Glycosylation analysis of transcription factor TFIIB using bioinformatics and experimental methods.

Abstract

Transcription is a fundamental process involving the interaction of RNA polymerase II and related transcription factors. TFIIB is a transcription factor that plays a significant role in the formation and stability of the preinitiation complex in a precise orientation, as well as in the control of initiation and pre-elongation steps. At the initiation step, TFIIB interacts with three structures: the end of the TATA-binding protein, a GC-rich DNA sequence followed by the TATA box, and the C-terminal domain of RNA polymerase II. It is known that RNA polymerase II is a glycoprotein and contains O-GlcNAc sugar at the C-terminal domain during the initiation stage of transcription. However, it is unclear whether the transcription factors interacting with RNA polymerase II are glycoproteins or not. The study aims to determine the glycosylation (N- and/or O-linked glycosylations) of TFIIB by using bioinformatics in one invertebrate and seven vertebrate species and experimental methods in the sea urchin Paracentrotus lividus oocyte. Both bioinformatics and experimental analysis have shown that TFIIB is a glycoprotein. In addition, PNGase-F enzyme treatment, lectin blotting, and colloidal-gold conjugated lectin labeling results revealed that TFIIB contains O-linked GalNAc, mannose, GlcNAc, and α-2,3-linked sialic acid. Based on our results, we suggest that glycosylation modification may be involved in the transcription mechanism of the TFIIB protein.