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

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

Research output: Contribution to journalArticle

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 languageEnglish (US)
Pages (from-to)6948-6958
Number of pages11
JournalNucleic acids research
Volume36
Issue number22
DOIs
StatePublished - Dec 1 2008

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Methanocaldococcus
Protein Binding
TATA Box
Transcription Factors
Binding Sites
Genome
TATA-Box Binding Protein
Small Untranslated RNA
Peptide Initiation Factors
Sequence Alignment
Electrophoretic Mobility Shift Assay
Transfer RNA
Genes
DNA
Proteins

ASJC Scopus subject areas

  • Genetics

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A whole-genome approach to identifying protein binding sites : Promoters in Methanocaldococcus (Methanococcus) jannaschii. / Li, Enhu; Reich, Claudia I.; Olsen, Gary J.

In: Nucleic acids research, Vol. 36, No. 22, 01.12.2008, p. 6948-6958.

Research output: Contribution to journalArticle

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