Identification of a calmodulin-binding NAC protein as a transcriptional repressor in Arabidopsis.
Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of proteins. However, little is known about how CaM directly regulates transcription. Screening of an Arabidopsis cDNA expression library using horseradish peroxidase-conjugated calmodulin as a probe identified a calmodulin-binding NAC protein (CBNAC). Using gel overlay assays, a Ca2+-dependent CaM-binding domain was identified in the C terminus of this protein. Specific binding of CaM to CaM-binding domain was corroborated by site-directed mutagenesis and a split-ubiquitin assay. Using a PCR-mediated random binding site selection method, we identified a DNA-binding sequence (CBNACBS) for CBNAC, which consisted of a GCTT core sequence flanked on both sides by other frequently repeating sequences (TTGCTTANNNNNNAAG). CBNAC was able to bind to CBNACBS, which resulted in the repression of transcription in Arabidopsis protoplasts. Interestingly, the transcriptional repression mediated by CBNAC was enhanced by CaM. These results suggest that CBNAC may be a CaM-regulated transcriptional repressor in Arabidopsis.
Kim
Ho Soo
HS
Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Korea.
Park
Byung Ouk
BO
Yoo
Jae Hyuk
JH
Jung
Mi Soon
MS
Lee
Sang Min
SM
Han
Hay Ju
HJ
Kim
Kyung Eun
KE
Kim
Sun Ho
SH
Lim
Chae Oh
CO
Yun
Dae-Jin
DJ
Lee
Sang Yeol
SY
Chung
Woo Sik
WS
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D020218:Response Elements / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology
D029681:Arabidopsis Proteins; C577034:CBNAC protein, Arabidopsis; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins