Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.).
Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca(2+)-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5'-TWCG(C/T)GTKKKKTKCG-3' (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate beta-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.
Choi
Man Soo
MS
Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Korea.
Kim
Min Chul
MC
Yoo
Jae Hyuk
JH
Moon
Byeong Cheol
BC
Koo
Sung Cheol
SC
Park
Byung Ouk
BO
Lee
Ju Huck
JH
Koo
Yoon Duck
YD
Han
Hay Ju
HJ
Lee
Sang Yeol
SY
Chung
Woo Sik
WS
Lim
Chae Oh
CO
Cho
Moo Je
MJ
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000235:genetics; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D015723:Gene Library; D005966:Glucuronidase / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000737:chemistry* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016415:Sequence Alignment; D020080:Tandem Repeat Sequences; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D018744:DNA, Plant; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D005966:Glucuronidase