The gatekeeper residue controls autoactivation of ERK2 via a pathway of intramolecular connectivity.

Proc Natl Acad Sci U S A. 2006 Nov 28; 103:18101-18106.

Emrick MA, Lee T, Starkey PJ, Mumby MC, Resing KA, Ahn NG.

Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309, USA.

Studies of protein kinases have identified a "gatekeeper" residue, which confers selectivity for binding nucleotides and small-molecule inhibitors. We report that, in the MAP kinase ERK2, mutations at the gatekeeper residue unexpectedly lead to autoactivation due to enhanced autophosphorylation of regulatory Tyr and Thr sites within the activation lip that control kinase activity. This occurs through an intramolecular mechanism, indicating that the gatekeeper residue indirectly constrains flexibility at the activation lip, precluding access of the phosphoacceptor residues to the catalytic base. Other residues that interact with the gatekeeper site to form a hydrophobic cluster in the N-terminal domain also cause autoactivation when mutated. Hydrogen-exchange studies of a mutant within this cluster reveal perturbations in the conserved DFG motif, predicting a route for side chain connectivity from the hydrophobic cluster to the activation lip. Mutations of residues along this route support this model, explaining how information about the gatekeeper residue identity can be transmitted to the activation lip. Thus, an N-terminal hydrophobic cluster that includes the gatekeeper forms a novel structural unit, which functions to maintain the "off" state of ERK2 before cell signal activation.

B2 TFIIA changes the conformation of the DNA in TBP/TATA complexes and increases their kinetic stability.

J Mol Biol. 2007 Sep 21; 372:619-632.

Hieb AR, Halsey WA, Betterton MD, Perkins TT, Kugel JF, Goodrich JA.

Department of Chemistry and Biochemistry, University of Colorado at Boulder, 215 UCB, Boulder, Colorado 80309-0215, USA.

Eukaryotic mRNA transcription by RNA polymerase II is a highly regulated complex reaction involving numerous proteins. In order to control tissue and promoter specific gene expression, transcription factors must work in concert with each other and with the promoter DNA to form the proper architecture to activate the gene of interest. The TATA binding protein (TBP) binds to TATA boxes in core promoters and bends the TATA DNA. We have used quantitative solution fluorescence resonance energy transfer (FRET) and gel-based FRET (gelFRET) to determine the effect of TFIIA on the conformation of the DNA in TBP/TATA complexes and on the kinetic stability of these complexes. Our results indicate that human TFIIA decreases the angle to which human TBP bends consensus TATA DNA from 104 degrees to 80 degrees when calculated using a two-kink model. The kinetic stability of TBP/TATA complexes was greatly reduced by increasing the KCl concentration from 50 mM to 140 mM, which is more physiologically relevant. TFIIA significantly enhanced the kinetic stability of TBP/TATA complexes, thereby attenuating the effect of higher salt concentrations. We also found that TBP bent non-consensus TATA DNA to a lesser degree than consensus TATA DNA and complexes between TBP and a non-consensus TATA box were kinetically unstable even at 50 mM KCl. Interestingly, TFIIA increased the calculated bend angle and kinetic stability of complexes on a non-consensus TATA box, making them similar to those on a consensus TATA box. Our data show that TFIIA induces a conformational change within the TBP/TATA complex that enhances its stability under both in vitro and physiological salt conditions. Furthermore, we present a refined model for the effect that TFIIA has on DNA conformation that takes into account potential changes in bend angle as well as twist angle.

Figure. A two-kink model used to evaluate the bending of DNA by TBP. (a) FRET increases as TBP is titrated into reactions containing TATA-14. TBP was titrated from 0.08 nM to 60 nM using 1 nM TATA-14. Reactions were analyzed in a 384 well borosilicate microplate using a Typhoon fluorimager. Donor (green), FRET (red), and the pseudo-color overlay of the FRET and donor signals are shown. (b) FRET efficiency increases due to an increase in the concentration of TBP. FRET efficiency was calculated at each concentration of TBP. Data points are the average of three experiments and error bars represent one standard deviation. (c) Two-kink bend model used to calculate the angle (α) to which TBP bends TATA DNA. The DNA is shown as a red rod consisting of three segments with lengths (L1, L2, and L3). The upper panel shows linear DNA and the sequence of the DNA construct. Ru is the measured end-to-end length of the unbound DNA. The lower panel shows bent DNA. Rb is the measured end-to-end length of the bound DNA.

Metal ion dependence, thermodynamics, and kinetics for intramolecular docking of a GAAA tetraloop and receptor connected by a flexible linker.

Biochemistry. 2006 Mar 21; 45:3664-3673.

Downey CD, Fiore JL, Stoddard CD, Hodak JH, Nesbitt DJ, Pardi A.

Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309, USA.

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH(3))(6)(3+)] << [Ca(2+)], [Mg(2+)], [Mn(2+)] << [Na(+)], [K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.

Figure. Specific inhibition of GAAA tetraloop-receptor docking for the A14 construct. (a) Schematic of inhibition of docking by a dT14 or rU14 oligonucleotide complementary to the single-stranded linker. (b) Ensemble FRET measurements of titration of the A14 construct with dT14 () and rU14 (). Error bars are approximately as large as the symbols. The solid lines represent fits to a binding isotherm (eq 2). (c) Single-molecule raster-scanned images of the A14 construct at 10 mM Mg2+, plotted in false color with donor fluorescence in green and acceptor fluorescence in red. The same RNA molecules are being observed in each image. The RNAs are primarily docked in the absence of inhibitor (left), undocked after flushing with 10 M dT14 (center), and docked again after flushing with 10 M dA14 (right).

Structure of the S-adenosylmethionine riboswitch regulatory mRNA element.

Nature. 2006 Jun 29;441(7097):1172-5.

Montange RK, Batey RT.

Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309, USA.

Riboswitches are cis-acting genetic regulatory elements found in the 5'-untranslated regions of messenger RNAs that control gene expression through their ability to bind small molecule metabolites directly. Regulation occurs through the interplay of two domains of the RNA: an aptamer domain that responds to intracellular metabolite concentrations and an expression platform that uses two mutually exclusive secondary structures to direct a decision-making process. In Gram-positive bacteria such as Bacillus species, riboswitches control the expression of more than 2% of all genes through their ability to respond to a diverse set of metabolites including amino acids, nucleobases and protein cofactors. Here we report the 2.9-angstroms resolution crystal structure of an S-adenosylmethionine (SAM)-responsive riboswitch from Thermoanaerobacter tengcongensis complexed with S-adenosylmethionine, an RNA element that controls the expression of several genes involved in sulphur and methionine metabolism. This RNA folds into a complex three-dimensional architecture that recognizes almost every functional group of the ligand through a combination of direct and indirect readout mechanisms. Ligand binding induces the formation of a series of tertiary interactions with one of the helices, serving as a communication link between the aptamer and expression platform domains.

Figure.Side (a) and top (c) views of the group I intron with the two principal conserved domains P4ÐP6 and P3ÐP7 coloured in blue and green, respectively. The active site is highlighted by the two red nucleotides, which represent the site of cleavage. Side (b) and top (d) views of the SAM-I riboswitch in the same orientation as the group I intron, with the two primary domains P1/P4 and P2/P3 coloured in blue and green, respectively.

Identifying pattern-defined regulatory islands in mammalian genomes.

Proc Natl Acad Sci U S A. 2007 Jun 12;104(24):10116-21.

Cheung TH, Barthel KK, Kwan YL, Liu X.

Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215, USA.

Identifying cis-regulatory regions in mammalian genomes is a key challenge toward understanding transcriptional regulation. However, identification and functional characterization of those regulatory elements governing differential gene expression has been hampered by the limited understanding of their organization and locations in genomes. We hypothesized that genes that are conserved across species will also display conservation at the level of their transcriptional regulation and that this will be reflected in the organization of cis-elements mediating this regulation. Using a computational approach, clusters of transcription factor binding sites that are absolutely conserved in order and in spacing across human, rat, and mouse genomes were identified. We term these regions pattern-defined regulatory islands (PRIs). We discovered that these sequences are frequently active sites of transcriptional regulation. These PRIs occur in approximately 1.1% of the half-billion base pairs covered in the search and are located mainly in noncoding regions of the genome. We show that the premise of PRIs can be used to identify previously known and novel cis-regulatory regions controlling genes regulated by myogenic differentiation. Thus, PRIs may represent a fundamental property of the architecture of cis-regulatory elements in mammalian genomes, and this feature can be exploited to pinpoint critical transcriptional regulatory elements governing cell type-specific gene expression.

Figure. PRIs with MEF2 binding sites and/or E-boxes from genes regulated during myogenesis are bound by these transcription factors. (A) Real-time PCR analysis of genes with PRIs selected for study. Fold change in transcript level of the indicated genes relative to the control gene EPB7.2 was determined by real-time PCR. Actual fold change values are reported over a heat map. (B) MEF2 and MYOD bind PRIs containing their corresponding binding sites during myogenesis but they do not bind consensus sites not found within PRIs. Formaldehyde-fixed, sonicated C2C12 chromatin extracts (GM and DM D1) were immunoprecipitated with anti-MEF2 or anti-MYOD antibodies. PRIs associated with the indicated genes or regions containing binding sites not within PRIs were analyzed by PCR. PRI from MYOG (indicated by *) served as a positive control. The input lane refers to PCR amplification of 0.1% of the input lysate (preimmunoprecipitation) with the same primer pairs. No primary antibody was added to the no antibody lanes (No Ab).