specific DNA binding.     

AraC-DNA looping: orientation and distance-dependent loop breaking by the cyclic AMP receptor protein.

The arabinose operon promoter, pBAD, is negatively regulated in the absence of arabinose by AraC protein, which forms a DNA loop by binding to two sites separated by 210 base-pairs, araO2 and araI1. pBAD is also positively regulated by AraC-arabinose and the cyclic AMP receptor protein, CRP. We provide evidence that CRP breaks the araO2-araI1 repression loop in vitro. The ability of CRP to break the loop in vitro and to activate pBAD in vivo is dependent upon the orientation and distance of the CRP binding site relative to araI1. An insertion of one DNA helical turn, 11 base-pairs, between CRP and araI only partially inhibits CRP loop breaking and activation of pBAD, while an insertion of less than one DNA helical turn, 4 base-pairs, not only abolishes CRP activation and loop breaking, but actually causes CRP to stabilize the loop and increases the araO2-mediated repression of pBAD. Both integral and non-integral insertions of greater than one helical turn completely abolish CRP activation and loop breaking in vitro.     

AraC proteins with altered DNA sequence specificity which activate a mutant promoter in Escherichia coli

We examined the recognition of the araBAD promoter by the AraC protein in the Escherichia coli arabinose operon. A mutant promoter, with base substitutions at positions contacted by AraC, was used to isolate suppressor mutations in araC by direct selection. Two hydroxylamine-induced araC mutations were isolated repeatedly; each contained a single amino acid substitution. When tested against a set of base substitution promoter mutants, one revertant, an Arg to His substitution at residue 250, displayed altered base specificity for a single position within the araBAD promoter. The other revertant, a Cys to Tyr substitution at residue 204, did not show consistent base-specific suppression. Neither demonstrated a higher affinity than the wild type protein for the mutant promoter in vitro. Both proteins suppress mutant sequences by a mechanism that does not appear to involve the formation of new net favorable contacts with the mutant base pairs of the promoter.     

Refined 1.8 A crystal structure of the lambda repressor-operator complex.

The crystal structure of the lambda repressor-operator complex has been refined to an R-factor of 18.9% at 1.8 A resolution. This refinement, using data collected at low temperature, has revealed the structure of the N-terminal arm and shows that the interactions of repressor with the two halves of the pseudo-symmetric operator site are significantly different. The two halves of the complex are most similar near the outer edge of the operator site (in a region where the lambda and 434 repressors make similar contacts), but they become increasingly different toward the center of the operator. There are striking differences near the center of the site where it appears that the arm makes significant contacts to only one half of the DNA site. This suggested a new way of aligning the operator sites in phage lambda. The high resolution structure confirms many of the previously noted features of the complex, but also reveals a number of new protein-DNA contacts. It also gives a better view of the extensive H-bonding networks that couple contacts made by different residues and different regions of the protein, and reveals important new details about the helix-turn-helix (HTH) region, and the positions of many water molecules in the complex.