TATA Binding Proteins Can Recognize Nontraditional DNA Sequences

Abstract

We demonstrate an accurate, quantitative, and label-free optical technology for high-throughput studies of receptor-ligand interactions, and apply it to TATA binding protein (TBP) interactions with oligonucleotides. We present a simple method to prepare single-stranded and double-stranded DNA microarrays with comparable surface density, ensuring an accurate comparison of TBP activity with both types of DNA. In particular, we find that TBP binds tightly to single-stranded DNA, especially to stretches of polythymine (poly-T), as well as to the traditional TATA box. We further investigate the correlation of TBP activity with various lengths of DNA and find that the number of TBPs bound to DNA increases >7-fold as the oligomer length increases from 9 to 40. Finally, we perform a full human genome analysis and discover that 35.5% of human promoters have poly-T stretches. In summary, we report, for the first time to our knowledge, the activity of TBP with poly-T stretches by presenting an elegant stepwise analysis of multiple techniques: discovery by a novel quantitative detection of microarrays, confirmation by a traditional gel electrophoresis, and a full genome prediction with computational analyses.

Interferometric Reflectance Imaging Sensor (IRIS)—A Platform Technology for Multiplexed Diagnostics and Digital Detection

“…A deeper understanding of these interactions involving TFs can shed light on the eukaryotic transcription initiation processes, hence the resultant gene expression regulations. With this goal in mind, IRIS has been used to study those interactions in [ 19 ]. Specifically, the study explores TATA binding protein (TBP), a key TF that takes part in the initiation of eukaryotic transcription.…”

Multiple novel promoter-architectures revealed by decoding the hidden heterogeneity within the genome

“…But as per our knowledge, this feature has not been detected in the human genome at this scale. ( 38 ) showed that TATA-binding proteins can bind poly- tracts and that a small fraction of human core promoters contain a TATA-box followed by a -rich region upstream of the TSS.…”

A High-Throughput Method to Examine Protein-Nucleotide Interactions Identifies Targets of the Bacterial Transcriptional Regulatory Protein Fur

“…To define the gonococcal Fur regulon more efficiently, we developed a microarray-based, label-free quantitative method utilizing IRIS to screen sequence-specific interactions of Fur to dsDNA in vitro [5], [34]. Combining this approach with bioinformatical analysis of the gonococcal genome and transcriptional analysis of N. gonorrhoeae wild type, fur mutant and fur complemented strains, we identified new Fur regulated genes.…”

“…IRIS can be broadly applied to characterize many types of biomolecular interactions, such as DNA-DNA hybridization, protein-protein binding, and protein-DNA interaction in a quantitative format [1] , [2] , [3] , [4] . Specifically, this technique has previously demonstrated utility for probing transcription factor interactions with arrayed oligonucleotides [5] . Bacterial transcription factors control tight regulation of gene expression in response to host specific environmental niches in a number of human pathogenic bacteria.…”

“…The Fur regulon of N. gonorrhoeae has not been well defined due to the lack of a fur mutant strain and an efficient and rapid method to characterize the interactions between the gonococcal Fur protein and predicted Fur binding sequences. To define the gonococcal Fur regulon more efficiently, we developed a microarray-based, label-free quantitative method utilizing IRIS to screen sequence-specific interactions of Fur to dsDNA in vitro [5] , [34] . Combining this approach with bioinformatical analysis of the gonococcal genome and transcriptional analysis of N. gonorrhoeae wild type, fur mutant and fur complemented strains, we identified new Fur regulated genes.…”