An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy transfer for a large number of combinations of commonly used fluorophores and quenchers. We have also measured the quenching effect of nucleotides on the fluorescence of each fluorophore. Quenching efficiencies were measured for both the resonance energy transfer and the static modes of quenching. We found that, in addition to their photochemical characteristics, the tendency of the fluorophore and the quencher to bind to each other has a strong influence on quenching efficiency. The availability of these measurements should facilitate the design of oligonucleotide probes that contain interactive fluorophores and quenchers, including competitive hybridization probes, adjacent probes, TaqMan probes and molecular beacons.

The mechanism of transport of mRNA-protein (mRNP) complexes from transcription sites to nuclear pores has been the subject of many studies. Using molecular beacons to track single mRNA molecules in living cells, we have characterized the diffusion of mRNP complexes in the nucleus. The mRNP complexes move freely by Brownian diffusion at a rate that assures their dispersion throughout the nucleus before they exit into the cytoplasm, even when the transcription site is located near the nuclear periphery. The diffusion of mRNP complexes is restricted to the extranucleolar, interchromatin spaces. When mRNP complexes wander into dense chromatin, they tend to become stalled. Although the movement of mRNP complexes occurs without the expenditure of metabolic energy, ATP is required for the complexes to resume their motion after they become stalled. This finding provides an explanation for a number of observations in which mRNA transport appeared to be an enzymatically facilitated process.gene expression ͉ live cell imaging ͉ mRNA export ͉ nuclear viscosity A fter mRNAs are synthesized, processed, and become associated with a number of different proteins at the transcription site, they are released into the nucleoplasm (1). The mechanism by which these large mRNA-protein (mRNP) complexes then move through dense nucleoplasm to reach the nuclear pores has been the subject of intense study and speculation (2, 3). Early workers proposed that mRNP complexes are transferred along a chain of receptors until they reach a nuclear pore, expending metabolic energy in the process (4). This solid-state transport model is supported by observations made in fixed nuclei that show some transcripts distributed along tracks that originate from the locus of the parent gene (5, 6). A second theory, called the ''gene-gating'' hypothesis, proposes that active genes are situated near the nuclear periphery and that mRNAs exit the nucleus through the nearest pores (7). This idea is supported by observations that certain mRNAs exit from one side of the nucleus (8) and that, in yeast, many transcriptionally active gene loci are located near the nuclear periphery (9). By contrast, a number of other studies have found that mRNP complexes move quite freely within the nucleus (10-16). This view is supported by studies of the distribution of newly synthesized Balbiani ring RNA in the salivary gland cells of insects (11), fluorescence recovery after photobleaching and fluorescence correlation spectroscopy studies of probes that bind to the poly(A) tails of mRNAs (12-15), and from single-particle analysis of mRNP complexes bound to GFP-linked proteins (16).Although the latter studies found that mRNP complexes are able to diffuse within the nuclear matrix, there was a paradoxical active transport component to their motility, because both a reduction in temperature and ATP depletion curtailed the mobility of the complexes (14-16). To better understand the nature of mRNP mobility, we have developed a system of fluorogenic probes and mRNA constructs that allo...

In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron‐dependent regulator), an iron‐responsive DNA‐binding protein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI), aromatic amino acids (pheA, hisE, hisB‐like) and others annotated to be involved in the synthesis of iron‐storage proteins (bfrA, bfrB). Some putative IdeR‐regulated genes identified in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)‐like molecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtA–mbtB, mbI, rv3402c and bfrA–bfd, for interaction with IdeR and for iron‐dependent expression. Gel retardation experiments and DNase footprinting analyses with purified IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the −10 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd are induced 14‐ to 49‐fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that IdeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR‐ and iron‐repressed genes, mbtB, mbtI and rv3402c, were induced during M. tuberculosis infection of human THP‐1 macrophages.

We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light are assigned to two separate fluorophores in the same probe. These probes contain a harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic light source, an emitter fluorophore of the desired emission color, and a nonfluorescent quencher. In the absence of complementary nucleic acid targets, the probes are dark, whereas in the presence of targets, they fluoresce-not in the emission range of the harvester fluorophore that absorbs the light, but rather in the emission range of the emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed energy from the harvester fluorophore to the emitter fluorophore by fluorescence resonance energy transfer, and it only takes place in probes that are bound to targets. Wavelength-shifting molecular beacons are substantially brighter than conventional molecular beacons that contain a fluorophore that cannot efficiently absorb energy from the available monochromatic light source. We describe the spectral characteristics of wavelength-shifting molecular beacons, and we demonstrate how their use improves and simplifies multiplex genetic analyses.

Summary It is believed that the introns are removed from pre-mRNAs during transcription, while the pre-mRNA is still tethered to the gene locus via RNA polymerase. However, during alternative splicing it is important that splicing be deferred until all of the exons and introns involved in the choice have been synthesized. We have developed an in situ RNA imaging method with single-molecule sensitivity to define the intracellular sites of splicing. Using this approach, we found that the normally tight coupling between transcription and splicing is broken in situations where the intron’s polypyrimidine tract is sequestered within strong secondary structures. We also found that in two cases of alternative splicing, in which certain exons are skipped due to the activity of the RNA binding proteins Sxl and PTB, splicing is uncoupled from transcription. This uncoupling occurs only on the perturbed introns, while the preceding and succeeding introns are removed co-transcriptionally.