Specificity and Affinity of Lac Repressor for the Auxiliary Operators O2 and O3 Are Explained by the Structures of Their Protein–DNA Complexes

Abstract

The structures of a dimeric mutant of the Lac repressor DNA-binding domain complexed with the auxiliary operators O2 and O3 have been determined using NMR spectroscopy and compared to the structures of the previously determined Lac-O1 and Lac-nonoperator complexes. Structural analysis of the Lac-O1 and Lac-O2 complexes shows highly similar structures with very similar numbers of specific and nonspecific contacts, in agreement with similar affinities for these two operators. The left monomer of the Lac repressor in the Lac-O3 complex retains most of these specific contacts. However, in the right half-site of the O3 operator, there is a significant loss of protein-DNA contacts, explaining the low affinity of the Lac repressor for the O3 operator. The binding mode in the right half-site resembles that of the nonspecific complex. In contrast to the Lac-nonoperator DNA complex where no hinge helices are formed, the stability of the hinge helices in the weak Lac-O3 complex is the same as in the Lac-O1 and Lac-O2 complexes, as judged from the results of hydrogen/deuterium experiments.

Using Student-Generated UV-Induced Escherichia coli Mutants in a Directed Inquiry Undergraduate Genetics Laboratory

“…The MacConkey screen is not suitable for identification of lacI null alleles that do not bind lac operator DNA: mutants of this type are typically Lac 1 on MacConkey medium and evade detection. However, one may generate lacI alleles that bind lac operator DNA but that do not bind lactose or IPTG (Romanuka et al 2009), and these mutants could appear Lac À or weakly Lac 1 using the MacConkey screen. We suspect they would have variable b-galactosidase activities depending on their DNA binding kinetics but predict that b-galactosidase activities with these mutants would be identical regardless of whether they were grown in the presence or absence of IPTG.…”

Nuclear magnetic resonance analysis of protein–DNA interactions

“…In most cases, only one partner is labelled while the other remains unlabelled, which has the advantage of allowing the number of observable NMR signals to be selected. Two samples can be prepared: in the first one, which is the most frequently encountered, the protein is 15 N and/or 13 C labelled while the DNA is unlabelled, and in the other one, which is optional, the DNA is 15 N and/or 13 C labelled and the protein unlabelled.…”

“…This strategy has also been used for protein -DNA complexes ( [18,[36][37][38]; [39, p. 423] C-filtered NOESY was first proposed using a filter based on the chemical shift-optimized adiabatic 13 C inversion pulse [40] and was then optimized using 1 J HC coupling constant properties [15], providing a sensitivity gain of up to 40 per cent in the most favourable case. Once DNA proton frequencies are assigned, intermolecular NOEs can be detected using two-dimensional C edited-NOESY-HSQC experiments [40], it has been possible to detect intermolecular NOE positions on a three-dimensional experiment.…”

“…Once DNA proton frequencies are assigned, intermolecular NOEs can be detected using two-dimensional C edited-NOESY-HSQC experiments [40], it has been possible to detect intermolecular NOE positions on a three-dimensional experiment. Briefly, all the protons are excited but for 13 C-linked protons, then the mixing time permits DNA protons to perform cross relaxation with surrounding protons; finally, magnetization is transferred to 13 C atoms and back to 1 H before detection. Obtaining intermolecular distance restraints remains difficult and time-consuming, but they permit the interface of protein -DNA complexes to be defined with high accuracy and precision to enable structure calculations.…”

Structural insights into transcription complexes

“…The Lac HP bound to a non-operator DNA (NOD) fragment is different: a major difference is that the hinge helices, which play an important role in the strong cooperative operator binding of the Headpieces are not formed (Kalodimos et al, 2002) and that of the Lac HP bound to a non-operator DNA (NOD) fragment (Kalodimos et al, 2004). The analysis of these complexes helps to understand how the Lac repressor recognizes its operators and can explain the significant differences in operator affinity (Romanuka et al, 2009).…”