Determination of the transcription factor TFIIB glycosylation profile by using bioinformatics and experimental methods

Transcription is the universal and key mechanism of life that occurs in all prokaryotes and eukaryotes. RNA polymerase II and transcription factors that interact with it are involved in transcription of all eukaryotic organism. Transcription factor TFIIB interacts with three structures at the initiation stage of transcription: the single end of TATA binding protein, TATA box followed by GC-rich DNA sequence, and C-terminal domain tail of RNA polymerase II. It has been known that RNA polymerase II is a glycoprotein and it contains O-GlcNAc sugar in the C-terminal domain at the initiation stage of transcription. However, it is not clear whether the transcription factor TFIIB interacting with RNA polymerase II is a glycoprotein or not. Also, data concerning the role of glycans in this interaction is quite limited. Thus, the first aim of the study is to determine the potential glycosylation positions (N- and O-linked glycosylation) of transcription factor TFIIB of nucleus proteins in various invertebrate and vertebrate species and to reveal conserved/differentiated amino acid sequences as well as their potential glycosylation profile amongst the species via bioinformatics. Taking into account the data obtained from bioinformatics, the second aim of the study is to determine experimentally of the potential glycosylation profiles (N- and/or O-) of TFIIB in the model organism sea urchin Paracentrotus lividus by using western/lectin blotting methods and TEM. For this purpose, TFIIB amino acid sequences of sea urchin Strongylocentrotus purpuratus, fish Oryzias latipes, frog Xenopus laevis, rat Rattusnorvegicus, mouse Mus musculus, bovine Bos taurus, orangutan Pongo pygmaeus abelii, and human Homo sapiens were used in bioinformatic studies. The construction of the secondary and three dimensional structure predictions of TFIIB protein, determination of the potential glycosylation positions and their accuracy analyzes, and the visualization of the relevant positions on the protein structure were performed using bioinformatic databases and servers. In experimental studies, egg cells from mature individuals of sea urchin P. lividus were used and western/lectin blotting and immune/lectin labelling by TEM were performed using the nuclear extract obtained. In addition to the determination of mucin type O-linked glycosylation, O-α-GlcNAcylation, O-β-GlcNAcylation, and ying-yang positions in the TFIIB protein of all studied species, N-linked glycosylation positions were also found in the TFIIB protein of sea urchin and frog as a result of bioinformatic studies. Furthermore, in the secondary and three dimensional structure predictions, changing in the protein structure from invertebrates to vertebrates was found. N-acetylglucosamine, Galactose, Mannose, and α2-3 linked Sialic acid were determined as a terminal glycan unit of the TFIIB protein in lectin blotting. The presence of α2-3 and α2-6 linked sialic acids in the TFIIB protein were found by using lectin labelling against sialic acids in TEM. With the findings obtained, it has been demonstrated for the first time that transcription factor TFIIB is a glycoprotein. It has also been determined that nucleocytoplasmic proteins may exhibit O- and/or N-linked glycosylation modifications as well as O-GlcNAc modification. With this finding, a new approach has been adopted to the glycosylation of nucleocytoplasmic proteins and prompted a new research question in the field of glycobiology. Thus, an alternative approach is presented for the classical viewpoint of molecular biology. Furthermore, a very practical and new method has been developed by us applying directly on the grid without sectioning in order to be used in TEM and used for the first time in this thesis.