Investigation On The Roles Of A SAGA Component, Tra1, In Transcriptional Initiation

In the nucleus of eukaryotic cells, DNA associates with histone proteins to form nucleosomes, which are the fundamental repeating unit of chromatin. Nucleosomes have the potential to inhibit transcription mainly by blocking binding of transcription factors to their cognate DNA sites. Thus, transcription often needs some factors including coactivators to overcome a formidable obstacle posed by nucleosomes. At first, transcription factors bind to target gene's upstream activation sequences, and then recruit coactivators such as SAGA, SWI/SNF to facilitate TBP, general transcription factors, RNA polII binding to promoter and forming PIC. It is thought that Tral , a subunit of SAGA complex , can bridge SAGA complex and transcription factor. Based on our previous experimental results, we introduced point mutations and deletion mutations in the domain of Tral protein possibly involved in the interaction with transcription factor Gcn4. This work sets the background to continue our investigation on the target genes regulated by Tral and the physiological effects influenced by Tral protein on the cellular processes such as cell growth, cell cycle, and DNA repair. Our experiment show that two of the point mutations have vast influenced to cell growth, and they are temperature sensitive. We cloned two yeast genes which are regulated by Tral in human, and we cloned and expressed 11 genes of SGAG and NuA4 complxes. With the availability of these Tral mutations, two target genes and 11 expressed proteins we will perform some biochemical studies to examine the roles of Tral in the assembly of SAGA complex and in transcriptional initiation.