A whole-genome approach to identifying protein binding sites: Promoters in Methanocaldococcus (Methanococcus) jannaschii

Enhu Li; Claudia I. Reich; Gary J. Olsen

doi:10.1093/nar/gkm499

A whole-genome approach to identifying protein binding sites: Promoters in Methanocaldococcus (Methanococcus) jannaschii

Enhu Li, Claudia I. Reich, Gary J. Olsen

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Abstract

We have adapted an electrophoretic mobility shift assay (EMSA) to isolate genomic DNA fragments that bind the archaeal transcription initiation factors TATA-binding protein (TBP) and transcription factor B (TFB) to perform a genome-wide search for promoters. Mobility-shifted fragments were cloned, tested for their ability to compete with known promoter-containing fragments for a limited concentration of transcription factors, and sequenced. We applied the method to search for promoters in the genome of Methanocaldococcus jannaschii. Selection was most efficient for promoters of tRNA genes and genes for several presumed small noncoding RNAs (ncRNA). Protein-coding gene promoters were dramatically underrepresented relative to their frequency in the genome. The repeated isolation of these genomic regions was partially rectified by including a hybridization-based screening. Sequence alignment of the affinity-selected promoters revealed previously identified TATA box, BRE, and the putative initiator element. In addition, the conserved bases immediately upstream and downstream of the BRE and TATA box suggest that the composition and structure of archaeal natural promoters are more complicated.

Original language	English (US)
Pages (from-to)	6948-6958
Number of pages	11
Journal	Nucleic acids research
Volume	36
Issue number	22
DOIs	https://doi.org/10.1093/nar/gkm499
State	Published - Dec 2008

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Genetics

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title = "A whole-genome approach to identifying protein binding sites: Promoters in Methanocaldococcus (Methanococcus) jannaschii",

abstract = "We have adapted an electrophoretic mobility shift assay (EMSA) to isolate genomic DNA fragments that bind the archaeal transcription initiation factors TATA-binding protein (TBP) and transcription factor B (TFB) to perform a genome-wide search for promoters. Mobility-shifted fragments were cloned, tested for their ability to compete with known promoter-containing fragments for a limited concentration of transcription factors, and sequenced. We applied the method to search for promoters in the genome of Methanocaldococcus jannaschii. Selection was most efficient for promoters of tRNA genes and genes for several presumed small noncoding RNAs (ncRNA). Protein-coding gene promoters were dramatically underrepresented relative to their frequency in the genome. The repeated isolation of these genomic regions was partially rectified by including a hybridization-based screening. Sequence alignment of the affinity-selected promoters revealed previously identified TATA box, BRE, and the putative initiator element. In addition, the conserved bases immediately upstream and downstream of the BRE and TATA box suggest that the composition and structure of archaeal natural promoters are more complicated.",

author = "Enhu Li and Reich, {Claudia I.} and Olsen, {Gary J.}",

note = "Funding Information: We thank Ying Jiang for sharing her estimates of in vivo transcription factor concentrations. We thank Jian Zhang, Ying Jiang and other members of the laboratory for helpful discussions. We thank two anonymous referees for their insightful and helpful critiques. This work was supported by grants from the US Department of Energy (DE-FG02-01ER63201) and NASA (NAG 5-12334) to G.J.O. We thank Dr. Carl R. Woese for his help in supporting E.L. by sharing funds from the Stanley O. Ikenberry Chair, a position that he holds at the University of Illinois. Funding to pay the Open Access publication charges for this article has been waived by Oxford University Press.",

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A whole-genome approach to identifying protein binding sites: Promoters in Methanocaldococcus (Methanococcus) jannaschii

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