Yon W. Ebright

HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′ terminal nucleotide

microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants bear a methyl group on the ribose of the 3′ terminal nucleotide. We showed previously that the methylation of miRNAs and siRNAs requires the protein HEN1 in vivo and that purified HEN1 protein methylates miRNA/miRNA* duplexes in vitro. In this study, we show that HEN1 methylates both miRNA/miRNA* and siRNA/siRNA* duplexes in vitro with a preference for 21–24 nt RNA duplexes with 2 nt overhangs. We also demonstrate that HEN1 deposits the methyl group on to the 2′ OH of the 3′ terminal nucleotide. Among various modifications that can occur on the ribose of the terminal nucleotide, such as 2′-deoxy, 3′-deoxy, 2′-O-methyl and 3′-O-methyl, only 2′-O-methyl on a small RNA inhibits the activity of yeast poly(A) polymerase (PAP). These findings indicate that HEN1 specifically methylates miRNAs and siRNAs and implicate the importance of the 2′-O-methyl group in the biology of RNA silencing.

Translocation of σ70 with RNA Polymerase during Transcription

Using fluorescence resonance energy transfer, we show that, in the majority of transcription complexes, sigma(70) is not released from RNA polymerase upon transition from initiation to elongation, but, instead, remains associated with RNA polymerase and translocates with RNA polymerase. The results argue against the presumption that there are necessary subunit-composition differences, and corresponding necessary mechanistic differences, in initiation and elongation. The methods of this report should be generalizable to monitor movement of any molecule relative to any nucleic acid.

Opening and Closing of the Bacterial RNA Polymerase Clamp

Clamping Down Crystal structures of RNA polymerase show that a “clamp” region which surrounds the DNA binding site can adopt conformations ranging from a closed to an open state. Chakraborty et al. (p. 591) used single-molecule fluorescence energy transfer experiments to detect the clamps conformational changes in solution during the transcription cycle. The results support a model in which a clamp opening allows DNA to be loaded into the active-center cleft and unwound. Direct interactions with DNA likely trigger clamp closure upon formation of a catalytically competent transcription initiation complex. Single-molecule fluorescence measurements define the clamp conformation during transcription initiation and elongation. Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.

Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP) I. Saturation and alanine-scanning mutagenesis☆

It has been proposed that the surface loop consisting of amino acid residues 152 to 166 of the catabolite gene activator protein (CAP) of Escherichia coli makes direct protein-protein contact with RNA polymerase at the lac promoter. In this work, we have used targeted saturation mutagenesis of codons 152 to 166 of the gene encoding CAP, followed by a screen, to isolate more than 200 independent mutants of CAP defective in transcription activation but not defective in DNA binding. All isolated single-substitution mutants map to just eight amino acid residues; 156, 157, 158, 159, 160, 162, 163 and 164. We propose that these residues define the full extent of the epitope on CAP for the proposed CAP-RNA polymerase interaction. In addition, we have constructed alanine substitutions at each position from residue 152 to 166 of CAP, and we have analyzed the effects on transcription activation at the lac promoter and on DNA binding. Alanine substitution of Thr158 results in an approximately eightfold specific defect in transcription activation. In contrast, alanine substitution of no other residue tested results in a more than twofold specific defect in transcription activation. We conclude that, for Thr158, side-chain atoms beyond C beta are essential for transcription activation at the lac promoter, and we propose that Thr158 OH7 gamma makes direct contact with RNA polymerase in the ternary complex of lac promoter, CAP and RNA polymerase. We conclude further that for no residue other than Thr158 are side-chain atoms beyond C beta essential for transcription activation at the lac promoter.

Derivatives of CAP having no solvent-accessible cysteine residues, or having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif.

The Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein. CAP contains a unique solvent-accessible cysteine residue at amino acid 10 of the helix-turn-helix motif. In published work, we have constructed a prototype semi-synthetic site-specific DNA cleavage agent from CAP by use of cysteine-specific chemical modification to incorporate a nucleolytic chelator-metal complex at amino acid 10 of the helix-turn-helix motif [Ebright, R., Ebright, Y., Pendergrast, P.S. and Gunasekera, A., Proc. Natl. Acad. Sci. USA 87, 2882-2886 (1990)]. Construction of second-generation semi-synthetic site-specific DNA cleavage agents from CAP requires the construction of derivatives of CAP having unique solvent-accessible cysteine residues at sites within CAP other than amino acid 10 of the helix-turn-helix motif. In the present work, we have constructed and characterized two derivatives of CAP having no solvent-accessible cysteine residues: [Ser178]CAP and [Leu178]CAP. In addition, in the present work, we have constructed and characterized one derivative of CAP having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif: [Cys170;Ser178]CAP.

Fluorescence Resonance Energy Transfer (FRET) in Analysis of Transcription-Complex Structure and Function

Publisher Summary This chapter discusses the Fluorescence Resonance Energy Transfer (FRET) to monitor movement of RNA polymerase (RNAP) relative to DNA during transcription and to define three-dimensional structures of transcription complexes in solution. FRET is a physical phenomenon that permits measurement of distances. FRET occurs in a system where a fluorescent probe serves as a donor and a second fluorescent probe serves as an acceptor, where the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. The chapter presents protocols for FRET experiments that permit measurement of distances between positions on upstream DNA and positions within RNAP (“trailing-edge FRET”), distances between positions on downstream DNA and positions within RNAP (“leading-edge FRET”), and distances between positions on RNAP core and positions within σ 70 (“core- σ 70 FRET”). Later the preparation of DNA fragments, sigma σ 70 , RNAP core, RNAP holoenzyme, and RNA polymerase-promoter open complex (RP o ) is discussed. DNA–RNAP FRET permits monitoring of movement of RNAP relative to DNA during promoter escape and elongation and provides information about three-dimensional structures of RP o and RD e . The approach involves two complementary sets of experiments: trailing-edge-FRET experiments, which monitor the distance between a fluorescent probe in RNAP and a fluorescent probe on DNA upstream of RNAP; and leading-edge-FRET experiments, which monitor the distance between a fluorescent probe in RNAP, and a fluorescent probe on DNA downstream of RNAP.

Transcription inhibition by the depsipeptide antibiotic salinamide A

We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center ‘bridge-helix cap’ comprising the ‘bridge-helix N-terminal hinge’, ‘F-loop’, and ‘link region’. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge. DOI: http://dx.doi.org/10.7554/eLife.02451.001

GE23077 binds to the RNA polymerase 'i' and 'i+1' sites and prevents the binding of initiating nucleotides.

Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center ‘i’ and ‘i+1’ nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance. DOI: http://dx.doi.org/10.7554/eLife.02450.001

AZIDE-SPECIFIC LABELING OF BIOMOLECULES BY STAUDINGER- BERTOZZI LIGATION: PHOSPHINE DERIVATIVES OF FLUORESCENT PROBES SUITABLE FOR SINGLE-MOLECULE FLUORESCENCE SPECTROSCOPY

We describe the synthesis of phosphine derivatives of three fluorescent probes that have a brightness and photostability suitable for single-molecule fluorescence spectroscopy and microscopy: Alexa488, Cy3B, and Alexa647. In addition, we describe procedures for use of these reagents in azide-specific, bioorthogonal labeling through Staudinger-Bertozzi ligation, as well as procedures for the quantitation of labeling specificity and labeling efficiency. The reagents and procedures of this report enable chemoselective, site-selective labeling of azide-containing biomolecules for single-molecule fluorescence spectroscopy and microscopy.

Identification of amino acid-base contacts in the Myc-DNA complex by site-specific bromouracil mediated photocrosslinking.

Myc binds to a 6 bp 2‐fold symmetric DNA site: 5′‐C‐3A‐2C‐1G+1T+2G+3‐3′. Using site‐specific 5‐bromouracil mediated photocrosslinking, we show that His336 of Myc contacts, or is close to, the thymine 5‐methyl group at 2‐fold symmetry‐related positions ‐2 and +2 of the DNA site in the Myc‐DNA complex. Our results strongly suggest that homologous amino acids of Myc and Max make equivalent contacts in the respective protein‐DNA complexes.