Biochemical characterization of recombinant gene products following a phylogenetic analysis of the UDPglucosyltransferase (UGT) multigene family of Arabidopsis has identified one enzyme (UGT84B1) with high activity toward the plant hormone indole-3-acetic acid (IAA) and three related enzymes (UGT84B2, UGT75B1, and UGT75B2) with trace activities. The identity of the IAA conjugate has been confirmed to be 1-O-indole acetyl glucose ester. A sequence annotated as a UDPglucose:IAA glucosyltransferase (IAA-UGT) in the Arabidopsis genome and expressed sequence tag data bases given its similarity to the maize iaglu gene sequence showed no activity toward IAA. This study describes the first biochemical analysis of a recombinant IAA-UGT and provides the foundation for future genetic approaches to understand the role of 1-O-indole acetyl glucose ester in Arabidopsis.

A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k off ‫؍‬ 2.9 s ؊1 ) of T7 RNAP and promoter DNA, the transition to the elongation mode (k for ‫؍‬ 0.36 s ؊1 ), and the processive synthesis (k pol ‫؍‬ 43 nt s ؊1 ) and release of a gene-length RNA transcript (ϳ1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k off > k for ), identifying a rate-limiting step between promoter DNA binding and promoter escape. Transcription initiation by RNAP1 is an important regulatory step for gene expression in vivo. However, the details of transcription initiation are difficult to elucidate using conventional solution methods because (i) initiation consists of a series of transient intermediate steps between promoter binding and elongation, and (ii) within a population of actively transcribing RNAP molecules, only a small fraction is engaged in initiation at any given time. Most biochemical studies of transcription initiation utilize solution conditions which allow only a single enzymatic turnover, e.g. RNAP halted at a known position in the DNA sequence by ribonucleotide starvation or rapid mixing/quenching of the transcription assay. An attempt has been made to synchronize a population of T7 RNAP molecules in solution (1); however, synchrony is rapidly lost as the transitions between states are probabilistic events. The challenges associated with synchronizing a population of molecules can be avoided by making measurements on a single molecule. Previous single-molecule studies of transcription by Escherichia coli RNAP have only allowed DNA binding (2), elongation (artificially halted by ribonucleotide starvation) (3-8), and termination (9) to be observed in isolation from the other transcriptional states. No previous single-molecule transcription assay has allowed promoter recognition and the transition from initiation to elongation to be observed.T7 RNAP is a common paradigm for studies of transcription initiation, as it is a single-subunit enzyme sharing many of the biochemical characteristics of the more complex multi-subunit RNAPs from prokary...

DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1.

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A method is described for the purification of native hexokinases P-I and P-I1 from yeast using preparative isoelectric focussing to separate the isozymes.The binding of glucose to hexokinase P-11, and the effect of this on the monomer-dimer association -dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation.Association constants for the monomer -dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupied in the dimer.At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme . glucose complex was 3 x moll-' and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength ( I = 0.02 mol l-', pH < 7.5) was also independent of enzyme concentration (in the range 10-1000 pg ml-') but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative ; values of the intrinsic dissociation constant varied from 2 . 5~ Under intermediate conditions (pH 7.0, ionic strength = 0.1 5 mol l-'), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperativity (Hill coefficient 1.2) ; in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations.The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-I1 does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.