Identification and Characterization of Preferred DNA-Binding Sites for the Thermus thermophilus HB8 Transcriptional Regulator TTHA0973.

James Shell Cox,Michael W Van Dyke,Mykala Mckinnes,Kristi Moncja

doi:10.3390/ijms20133336

Abstract

Advances in genomic sequencing have allowed the identification of a multitude of genes encoding putative transcriptional regulatory proteins. Lacking, often, is a fuller understanding of the biological roles played by these proteins, the genes they regulate or regulon. Conventionally this is achieved through a genetic approach involving putative transcription factor gene manipulation and observations of changes in an organism’s transcriptome. However, such an approach is not always feasible or can yield misleading findings. Here, we describe a biochemistry-centric approach, involving identification of preferred DNA-binding sequences for the Thermus thermophilus HB8 transcriptional repressor TTHA0973 using the selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), massively parallel sequencing, and bioinformatic analyses. We identified a consensus TTHA0973 recognition sequence of 5′–AACnAACGTTnGTT–3′ that exhibited nanomolar binding affinity. This sequence was mapped to several sites within the T. thermophilus HB8 genome, a subset of which corresponded to promoter regions regulating genes involved in phenylacetic acid degradation. These studies further demonstrate the utility of a biochemistry-centric approach for the facile identification of potential biological functions for orphan transcription factors in a variety of organisms.

Highlights

Transcriptional regulation is the primary means by which most organisms, both prokaryotic and eukaryotic, control gene expression
We investigated the T. thermophilus HB8 transcription factor TTHA0973, which had been previously investigated by more conventional approaches [11]
Following REPSA selection, we isolated a library of DNA sequences that exhibited substantial TTHA0973-dependent cleavage inhibition by the type IIS restriction endonuclease BpmI. Sequencing these and performing Multiple Em for Motif Elicitation (MEME) motif elicitation yielded consensus sequences that were merged to a single consensus sequence, 5 –AACnAACGTTnGTT–3

Summary

Introduction

Transcriptional regulation is the primary means by which most organisms, both prokaryotic and eukaryotic, control gene expression. Scanning for consensus sequences throughout an organism’s genome, those intergenic regions most likely involved in promoting transcription, can yield a panel of genes potentially regulated by a particular transcription factor Bioinformatic analyses of these genes and downstream members of their operons, ranging from identification of homologous domains with known functions in their encoded proteins to phenotypic changes in response to environmental changes, can provide important clues as to the biological roles of an unknown transcription factor. We use the combinatorial selection method, Restriction Endonuclease Protection, Selection and Amplification (REPSA), massively parallel sequencing, and de novo motif discovery to determine a consensus DNA-binding sequence for a putative transcription factor [6,7,8] This is followed by motif scanning within the subject genome and bioinformatic analyses to identify potential regulated genes and their possible biological functions. This study provides us with a better understanding of the strengths and weaknesses of a biochemistry-centric approach for investigating transcription factors and will help shape future studies on other uncharacterized, orphan transcription regulatory proteins

REPSA Selection of TTHA0973-Binding DNAs

Discussion

Oligonucleotides

Protein Expression and Purification

Transcription Factor Consensus Sequence Determination

Protein-DNA Binding Assays

Bioinformatic Determination of Candidate Regulated Genes