Structures of complexes comprised of Fischerella transcription factor HetR with Anabaena DNA targets

Abstract

HetR is an essential regulator of heterocyst development in cyanobacteria. Many mutations in HetR render Anabaena incapable of nitrogen fixation. The protein binds to a DNA palindrome upstream of hetP and other genes. We have determined the crystal structures of HetR complexed with palindromic DNA targets, 21, 23, and 29 bp at 2.50-, 3.00-, and 3.25-Å resolution, respectively. The highest-resolution structure shows fine details of specific protein-DNA interactions. The lower-resolution structures with longer DNA duplexes have similar interaction patterns and show how the flap domains interact with DNA in a sequence nonspecific fashion. Fifteen of 15 protein-DNA contacts predicted on the basis of the structure were confirmed by single amino acid mutations that abolished binding in vitro and complementation in vivo. A striking feature of the structure is the association of glutamate 71 from each subunit of the HetR dimer with three successive cytosines in each arm of the palindromic target, a feature that is conserved among all known heterocyst-forming cyanobacteria sequenced to date.heterocyst differentiation | mutagenesis | X-ray crystallography

The HetR-binding site that activates expression of patA in vegetative cells is required for normal heterocyst patterning in Anabaena sp. PCC 7120

“…Heterocysts and proheterocysts are indicated by arrowheads 192-201 197 for binding; the five base pairs that are present between GGG and CCC appear to have little importance. Beyond the core sequence, four other base pairs have roles in the interaction [18]. The hetP site has at least 10 base pairs involved in contacts with HetR and is therefore the most preferred HetR-recognition site.…”

“…HetR binds to consensus recognition sites upstream of hetP [17] and hetZ [10], two genes required for heterocyst differentiation and up-regulated in developing heterocysts. The crystal structure of the HetR-DNA complex has been reported, showing the importance of GGG(n5)CCC and several flanking base pairs in the interaction [18]. More recently, HetR was shown to regulate hepA, a gene involved in formation of the polysaccharide layer of heterocyst envelope, via a lower affinity recognition site in its promoter [19].…”

ABC Transporter Required for Intercellular Transfer of Developmental Signals in a Heterocystous Cyanobacterium

“…HetR is a transcription factor that promotes differentiation. In addition to regulating the transcription of a large number of morphogenesis and metabolism genes, HetR positively autoregulates its own production and is necessary for induction of transcription of patS and hetN (16)(17)(18). The full-length 17-amino-acid (17-aa) patS-encoded peptides and 287-aa hetN-encoded peptides do not appear to move from cell to cell, and the presumed fragments that do are unknown (3,12).…”

Expanding the Direct HetR Regulon in Anabaena sp. Strain PCC 7120

“…Heterocysts are morphologically distinct cells that develop at semiregular intervals and are separated by approximately 10 to 20 photosynthetic vegetative cells, resulting in a 1-dimensional pattern along filaments. This differentiation process results in a change in the transcription of roughly 1,500 genes, which is facilitated by HetR, the master regulator of differentiation (12). A hetR deletion in Anabaena results in the inability to develop heterocysts, whereas overexpression yields supernumerary heterocysts even under nitrogen-replete conditions (13,14).…”

“…The N termini create a DNA-binding domain containing helix-turn-helix motifs. Recently, cocrystallization of Fischerella HetR bound to a 21-bp DNA fragment based on that from the hetP promoter has identified the necessary protein-DNA interactions that confer DNA binding specificity to HetR (12). Most strikingly, the interaction of Glu71 with three consecutive cytosines during DNA binding defines the requirement of an inverted repeat-containing sequence with CCC-N 5 -GGG at its core.…”