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

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 (∆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 ∆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.

Structural Characterization of DNA Binding Domain of Essential Mammalian Protein TTF 1

“…OriginPro software was used to map and evaluate the same (Signorelli et al, 2017). Protocol as described by Maiti et al, 2004 was used for curve fitting of the amide I band and its assignment to the structural components using the Origin software (Tiwari et al, 2023).…”

“…Terminator proteins that mediate transcription termination have been found in a variety of taxa which includes mammalian TTF1, Nsi1 [also known as yeast transcription terminator1 (Ytt1)] of S. cerevisiae, Reb1 (RNA polymerase I enhancer binding protein) of Schizosaccharomyces pombe, and Rib2 of Xenopus (Jaiswal et al, 2016) Transcription Termination Factor 1 (TTF1) is a multifunctional nucleolar DNA-binding protein that is involved in various processes, such as Pol I-mediated transcription initiation (Németh et al, 2008) and termination (Grummt et al, 1986), pre-rRNA processing, chromatin remodelling, DNA damage repair (Tiwari. K. et al, 2023), and polar replication fork arrest (Németh et al, 2013).…”

“…Some important TTF1 interacting protein are Cockayne Syndrome group B (CSB), Murine Double Minute 2 (MDM2), Alternative Reading Frame (ARF p19 ARF in mouse and p14 ARF in human), TTF1 interacting protein 5 (Tip5), Nucleosome remodelling and histone deacetylase complex (NuRD), p300/CBP associated factor (PCAF), Polymerase 1 and transcript release factor (PTRF), and nucleophosmin NPM/ B23. Our lab has recently identified DDB1 as a novel interacting partner of TTF1 and its role in UV-mediated DNA damage sensing (Németh et al, 2013;Tiwari. K. et al, 2023)The C-terminal region, besides being highly conserved, plays an important role in DNA binding and interacts with various proteins to perform the essential functions required for cell growth and proliferation.…”