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.     

Intracellular localization of GBF proteins and blue light-induced import of GBF2 fusion proteins into the nucleus of cultured Arabidopsis and soybean cells

The G-box is an important regulatory element found in the promoters of many different genes. Four members of an Arabidopsis gene family encoding basic leucine zipper proteins (GBFs) which bind the G-box have previously been cloned. To study GBFs, a polyclonal antibody was raised against GBF1 expressed in bacteria. This antibody also recognized GBF2 and GBFS. Immunoblot analysis of nuclear and cytoplasmic fractions from Arabidopsis and soybean (SB-M) cell cultures indicated that over 90% of proteins detected with anti-GBF1 were cytoplasmic. Electrophoretic mobility shift assays indicated that over 90% of G-box binding activity was cytoplasmic. DMA affinity chromatography demonstrated that each protein detected with anti-GBF1 specifically bound the G-box. To study individual GBFs, DNA constructs fusing GBF1, GBF2 and GBF4 to GUS were made and assayed by transient expression in SB-M protoplasts. Of GUS:GBF1 proteins, 50–62% were localized in the cytoplasm under all conditions tested, while 97% of GUS:GBF4 was localized in the nucleus. By contrast, whereas about 50% of GUS:GBF2 was found in the cytoplasm of dark-grown cells, over 80% of this protein was found in the nucleus in cells cultured under blue light. Deletion analysis of GBF1 identified a region between amino acids 112 and 164 apparently required for cytoplasmic retention. These results suggest the intriguing possibility that limitation of nuclear access may be an important control on GBF activity. In particular, GBF2 is apparently specifically imported into the nucleus in response to light.     

Heterodimerization between light-regulated and ubiquitously expressed Arabidopsis GBF bZIP proteins.

The promoters of a variety of plant genes are characterized by the presence of a G-box (CCACGTGG) or closely related DNA motifs. These genes often exhibit quite diverse expression characteristics and in many cases the G-box sequence has been demonstrated to be essential for expression. The G-box of the Arabidopsis rbcS-1A gene is bound by a protein, GBF, identified in plant nuclear extracts. Here we report the isolation of three Arabidopsis thaliana cDNA clones encoding GBF proteins referred to as GBF1, GBF2 and GBF3. GBF1 and GBF2 mRNA is present in light and dark grown leaves as well as in roots. In contrast, GBF3 mRNA is found mainly in dark grown leaves and in roots. The deduced amino acid sequences of the three cDNAs indicate that each encodes a basic/leucine zipper protein. In addition, all three proteins are characterized by an N-terminal proline-rich domain. Homodimers of the three proteins specifically recognize the G-box motif, with GBF1 and GBF3 binding symmetrically to this palindromic sequence. In contrast, GBF2 binds to the symmetrical G-box sequence in such a way that the juxtaposition of the protein and the DNA element is clearly asymmetric and hence distinct from that observed for the other two proteins. The fact that GBF1, GBF2 and GBF3 possess both distinct DNA binding properties and expression characteristics prompt us to entertain the notion that these proteins may individually mediate distinct subclasses of expression properties assigned to the G-box. Furthermore, we demonstrate that GBF1, GBF2 and GBF3 heterodimerize and these heterodimers also interact with the G-box, suggesting a potential mechanism for generating additional diversity from these GBF proteins.     

利用酵母双杂交技术筛选与AtbZIP1相互作用的蛋白质

碱性亮氨酸拉链bZIP类转录因子在植物的生长发育、光形态建成、光信号传导及非生物胁迫反应中发挥重要的作用.为研究AtbZIP1基因的作用机理,本研究首先验证了该基因的自激活转录活性,通过缺失突变确定了该转录因子的转录激活结构域;以AtbZIP1缺失突变体AtbZ3为诱饵蛋白,采用Matchmaker Gold Yeast Two-Hybrid System(Clonetch),共筛选获得5个与诱饵蛋白相互作用的蛋白质;并通过AbA(Aureobasidin A)抗生素标记基因,His营养缺陷和LacZ蓝白斑检测验证了阳性克隆.亚细胞定位分析发现,AtbZIP1蛋白除了定位于细胞核外,还定位于叶绿体细胞.通过分析这些靶蛋白的已知功能,为研究AtbZIP1蛋白的未知生物学功能提供重要信息.     

Viral SELEX reveals individual and cooperative roles of the C-box and G-box in HIV-2 replication

The 5' UTR of HIV-2 genomic RNA contains signaling motifs that regulate specific steps of the replication cycle. Two motifs of interest are the C-box and the G-box. The C-box is found in the 5' untranslated region upstream of the primer binding site, while the G-box is found downstream from the major splice donor site, encompassing the gag start codon and flanking nucleotides. Together the C-box and the G-box form a long-range base-pairing interaction called the CGI. We and others have previously shown that formation of the CGI affects RNA dimerization in vitro and the positions of the C-box and the G-box are suggestive of potential roles of the CGI in other steps of HIV-2 replication. Therefore, we attempted to elucidate the role of the CGI using a viral SELEX approach. We constructed proviral DNA libraries containing randomized regions of the C-box or G-box paired with wild-type or mutant base-pairing partners. These proviral DNA libraries were transfected into COS-7 cells to produce viral libraries that were then used to infect permissive C8166 cells. The "winner" viruses were sequenced and further characterized. Our results demonstrate that there is strong selective pressure favoring viruses that can form a branched CGI. In addition, we show that the mutation of the C-box alone can enhance RNA encapsidation, and mutation of the G-box can alter the levels of Gag protein isoforms. These results suggest coordinated regulation of RNA translation, dimerization, and encapsidation during HIV-2 replication.