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Selective enrichment of RNA species for tight binding to Escherichia coli rho factor
Author(s) -
Schneider Dan,
Gold Larry,
Platt Terry
Publication year - 1993
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.7.1.7678562
Subject(s) - rna , terminator (solar) , escherichia coli , population , biology , binding site , dissociation constant , nucleotide , termination factor , stem loop , systematic evolution of ligands by exponential enrichment , microbiology and biotechnology , chemistry , biophysics , biochemistry , genetics , rna polymerase , gene , physics , receptor , ionosphere , demography , astronomy , sociology
We have applied the SELEX procedure (systematic evolution of ligands by exponential enrichment) to obtain RNA molecules that bind tightly to the Escherichia coli transcription termination factor rho. The starting pool was a population of RNA molecules 77 nucleotides (nt) long, in which was embedded a cassette of 30 nt of randomized sequence. The apparent dissociation constant of this RNA pool for hexameric rho factor was about 1 μ M. After eight rounds of selection by filter binding, with RNA in either 10‐fold or 40 to 100‐fold excess at each step, the dissociation constant of the selected RNA had dropped by more than 500‐fold to about 1 nM. Analysis of 29 clonal isolates from the population revealed that five had K DS substantially weaker than 10 nM (presumably background carryover), 40% were C‐rich (as might have been predicted from rho's known substrate binding), and 40% had a strikingly preserved potential hairpin, in most cases of 6 base pairs with a 3 nt CAA loop and preceded by a CCCCA consensus. The rhodependent trp t' terminator region includes a related potential hairpin structure; however, it is energetically unfavorable. The implications of the sequence findings for elucidating both static and dynamic aspects of rho factor recognition and response to its RNA target site are discussed.— Schneider, D., Gold, L., Platt, T. Selective enrichment of RNA species for tight binding to Escherichia coli rho factor. FASEB J. 7: 201‐207; 1993.