Effect Of The Sencond Domain Deletion On The Interaction Between Yelp₃ And Elp₄ And Other Proteins

Eukaryotic RNA polymerase II holoenzyme to exercise their functions, need many proteins present together in a large complex. In 1999 Elongator complex was first isolated from a high degree of phosphorylation of RNA polymerase II. This complex has been shown to be important for transcription elongation through a chromatin template. It consists of six subunits, namely elp1, elp2, elp3, elp4, elp5, elp6. The most striking feature of Elp3 is its histone acetyltransferase activity, it belongs to the GCN5-related N-acetyltransferase family. Deletion of elp3 gene can lead to a variety of growth defects, many of them related with the transcription elongation. It was reported that Elp3 has a crucial role in neurobiology. Elongator complex can acetylate histone H3 lysine 14 and histone H4 lysine 8 and the conserved HAT domain of Elp3 is necessary for its function. Bioinformatics analysis shown that Elp3 has a N-terminal S-adenosyl-L-methionine kinase domain, it has the Radical SAM family of sequence homology, which includes Fe4S4 clusters, can be combined with S-adeno - glucoside methionine. According to reports, the structure domain-binding S-adenosylmethionine has changed since the clustering of the iron-sulfur EPR spectra, through the directed mutagenesis to determine whether the clustering of cysteine residues in content, to prove that it can be decomposed into S-adenosyl-methionine. So in addition to the HAT activity, Elp3 may also have an unknown function of the catalytic domain.Yeast Elp3, the important catalytic subunit Elp3, possessesing histone acetyltransferase (HAT) domain and SAM-like (SAM radical) in its N- and C- terminal, both of them is essential for Elongator function. Elp3, existing both in cell nucleus and cytoplasm, plays an important role in transcription elongating by acetylating the highly conserved lysine residues in N-terminal tails of histones in cell nucleus and takes part in exocytosis and tRNA modification in cytoplasm and relevant to acetylation of the a-tublin. The current opinion on the function of Elp3 N-terminal SAM like domain is not yet uniform. Paraskevopoulo? and others confirmed that the second domain of Elp3 can be combined SAM, that it may have histone demethylase activity predicted by bioinformatics analysis. While the Greenwood and others believe that the main function of the second domain, reflected in the structure shall be necessary for the integrity of Elongator complex, which by itself has no catalytic activity. Therefore, the function of second domain in Elp3 remains to be confirmed by further studies.Data has been shown that deletion of the second domain in yeast Elp3 N terminal can signifycantly affect its function. As we all know protein has no function without interaction with others and protein-protein interactions operate at almost every level of cellular functions. In this thesis we study the function of Elp3 second domain through detect the effect of deletion this domain on the Elp3-Elp4 or CTK1 interaction by yeast two-hybrid and Pull Down Assay. Our result shown that the second domain deletion Elp3 mutant lost the ability to interact with human and yeast Elp4, CTK1. It suggests that the second domain in Elp3 N-terminal, possessing structure function in conformation of holo-Elongator complex and interaction with other proteins, is essential for function of Elp3. This study adds new knowledge of Elp3 function and benefits for further study on the molecular mechanism of Elp3 function.