Because the signal was found in so many diverse groups of bacteria, the researchers thought it might represent a universal regulatory mechanism. The next question was whether the signal was promoting or repressing expression of Nrd genes.
The team observed that their signal always overlapped with the promoter signal, the region of DNA required for the initiation of the conversion of gene to protein. Molecules that promote transcription recognize and bind to this sequence, which lies just outside of the gene. Repressor signals commonly work by allowing other proteins to bind on top of the promoter sequence and physically block promoters. Therefore, the duo predicted that the NrdR-box functioned as a repressor sequence.
Next, the researchers identified the transcription factor protein that binds to the NrdR-box. To do this, they used a bioinformatics approach they call phylogenetic profiling, compiling a list of genomes that clearly contained the NrdR-box and those that clearly did not have it. Then they searched the proteomes of 63 bacteria species, looking for proteins that strictly followed the same present-or-absent pattern as the NrdR-box. Only one protein cluster matched the pattern, and it represented a family of proteins that shared traits of transcription factors.
To strengthen the prediction that these proteins were the transcription factors that bind the NrdR-box, the team used another comparative genomic tool called positional clustering. Positional clustering takes advantage of the fact that functionally related gene sequences (such as the genes for Nrd and its transcription factor) frequently inhabit the same 'neighborhood' of the chromosome.
"If you are looking in one genome, many genes will be neighbors by coincidence," Gelfand noted. "But if tw
Source:Howard Hughes Medical Institute