TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG.

Regulatory elements containing the sequence ACGT are found in several plant promoters and are recognized by various basic/leucine zipper (bZIP) proteins. The Arabidopsis G-box binding factor 1 (GBF1), initially identified by its ability to bind to the palindromic G-box (CCACGTGG), also interacts with the TGACGT motif if this hexamer sequence is followed by either the dinucleotide GG--as found in the Hex motif of the wheat histone 3 promoter--or GT. Here we describe the isolation of an Arabidopsis bZIP protein, denoted TGA1, that also recognizes ACGT-containing sequences. However, TGA1 differs from members of the GBF family in the spectrum of base pair permutations flanking the ACGT sequence that are required for DNA binding. TGA1 primarily requires a TGACG motif and preferentially binds to those pentamers that are followed by a T residue. We show that although both TGA1 and GBF1 bind to the Hex motif (TGACGTGG), this binding can be distinguished on the basis of their specific DNA-protein contacts. Furthermore, TGA1 also differs from members of the GBF family in that it apparently does not form heterodimers with any member of this family.     

粘虫核型多角体病毒凋亡抑制基因的定位,序列和启动子结构

以ＡｃＮＰＶ凋亡抑制基因ｐ３５为探针,与ＬｓＮＰＶＤＮＡ的限制性片段和ＬｓＮＰＶＤＮＡＥｃｏＲＶ片段杂交,发现ＥｃｏＲＶ５．５ｋｂ片段有强烈的杂交信号。将此片段亚克隆后,测定了１２４４ｂｐ序列,发现一个完整的ＯＲＦ,推导的３０２个氨基酸与ＡｃＮＰＶｐ３５蛋白有７０．４％的氨基酸同源性,证明所测ＯＲＦ为ＬｓＮＰＶ的ｐ３５基因。结构分析发现其５′端有早期基因启动子元件ＧＣ、ＡＣＧＴ和ＴＡＴＡｂｏｘ。有２２ｂｐ的顺向重复序列,包括由两个重叠的ＴＡＴＡｂｏｘ和上下游两个ＡＣＧＴｍｏｔｉｆ组成的两套启动子元件,这些结构特征与ＡｃＮＰＶ的凋亡抑制基因十分相似。     

In vitro binding of the tomato bZIP transcriptional activator VSF-1 to a regulatory element that controls xylem-specific gene expression

The bean grp1.8 gene is specifically expressed in vascular tissue. Monomers and multimers of a 28 bp regulatory element of the grp1.8 promoter ( vs-1 ) specifically activated both the −82 CaMV 35S and the −76/ grp1.8 minimal promoters in vascular tissue of transgenic tobacco plants. vs-1 partially overlaps with a negative regulatory element in the grp1.8 promoter that is necessary for restriction of gene expression to vascular tissue. Nuclear extracts from tobacco and tomato cells contain a factor that binds to vs-1 in vitro . To study the molecular basis of xylem-specific expression mediated by the vs-1 promoter element, a gene was isolated from tomato encoding a protein that binds to vs-1 in vitro . This protein, designated VSF-1, contains a bZIP motif close to the C-terminus. Mutated vs-1 elements were no longer bound by VSF-1 and also failed to activate the minimal −82 CaMV 35S promoter in vivo . Transient expression of VSF-1 in protoplasts stimulated vs-1 dependent activation of the −76/ grp1.8 minimal promoter. Binding studies and use of a polyclonal antiserum against VSF-1 provided further evidence that vs-1 is a potential in vivo target site, as VSF-1 was a part of the observed complex formed between vs-1 and nuclear protein extract. vs-1 does not contain the 5'-ACGT-3' core sequence that is part of known plant bZIP protein binding sites or another palindromic sequence. Based on the unusual binding specificity and a characteristic amino acid sequence in the bZIP domain we propose that VSF-1 and the partially homologous PosF21, a bZIP protein from Arabidopsis , belong to a new family of plant bZIP proteins.     

Depression of the xylanase-encoding cgxA gene of Chaetomium gracile in Aspergillus nidulans

Regulation of the Chaetomium gracile xylanase A gene (cgxA) was investigated using Aspergillus nidulans as an intermediate host. Deletion of a 185 bp DNA fragment from its promoter region led to higher levels of the cgxA gene expression, indicating that the 185 bp DNA fragment contains an element involved in repression of the gene. A nuclear extract was assayed for proteins which bind to the 185 bp DNA fragment. A protein designated AnRP bound sequence specifically to the DNA fragment. The minimum sequence required for AnRP binding, 5'TTGACAAAT-3', was determined by means of gel mobility shift assays with various double-stranded oligonucleotides. Furthermore, this sequence repressed the expression of the cgxA gene when inserted at the 5' end of the cgxA gene on pXAH, which was deleted for the repressive element from the promoter region.     

Autoregulation of the Pseudomonas aeruginosa protein PtxS occurs through a specific operator site within the ptxS upstream region

We have previously shown that the Pseudomonas aeruginosa toxA regulatory protein PtxS autoregulates its own synthesis by binding to a 52-bp fragment. The 3' end of the 52-bp fragment is located 58 bp 5' of the ptxS translation start site. We have identified a 14-bp palindromic sequence (TGAAACCGGTTTCA) within the 52-bp fragment. In this study, we used site-directed mutagenesis and promoter fusion experiments to determine if PtxS binds specifically to this palindromic sequence and regulates ptxS expression. We have also tried to determine the roles of specific nucleotides within the palindromic sequence in PtxS binding and ptxS expression. Initial promoter fusion experiments confirmed that the 52-bp fragment does not overlap with the region that carries the ptxS promoter activity. PtxS binding was eliminated upon the deletion of the 14-bp palindromic sequence from the 52-bp fragment. In addition, the deletion of the 14-bp sequence caused a significant enhancement in ptxS expression in the P. aeruginosa strain PAO1 and the ptxS isogenic mutant PAO::ptxS. Mutation of specific nucleotides within the 14-bp sequence eliminated, reduced, or had no effect on PtxS binding. However, mutations of several of these nucleotides produced a significant increase in ptxS expression in both PAO1 and PAO::ptxS. These results suggest that (i) the 14-bp palindromic sequence and specific nucleotides within it play a role in PtxS binding and (ii) deletion of the palindromic sequence or changing of certain nucleotides within it interferes with another mechanism that may regulate ptxS expression.     

Sinorhizobium meliloti fur-like (Mur) protein binds a fur box-like sequence present in the mntA promoter in a manganese-responsive manner

In Sinorhizobium meliloti, the Mur(Sm) protein, a homologue of the ferric uptake regulator (Fur), mediates manganese-dependent regulation of the MntABCD manganese uptake system. In this study, we analyzed Mur(Sm) binding to the promoter region of the S. meliloti mntA gene. We demonstrated that Mur(Sm) protein binds with high affinity to the promoter region of mntA gene in a manganese-responsive manner. Moreover, the results presented here indicate that two monomers, or one dimer, of Mur(Sm) binds the DNA. The binding region was identified by DNase I footprinting analysis and covers a region of about 30 bp long that contains a palindromic sequence. The Mur(Sm) binding site, present in the mntA promoter region, is similar to a Fur box; however, manganese-activated Mur(Sm) binds a canonical Fur box with very low affinity. Furthermore, the data obtained indicate that Mur(Sm) responds to physiological concentrations of manganese.     

High-resolution analysis of a histone H1 binding site in a rat albumin gene

Interaction of rat liver histone H1 fraction with the 5'-end of the rat serum albumin gene was localized within a 346 base pair (bp) restriction fragment. Sequence analysis of the fragment showed the fragment was 72 mol % adenosine-thymidine, which is significantly greater than the mole percent adenosine-thymidine composition of the rat genome. Gel retardation assays of the histone H1-DNA interaction indicate the complex formed behaves as previously characterized H1-DNA and shows a high-affinity H1 binding site within the enriched albumin restriction site. Deoxyribonuclease I (DNase I) protection assays on the H1 binding site define three protected regions only on the template strand of the DNA fragment. The three sites lie 55 and 110 bp apart (165 bp between the first and third binding site) with a consensus binding sequence of 5'-GA-ATA-CTGGCTT-C-TT-CTA-G-3'. The sequences between the protected DNA regions are highly enriched in adenosine-thymidine bases (79.3 and 86 mol % adenosine-thymidine, respectively). The functional significance is not understood.