RPL32-2 Of S.pombe Is A Potential Transcriptional Regulator

The ribosomal protein L32 is an extra-ribosomal function protein. It has become increasingly clear that appears to interact with three distinct RNA molecules to influence three different elements of RNA processing and function. We used the one-hybrid screen method rapidly screened a potential transcriptional regulator from cDNA library of S.pombe and used domain swap technique identified transcriptional regulator of yeast. It is ribosomal protein L32-2 (RPL32-2) of S.pombe has a DNA binding and potential transcriptional regulation function.1. We prepared enrichment mRNA of S.pombe and constructed a completed cDNA library in inserted pGADT7 vector. CaM was used to bait that was cloned from S.pombe and screen new CaM binding proteins from cDNA library to basic two-hybrid system. There are 64 clones were isolated primary and repeated co-transformation to confirm positive clones. We chose 10 clones and sequenced to verify putative positive clones. In these 10 positive clones, we found that contain three CaM binding protein genes known (glyceradehy-3-phosphate dehydrogenase, CaMK III or elongation factor 2 and phosphoglycerate mutase), a disturbed code Moel gene, and a RPL32-2 gene clone. Our date shown that RP132-2 could induce expression of reporter genes without CaM in yeast two-hybrid system. We blasted the amino acid sequence of RPL32-2 and found it has homology with few zinc finger proteins in a certain sense. We suggest that RPL32-2 has a new potential function in transcription regulation.2. The domain swap experiment shown that RPL32-2 has the functions both in binding DNA and transcriptional activation. These functions were lost by truncated its C or N terminal. This result suggested that RPL32-2 of S.pombe is not an independently DNA-binding domain or transcription-activating domain.3. We confirmed the sequence of RPL32-2 consensus binding site is GTTGGT by random PCR. The six nucleotides binding affinity was analyzed with the high concentration of the mutational site probes. The result of binding competition was...