Helicobacter pylori NikR protein exhibits distinct conformations when bound to different promoters

Helicobacter pylori NikR (HpNikR) is a ribbon-helix-helix (RHH) DNA-binding protein that binds to several different promoter regions. The binding site sequences are not absolutely conserved. The ability of HpNikR to discriminate specific DNA sites resides partly in its nine-amino acid N-terminal arm. Previously, indirect evidence indicated that the arm exists in different conformations when HpNikR is bound to the nixA and ureA promoters. Here, we directly examined HpNikR conformation when it was bound to nixA and ureA DNA fragments by tethering (S)-1{[bis(carboxymethyl)amino]methyl}-2-{4-[(2-bromoacetyl)amino]phenylethyl}(carboxymethyl)amino]acetic acid, iron(III) to different positions in the N-terminal arm and RHH DNA binding domain. Different cleavage patterns at each promoter directly demonstrated that both the RHH domain and the arm adopt different conformations on the nixA and ureA promoters. Additionally, the two RHH domain dimers of the HpNikR tetramer are in distinct conformations at ureA. Site-directed mutagenesis identified an interchain salt bridge (Lys(48)-Glu(47')) in the RHH domain remote from the DNA binding interface that is required for high affinity binding to ureA but not nixA. Finally, DNA affinity measurements of wild-type HpNikR and a salt bridge mutant (K48A) to hybrid nixA-ureA promoters demonstrated that inverted repeat half-sites, spacers, and flanking DNA are all required for sequence-specific DNA binding by HpNikR. Notably, the spacer region made the largest contribution to DNA affinity. HpNikR exhibits a substantially expanded regulon compared with other NikR proteins. The results presented here provide a molecular basis for understanding regulatory network expansion by NikR as well as other prokaryotic regulatory proteins.     

Identification of sequence elements in the human cytomegalovirus DNA polymerase gene promoter required for activation by viral gene products.

To determine the mechanisms involved in the regulation of human cytomegalovirus early gene expression, we have examined the gene that encodes the viral DNA polymerase (UL54, pol). Our previous studies demonstrated that sequences required for activation of the pol promoter by immediate-early proteins are contained within a region from -128 to +20 and that cellular proteins can bind to this activation domain. In this study, we demonstrate by competition analysis that binding of cellular proteins to pol is associated with an 18-bp region containing a single copy of a novel inverted repeat, IR1. Time course analysis indicated that viral infection increased the level of protein binding to IR1, concurrent with the activation of the pol promoter. Mutation of the IR1 element abrogated binding of cellular factors to the pol promoter and reduced by threefold the activation by immediate-early proteins. Similarly, mutation of IR1 rendered the promoter poorly responsive to activation by viral infection. Mutation of additional sequence elements in the pol promoter had little effect, indicating that IR1 plays the major role in pol promoter regulation. These studies demonstrate that the interaction between cellular factors and IR1 is important for the regulation of expression of the polymerase gene by viral proteins.     

Binding of activator SyrM to the site ofnodD3 P1 region ofRhizobium meliloti

The nodD3 gene ofRhizobium meliloti is transcribed via promoter P1 or P2. Gel retardation assay showed binding of SyrM to the P1 upstream region of nodD3. DNaseI footprint analysis demonstrated that the binding site of SyrM in nodD3 P1 region consists of two inverted repeat sequences arranged in tandem. SyrM seems to bind to DNA in the form of dimer or tetramer and requires the two inverted repeat sequences for binding.     

Assembly of the RFX complex on the MHCII promoter: role of RFXAP and RFXB in relieving autoinhibition of RFX5

The RFX complex is key component of a multi-protein complex that regulates the expression of the Major Histocompatibility Class II (MHCII) genes, whose products are essential for the initiation and development of the adaptive immune response. The RFX complex is comprised of three proteins--RFX5, RFXAP, and RFXB--all of which are required for expression of MHCII genes. We have used electrophoretic mobility shift assays to characterize the DNA binding of RFX5 and the complexes it forms with RFXB and RFXAP, to the proximal regulatory region of the MHCII promoter. DNA binding of RFX5 is inhibited by domains flanking its DNA binding domain, and both RFXAP and RFXB are required to overcome the inhibition of both domains. We provide evidence that a single RFX complex binds to the proximal regulatory region of the MHCII promoter and identify regions of the DNA that are important for high affinity binding of the RFX complex. Together, our results provide the most detailed view to date of the assembly of the RFX complex on the MHCII promoter and how its DNA binding is regulated.     

Analysis of genetic elements controlling Staphylococcus aureus lrgAB expression: potential role of DNA topology in SarA regulation

Penicillin-induced killing and murein hydrolase activity in Staphylococcus aureus are dependent on a variety of regulatory elements, including the LytSR two-component regulatory system and the virulence factor regulators Agr and Sar. The LytSR effects on these processes can be explained, in part, by the recent finding that a LytSR-regulated operon, designated lrgAB, affects murein hydrolase activity and penicillin tolerance. To examine the regulation of lrgAB expression in greater detail, we performed Northern blot and promoter fusion analyses. Both methods revealed that Agr and Sar, like LytSR, positively regulate lrgAB expression. A mutation in the agr locus reduced lrgAB expression approximately sixfold, while the sar mutation reduced lrgAB expression to undetectable levels. cis-acting regulatory elements involved in lrgAB expression were identified by fusing various fragments of the lrgAB promoter region to the xylE reporter gene and integrating these constructs into the chromosome. Catechol 2,3-dioxygenase assays identified DNA sequences, including an inverted repeat and intrinsic bend sites, that contribute to maximal lrgAB expression. Confirmation of the importance of the inverted repeat was achieved by demonstrating that multiple copies of the inverted repeat reduced lrgAB promoter activity, presumably by titrating out a positive regulatory factor. The results of this study demonstrate that lrgAB expression responds to a variety of positive regulatory factors and suggest that specific DNA topology requirements are important for optimal expression.     

Binding of the Tn3 transposase to the inverted repeats of Tn3

The transposase protein and the inverted repeat sequences of Tn3 are both essential for Tn3 cointegrate formation and transposition. We have developed two assays to detect site-specific binding of transposase to the inverted repeats: (1) a nitrocellulose filter binding assay in which transposase preferentially retains DNA fragments containing inverted repeat sequences, and (2) a DNase 1 protection assay in which transposase prevents digestion of the inverted repeats by DNase 1. Both assays show that transposase binds directly to linear, duplex DNA containing the inverted repeats. The right inverted repeat of Tn3 binds slightly more strongly than the left one. Site-specific binding requires magnesium but does not require a high energy cofactor.     

DNA binding activities of the Caenorhabditis elegans Tc3 transposase.

Tc3 is a member of the Tc1/mariner family of transposable elements. All these elements have terminal inverted repeats, encode related transposases and insert exclusively into TA dinucleotides. We have studied the DNA binding properties of Tc3 transposase and found that an N-terminal domain of 65 amino acids binds specifically to two regions within the 462 bp Tc3 inverted repeat; one region is located at the end of the inverted repeat, the other is located approximately 180 bp from the end. Methylation interference experiments indicate that this N-terminal DNA binding domain of the Tc3 transposase interacts with nucleotides on one face of the DNA helix over adjacent major and minor grooves.     

Binding of activator SyrM to the site of nodD3 P1 region of Rhizobium meliloti

The nodD3 gene ofRhizobium meliloti is transcribed via promoter P1 or P2. Gel retardation assay showed binding of SyrM to the P1 upstream region of nodD3. DNaseI footprint analysis demonstrated that the binding site of SyrM in nodD3 P1 region consists of two inverted repeat sequences arranged in tandem. SyrM seems to bind to DNA in the form of dimer or tetramer and requires the two inverted repeat sequences for binding. Project supported by the National Natural Science Foundation of China (Grant No. 39370027).     

Control of the adipsin gene in adipocyte differentiation. Identification of distinct nuclear factors binding to single- and double-stranded DNA

The mouse adipsin gene encodes a serine protease with complement factor D activity that is expressed during adipocyte differentiation and is deficient in several animal models of obesity. We have investigated the regulation of adipsin expression by transfecting preadipocytes and adipocytes with plasmids containing the 5'-flanking region of the adipsin gene linked to a reporter gene. Constructions containing a -950 to +35 segment of the adipsin promoter were preferentially expressed in adipose cells. Deletion experiments identified a region from -114 to -38 which contains a large inverted repeat sequence and negatively regulated gene expression in preadipocytes and positively regulated expression in fat cells. Exonuclease III protection and gel retardation assays indicated that this region of duplex DNA had multiple binding sites for nuclear factors, several of which were preadipose specific. In addition, we also identified two distinct factors that bound symmetrically and sequence specifically to the inverted repeat sequences only when they were in single-stranded form; one of these factors was induced during adipocyte differentiation. These results suggest that the control of the adipsin promoter in differentiation may involve an interplay of multiple regulated DNA-binding proteins, including two that have preferential affinity for single-stranded DNA.     

A sequence-specific single-strand DNA binding protein that contacts repressor sequences in the human GM-CSF promoter.

NF-GMb is a nuclear factor that binds to the proximal promoter of the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene. NF-GMb has a subunit molecular weight of 22 kDa, is constitutively expressed in embryonic fibroblasts and binds to sequences within the adjacent CK-1 and CK-2 elements (CK-1/CK-2 region), located at approximately -100 in the GM-CSF gene promoter. These elements are conserved in haemopoietic growth factor (HGF) genes. NF-GMb binding requires the presence of repeated 5'CAGG3' sequences that overlap the binding sites for positive activators. Surprisingly, NF-GMb was found to bind solely to single-strand DNA, namely the non-coding strand of the GM-CSF CK-1/CK-2 region. NF-GMb may belong to a family of single-strand DNA binding (ssdb) proteins that have 5'CAGG3' sequences within their binding sites. Functional analysis of the proximal GM-CSF promoter revealed that sequences in the -114 to -79 region of the promoter containing the NF-GMb binding sites had no intrinsic activity in fibroblasts but could, however, repress tumour necrosis factor-alpha (TNF-alpha) inducible expression directed by downstream promoter sequences (-65 to -31). Subsequent mutation analysis showed that sequences involved in repression correlated with those required for NF-GMb binding.