# A TATA-box-binding protein binds single-stranded DNA in two modes: To poly(G) tracts and to flexible DNA regions

## TL;DR

This study reveals that a unique TATA-box-binding protein from Giardia binds to single-stranded DNA in two distinct ways, offering new insights into eukaryotic transcription.

## Contribution

The study identifies two novel ssDNA-binding modes of a divergent TBP from Giardia, validated through sequence design and competition experiments.

## Key findings

- gTBP binds to ssDNA with four or more consecutive guanine bases as an oligomer (A mode).
- gTBP binds to flexible ssDNA regions as a monomer (B mode), dependent on base stacking energy.
- Designed DNA sequences with similar stacking energy profiles competed with natural sequences for gTBP binding.

## Abstract

The TATA-box-binding protein (TBP) homolog from Giardia intestinalis (gTBP) is highly divergent, lacking key phenylalanine residues crucial for binding and unwinding double-stranded DNA. Surprisingly, we determined that gTBP exhibits unconventional DNA-binding properties and preferentially binds to single-stranded DNA (ssDNA) using a DNA-binding pocket that is narrower relative to other eukaryotic TBPs. Additionally, we showed that gTBP binds in two distinct modes, which we call the A and B modes, that are dependent on ssDNA sequence and protein concentration. For the A mode, gTBP binds as an oligomer to ssDNA that contains four or more consecutive guanine bases. For the B mode, using base stacking energy potentials between adjacent dinucleotides as a simple proxy for per-nucleotide flexibility, gTBP binds as a monomer to ssDNA in a manner that is dependent on DNA structural properties. To validate the latter concept, we designed de novo DNA sequences with base stacking energy profiles comparable to two DNA sequences that bind gTBP and showed that these designed sequences can compete for gTBP binding against the two original sequences. Overall, we present a potential new perspective on eukaryotic transcription regulation based on our findings around unconventional gTBP-ssDNA binding. A comprehensive understanding of the binding modes of gTBP could yield insights into Giardia’s biology and eukaryotic transcription in general.