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bioRxiv · 10.1101/2022.08.20.504633

Biophysical Characterization of an Essential Mammalian Protein; Transcription Termination Factor I (TTF1)

Tiwari, K.·Singh, G.·Singh, S. K.
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Abstract

Mammalian Transcription Terminator Factor 1 (TTF1) is an essential protein which plays diverse cellular physiological functions like transcription regulation (both initiation and termination), replication fork blockage, chromatin remodelling, DNA damage repair etc. Hence, understanding the structure and mechanism conferred by its variable confirmations becomes significantly important. But so far, almost nothing is known about the structure of either the full-length protein or any of its domain in isolation. Moving towards achieving the above goals, our lab has codon optimised, expressed and purified N-terminal 190 amino acid deleted TTF1 ({Delta}N190TTF1) protein, since full length protein even after multiple trials could not be purified in soluble form. In this article, we have characterized this essential protein by studying its homogeneity, molecular size and secondary structure using tools like dynamic light scattering (DLS), circular dichroism (CD) spectroscopy, Raman spectroscopy and atomic force microscopy (AFM). By CD and DLS we have shown that the purified protein is homogenous and soluble. CD spectroscopy also revealed that {Delta}N190TTF1 is a helical protein which was further confirmed by analysis of Raman spectra and Amide I region deconvolution studies. AFM imaging data discovered the size of single protein molecule to be 94 nm which is in agreement with the size determined by the DLS study as well. Our structural and biophysical characterization of this essential protein will open avenues towards solving the structure to atomic resolution and also will encourage the research to investigate the mechanism behind its diverse functions attributed to its various domains.

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Tiwari, K., Singh, G., Singh, S. K.. 2022-08-21. Biophysical Characterization of an Essential Mammalian Protein; Transcription Termination Factor I (TTF1). https://doi.org/10.1101/2022.08.20.504633

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