Orientation of the Lac repressor DNA binding domain in complex with the leftlacoperator half site characterized by affinity cleaving

ABSTRACT The Escherichia coli l-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of l-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of l-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a ΔrhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.

Download Full-text

Mechanism of Lac repressor switch-off: orientation of the Lac repressor DNA-binding domain is reversed upon inducer binding

FEBS Letters ◽

10.1016/0014-5793(95)01153-6 ◽

1995 ◽

Vol 375 (1-2) ◽

pp. 27-30 ◽

Cited By ~ 14

Author(s):

Dimitrii E. Kamashev ◽

Natalia G. Esipova ◽

Konstantin K. Ebralidse ◽

A.D. Mirzabekov

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Lac Repressor ◽

Dna Binding Domain

Download Full-text

Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator O1 through alternative conformations of its DNA-binding domain

The EMBO Journal ◽

10.1093/emboj/cdf318 ◽

2002 ◽

Vol 21 (12) ◽

pp. 2866-2876 ◽

Cited By ~ 77

Author(s):

C. G. Kalodimos

Keyword(s):

Dna Binding ◽

Dna Recognition ◽

Binding Domain ◽

Lac Repressor ◽

Dna Binding Domain ◽

Natural Operator

Download Full-text

Dynamic Consequences of Specificity within the Cytidine Repressor DNA-Binding Domain

10.1101/2021.02.28.433298 ◽

2021 ◽

Author(s):

Colleen L Moody ◽

Jenaro Soto ◽

Vira Tretyachenko-Ladokhina ◽

Donald F Senear ◽

Melanie J Cocco

Keyword(s):

Dna Binding ◽

Hydrogen Exchange ◽

Intrinsically Disordered Protein ◽

Structural Features ◽

Binding Domain ◽

Free State ◽

Dna Binding Domain ◽

Intrinsically Disordered ◽

Cytidine Repressor ◽

Half Site

The E. coli cytidine repressor (CytR) is a member of the LacR family of bacterial repressors that regulates nine operons with distinct spacing and orientations of recognition sites. Understanding the structural features of the CytR DNA-binding domain (DBD) when bound to DNA is critical to understanding differential mechanisms of gene regulation. We previously reported the structure of the CytR DBD monomer bound specifically to half-site DNA and found that the DBD exists as a three-helix bundle containing a canonical helix-turn-helix motif, similar to other proteins that interact with DNA [Moody, et al (2011), Biochemistry 50:6622-32]. We also studied the free state of the monomer and found that since NMR spectra show it populates up to four distinct conformations, the free state exists as an intrinsically disordered protein (IDP). Here, we present further analysis of the DBD structure and dynamics in the context of full-site operator or nonspecific DNA. DBDs bound to full-site DNA show one set of NMR signals, consistent with fast exchange between the two binding sites. When bound to full-length DNA, we observed only slight changes in structure compared to the monomer structure and no folding of the hinge helix. Notably, the CytR DBD behaves quite differently when bound to nonspecific DNA compared to LacR. A dearth of NOEs and complete lack of protection from hydrogen exchange are consistent with the protein populating a flexible, molten state when associated with DNA nonspecifically, similar to fuzzy complexes. The CytR DBD structure is significantly more stable when bound specifically to the udp half-site substrate. For CytR, the transition from nonspecific association to specific recognition results in substantial changes in protein mobility that are coupled to structural rearrangements. These effects are more pronounced in the CytR DBD compared to other LacR family members.

Download Full-text

The DNA-binding domain of BenM reveals the structural basis for the recognition of a T-N11-A sequence motif by LysR-type transcriptional regulators

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913017320 ◽

2013 ◽

Vol 69 (10) ◽

pp. 1995-2007 ◽

Cited By ~ 27

Author(s):

Amer M. Alanazi ◽

Ellen L. Neidle ◽

Cory Momany

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Dna Recognition ◽

Transcriptional Regulators ◽

Binding Domain ◽

Structural Basis ◽

Binding Motif ◽

Sequence Motif ◽

Dna Binding Domain ◽

Half Site

LysR-type transcriptional regulators (LTTRs) play critical roles in metabolism and constitute the largest family of bacterial regulators. To understand protein–DNA interactions, atomic structures of the DNA-binding domain and linker-helix regions of a prototypical LTTR, BenM, were determined by X-ray crystallography. BenM structures with and without bound DNA reveal a set of highly conserved amino acids that interact directly with DNA bases. At the N-terminal end of the recognition helix (α3) of a winged-helix–turn–helix DNA-binding motif, several residues create hydrophobic pockets (Pro30, Pro31 and Ser33). These pockets interact with the methyl groups of two thymines in the DNA-recognition motif and its complementary strand, T-N11-A. This motif usually includes some dyad symmetry, as exemplified by a sequence that binds two subunits of a BenM tetramer (ATAC-N7-GTAT). Gln29 forms hydrogen bonds to adenine in the first position of the recognition half-site (ATAC). Another hydrophobic pocket defined by Ala28, Pro30 and Pro31 interacts with the methyl group of thymine, complementary to the base at the third position of the half-site. Arg34 interacts with the complementary base of the 3′ position. Arg53, in the wing, provides AT-tract recognition in the minor groove. For DNA recognition, LTTRs use highly conserved interactions between amino acids and nucleotide bases as well as numerous less-conserved secondary interactions.

Download Full-text