Of Schizosaccharomyces Pombe Trz1p Nuclear Localization Sequence Further Study

Endonuclease tRNase Z catalyzes the generation of the mature 3'end of tRNA precursors through specific endonucleolytic cleavage. tRNase Z enzymes belong to the super-family of metallo-β-lactamases (MBL). In all kinds of organisms, tRNase Z can be divided into long form (tRNase ZL) and short form (tRNase ZS). There are different kinds and numbers of tRNase Zs in different organisms. This subject mainly focuses on the tRNase Z's function in Schizosaccharomyces pombe. In S.pombe, there are two tRNase ZL genes:sptrzl+(SPAC1D4.10) and sp?rz2+(SPBC3D6.03c). We mainly further analyzes the nuclear localization signal (NLS) in this subject:using EGFP as a tracer protein, we cut the gene into a number of truncation mutants. Experiment results show that the fusion proteins containing KKRK sequence is localized in nucleus, but the fusion proteins not containing KKRK sequence is localized in nucleus and cytoplasm. So, we can conclude that the sptrzl is localized in nucleus and its function mostly plays in nucleus and show that 208KKRK211 in the N-terminal of SpTrzlp plays an important role in its nuclear import. In the same time, by synthesis oligonucleotide and truncated proteins, we identified the KKRK sequence is the key nuclear localization sequence in the sptrzl+and roughly identified the FKKRKHENINRG is the most likely sptrzl+nuclear localization sequence.The nuclear and mitochondrial tRNA processing is different. We have identified the sptrz2" is localized in mitochondria, and we mainly study the function of sptrz2+ In the other side, by generating a temperature-sensitive mutant, we wish to solve some questions in mitochondrial processing of tRNA 3'end. The pcdl+gene may be involved in apoptosis. However, there are more and more data to show that this protein seems not have connect with apoptosis but the cell development and the cell cycle, even causes some diseases, so, we wish to study the function of pcdl+ gene. We intend to generate a temperature-sensitive mutant to study its function. To our disappointed, we did not successfully generate a temperature-sensitive mutant caused by pcdl+mutation. In the other side, we know some appropriate conditions about how to easily generate a temperature-sensitive mutant, this also have significance for future work.