B Motif (b + motif)

Distribution by Scientific Domains

Selected Abstracts

The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif

Hironori Kawakami
Summary Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp. [source]

Identification of a novel nuclear factor-kappaB sequence involved in expression of urokinase-type plasminogen activator receptor

FEBS JOURNAL, Issue 11 2000
Yao Wang
We have previously defined the promoter of human urokinase-type plasminogen activator receptor (uPAR) gene in a 188-bp fragment between bases ,141 and +47 relative to the translation start site. Here, we report that a novel nuclear factor-kappaB (NF-,B)-like sequence (5,-GGGAGGAGTC-3,) at ,45 is located in the uPAR promoter and one of the two DNase I-protected regions, region I between bases ,51 and ,30. This NF-,B-like motif differs at positions 7,9 from the decameric consensus sequences of NF-,B (5,-GGGRNNYYCC-3, where R indicates A or G, Y indicates C or T, and N indicates any nucleotide) and at positions 1 and 7,9 from the ,B-like motifs (5,-HGGARNYYCC-3, where H indicates A, C, or T, R indicates A or G, Y indicates C or T, and N indicates any nucleotide). Nuclear extracts from HCT116 cells contain proteins that specifically bind to the NF-,B-like site at position ,45. Mutation of the NF-,B-like motif decreased the binding of transcription factor NF-,B and reduced the uPAR promoter activity in comparison with the wild-type sequences. Co-transfection with a dominant negative I-,B kinase-2 expression vector reduced uPAR promoter activity by 65,75%. These results demonstrate that a previously uncharacterized NF-,B motif is required for uPAR promoter activity. [source]

An intron enhancer activates the immunoglobulin-related Hemolin gene in Hyalophora cecropia

K. Roxström-Lindquist
Abstract Hemolin is the only insect member of the immunoglobulin (Ig) superfamily reported to be up-regulated during an immune response. In diapausing pupae of Hyalophora cecropia the gene is expressed in fat body cells and in haemocytes. Like the mammalian Ig , light chain gene, the Hemolin gene harbours an enhancer including a ,B motif in one of its introns. This motif binds the H. cecropia Rel factor Cif (Cecropia immunoresponsive factor). The Hemolin third intron also mediates transient reporter gene expression in immunoresponsive Drosophila mbn-2 cells. Co-transfections of Drosophila SL2 cells showed that the Drosophila Rel factor Dif (Dorsal-related immunity factor), transactivates reporter gene constructs through the intron. Moreover, a 4.8-fold synergistic activation was obtained when Dif is combined with the rat C/EBP (CCAAT/enhancer element-binding protein) and human HMGI (high mobility group protein I). This is the first report of an insect immune-related gene that is up-regulated by an enhancer activity conferred through an intron. [source]

Cloning, expression, purification and preliminary X-­ray crystallographic studies of Escherichia coli Hsp100 nucleotide-binding domain 2 (NBD2)

Jingzhi Li
Escherichia coli Hsp100 ClpB has been identified recently as playing critical roles in multi-chaperone systems. ClpB binds and disaggregates denatured polypeptides by employing ATP hydrolysis and allows other molecular chaperones such as Hsp70 DnaK and Hsp40 DnaJ to refold the non-native polypeptides. ClpB contains two nucleotide-binding domains (NBD1 and NBD2) in its primary sequence. Walker A and Walker B motifs exist in both nucleotide-binding domains. Therefore, ClpB belongs to the large ATPase family known as ATPase associated with various cellular activities (AAA). The mechanisms by which NBD1 and NBD2 function to support the ClpB molecular-chaperone activity are currently unknown. To investigate how NBD2 participates in ClpB function to disaggregate denatured proteins, ClpB NBD2 has been cloned and crystallized. The ClpB NBD2 crystals diffract X-rays to 2.5,Å using synchrotron X-ray sources. The crystals belong to space group P212121, with unit-cell parameters a = 99.57, b = 149.34, c = 164.69,Å. [source]