Genetic assays to define and characterize protein-protein interactions involved in gene regulation

Nickels, BE. 2009.

Journal:

Methods

Volume Number:

Pages:

53-62

Abstract:

Transcription can be regulated during initiation, elongation, and termination by an enormous variety of regulatory factors. A critical step in obtaining a mechanistic understanding of regulatory factor function is the determination of whether the regulatory factor exerts its effect through direct contact with the transcription machinery. Here I describe the application of a transcription activation-based bacterial two-hybrid assay that is useful for the identification and genetic dissection of protein-protein interactions involved in gene regulation. I provide examples of how this two-hybrid system can be adapted for the study of "global" regulatory factors, sequence-specific DNA-binding proteins, and interactions that occur between two subunits of RNA polymerase (RNAP). These assays facilitate the isolation and characterization of informative amino acid substitutions within both regulatory factors and RNAP. Furthermore, these assays often enable the study of substitutions in essential domains of RNAP that would be lethal in their natural context.

Notes:

Nickels, Bryce EResearch Support, Non-U.S. Gov'tReviewUnited StatesMethods (San Diego, Calif.)Methods. 2009 Jan;47(1):53-62. Epub 2008 Oct 24.

Related External URL:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18952173

Citation:: Nickels, BE. 2009. Genetic assays to define and characterize protein-protein interactions involved in gene regulation. Methods. 47:53-62.

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Recent Publications

Berdygulova, Z, Esyunina D, Miropolskaya N, Mukhamedyarov D, Kuznedelov K, Nickels BE, Severinov K, Kulbachinskiy A, Minakhin L. 2012. A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing.. Nucleic acids research. Abstractnucleic_acids_res_2012_berdygulova.pdf

Gp39, a small protein encoded by Thermus thermophilus phage P23-45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity.

Vvedenskaya, IO, Sharp JS, Goldman SR, Kanabar PN, Livny J, Dove SL, Nickels BE. 2012. Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs. Genes Dev. 26(13):1498-1507. Abstractgenes_dev_2012_vvedenskaya.pdf

Prokaryotic and eukaryotic RNA polymerases can use 2- to ~4-nt RNAs, ‘‘nanoRNAs,’’ to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.

Weiss, LA, Harrison PG, Nickels BE, Glickman MS, Campbell EA, Darst SA, Stallings CL. 2012. Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis. J Bacteriol. 194:5621-31. Abstractj_bacteriol_2012_weiss.pdfWebsite

Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP beta subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP beta interactions demonstrates that the CarD/RNAP beta association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP beta interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP beta interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding.

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