Mechanism of lac repressor function EMBO J 2011.pdfVIP

Mechanism of transcriptional repression at a bacterial promoter by analysis of single molecules Alvaro Sanchez1,4, Melisa L Osborne2, Larry J Friedman2, Jane Kondev3,* and Jeff Gelles2,* 1Graduate program in Biophysics and Structural Biology, Brandeis University, Waltham, MA, USA, 2Department of Biochemistry, Brandeis University, Waltham, MA, USA and 3Department of Physics, Brandeis University, Waltham, MA, USA The molecular basis for regulation of lactose metabolism in Escherichia coli is well studied. Nonetheless, the physi- cal mechanism by which the Lac repressor protein pre- vents transcription of the lactose promoter remains unresolved. Using multi-wavelength single-molecule fluorescence microscopy, we visualized individual com- plexes of fluorescently tagged RNA polymerase holoen- zyme bound to promoter DNA. Quantitative analysis of the single-molecule observations, including use of a novel statistical partitioning approach, reveals highly kinetically stable binding of polymerase to two different sites on the DNA, only one of which leads to transcription. Addition of Lac repressor directly demonstrates that bound repressor prevents the formation of transcriptionally productive open promoter complexes; discrepancies in earlier studies may be attributable to transcriptionally inactive polymer- ase binding. The single-molecule statistical partitioning approach is broadly applicable to elucidating mechanisms of regulatory systems including those that are kinetically rather than thermodynamically controlled. The EMBO Journal (2011) 30, 3940–3946. doi:10.1038/ emboj.2011.273; Published online 9 August 2011 Subject Categories: chromatin transcription Keywords: Lac promoter; open complexes; single-molecule imaging Introduction In all organisms, messenger RNAs are synthesized by a multi- subunit RNA polymerase (RNAP) that binds to promoter regions of DNA, separates the DNA strands to form an ‘open’ promoter complex and then escapes from the promoter, moving along the