When simple sequence comparison fails: the cryptic case of the shared domains of the bacterial replication initiation proteins DnaB and DnaD.

DnaD and DnaB are essential DNA-replication-initiation proteins in low-G+C content Gram-positive bacteria. Here we use sensitive Hidden Markov Model-based techniques to show that the DnaB and DnaD proteins share a common structure that is evident across all their structural domains, termed DDBH1 and DDBH2 (DnaD DnaB Homology 1 and 2). Despite strong sequence divergence, many of the DNA-binding and oligomerization properties of these domains have been conserved. Although eluding simple sequence comparisons, the DDBH2 domains share the only strong sequence motif; an extremely highly conserved YxxxIxxxW sequence that contributes to DNA binding. Sequence alignments of DnaD alone fail to identify another key part of the DNA-binding module, since it includes a poorly conserved sequence, a solvent-exposed and somewhat unstable helix and a mobile segment. We show by NMR, in vitro mutagenesis and in vivo complementation experiments that the DNA-binding module of Bacillus subtilis DnaD comprises the YxxxIxxxW motif, the unstable helix and a portion of the mobile region, the latter two being essential for viability. These structural insights lead us to a re-evaluation of the oligomerization and DNA-binding properties of the DnaD and DnaB proteins.

X-ray crystallographic study of DNA duplex cross-linking: simultaneous binding to two d(CGTACG)2 molecules by a bis(9-aminoacridine-4-carboxamide) derivative.

Acridine-4-carboxamides form a class of known DNA mono-intercalating agents that exhibit cytotoxic activity against tumour cell lines due to their ability to inhibit topoisomerases. Previous studies of bis-acridine derivatives have yielded equivocal results regarding the minimum length of linker necessary between the two acridine chromophores to allow bis-intercalation of duplex DNA. We report here the 1.7 A resolution X-ray crystal structure of a six-carbon-linked bis(acridine-4-carboxamide) ligand bound to d(CGTACG)2 molecules by non-covalent duplex cross-linking. The asymmetric unit consists of one DNA duplex containing an intercalated acridine-4-carboxamide chromophore at each of the two CG steps. The other half of each ligand is bound to another DNA molecule in a symmetry-related manner, with the alkyl linker threading through the minor grooves. The two crystallographically independent ligand molecules adopt distinct side chain interactions, forming hydrogen bonds to either O6 or N7 on the major groove face of guanine, in contrast to the semi-disordered state of mono-intercalators bound to the same DNA molecule. The complex described here provides the first structural evidence for the non-covalent cross-linking of DNA by a small molecule ligand and suggests a possible explanation for the inconsistent behaviour of six-carbon linked bis-acridines in previous assays of DNA bis-intercalation.

The interaction of RNA with TRAP: the role of triplet repeats and separating spacer nucleotides.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes in several Bacilli by binding to the leader region of the nascent trp mRNA, inhibiting continued transcription. The 11 subunit TRAP molecule is active in complex with tryptophan, and binds consequently an RNA target segment consisting of 11 (G/U)AG triplets, each separated by two or three non-conserved "spacer" nucleotides. Here, we report the first crystal structures of TRAP in a complex with RNA containing UAG triplets separated by two nucleotides and in a complex with RNA containing GAG triplets separated by three nucleotides. Comparison with known structures of TRAP-RNA complexes shows that both substitution of G-1 with U-1 in the triplet and addition of an extra spacer nucleotide lead to a more flexible complex. This suggests an explanation why, in the trp leader RNA, all three-nucleotide spacer regions are followed by a G-1 nucleotide. Taken together, the structures demonstrate that RNA binding to TRAP is mediated by specific interactions involving the A-2 and G-3 nucleotides of the triplet. This is accompanied by the disruption of stacking interactions between the bases of the other nucleotides, contributing to the increase in entropy that drives binding.

Specificity of TRAP-RNA interactions: crystal structures of two complexes with different RNA sequences.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes in bacilli by binding to the leader region of the nascent trp operon mRNA. When activated by binding tryptophan, the 11-subunit circular TRAP molecule binds to a target sequence consisting of 11 (G/U)AG repeats, separated by two or three variable 'spacer' nucleotides. Reported here are two crystal structures of TRAP bound to RNAs containing 11 GAG repeats separated by UU and CC spacer nucleotides, determined at 1.75 and 2.50 A resolution, respectively. These show the spacer regions of the RNA molecules to be highly flexible, making no direct hydrogen-bonding contacts with the protein. Comparison of these structures with the previous structure of TRAP bound to (GAGAU)(10)GAG RNA, in which the spacer nucleotides stack with each other close to the protein surface, shows that the RNA can adopt different conformations depending on the sequence of the spacer regions. This gives insight into the structural basis of the specificity of TRAP and into the mechanism of binding.