Human cyclin T1, a component of the P-TEFb kinase complex, was originally identified through its biochemical interaction with the Tat transactivator protein of human immunodeficiency virus type 1 (HIV-1). Current understanding suggests that binding of Tat to P-TEFb is required to promote efficient transcriptional elongation of viral RNAs. However, the dynamics and the subnuclear localization of this process are still largely unexplored in vivo. Here we exploit high resolution fluorescence resonance energy transfer (FRET) to visualize and quantitatively analyze the direct interaction between Tat and cyclin T1 inside the cells. We observed that cyclin T1 resides in specific subnuclear foci which are in close contact with nuclear speckles and that Tat determines its redistribution outside of these compartments. Consistent with this observation, strong FRET was observed between the two proteins both in the cytoplasm and in regions of the nucleus outside of cyclin T1 foci and overlapping with Tat localization. These results are consistent with a model by which Tat recruits cyclin T1 outside of the nuclear compartments where the protein resides to promote transcriptional activation.The human immunodeficiency virus type 1 (HIV-1) 1 transactivator protein Tat is a small polypeptide (86 -101 amino acids, according to the viral strains) essential for efficient transcription of viral genes. The protein is a highly unusual transcription factor since, at the HIV LTR promoter, it interacts with a cis-acting RNA element (trans-activation-responsive region, TAR) present at the 5Ј-end of each viral transcript (1).Through this interaction, Tat activates HIV-1 transcription by promoting the assembly of transcriptionally active complexes at the LTR by multiple protein-protein interactions (for a recent review, see Ref. 2). Over the last few years a number of cellular proteins have been reported to interact with Tat and to mediate or modulate its activity. These include general transcription factors, among which TBP, TAFII250, TFIIB, TFIIH (3-7); RNA polymerase II (8); transcription factor Sp1 (9); the transcriptional co-activators and histone acetyltransferases p300/CBP and P/CAF (10 -12); and the cyclin subunit of the positive transcription elongation factor complex (P-TEFb), cyclin T1 (13-16).The finding that Tat biochemically and functionally interacts with several cellular proteins raises some fundamental questions. Does Tat directly interact with its partners inside live cells? Which is the subcellular compartment of these interactions? Are they occurring simultaneously or consecutively? Some of these questions can be successfully addressed by taking advantage of fluorescence resonance energy transfer (FRET) measurements (17), allowing investigation of direct interaction of proteins labeled with optically matched fluorophores. FRET exploits radiationless energy transfer driven by dipole-dipole interaction occurring from a fluorophore (the donor) in the excited state to another fluorophore (the acceptor) when in close proximity...

A single-biomolecule optical toggle-switch is demonstrated based on a mutated green fluorescent protein (GFP). We have exploited molecular biology techniques to tailor GFP molecular structure and photophysical properties and give it optically-controlled bistability between two distinct states. We present optical control of the fluorescence dynamics with two laser beams at 476 and 350 nm down to the ultimate limit of single molecules. These results indicate that GFP-class fluorophores are promising candidates for the realization of biomolecular devices such as volumetric optical memories and optical switches.

The coherent dynamics of vibronic wave packets in the green fluorescent protein is reported. At room temperature the nonstationary dynamics following impulsive photoexcitation displays an oscillating optical transmissivity pattern with components at 67 fs (497 cm(-1)) and 59 fs (593 cm(-1)). Our results are complemented by ab initio calculations of the vibrational spectrum of the chromophore. This analysis shows the interplay between the dynamics of the aminoacidic structure and the electronic excitation in the primary optical events of green fluorescent proteins.

The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein-protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe-64-->Leu, Ser-65-->Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single-protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher-intensity emission after blue-light excitation with respect to wild-type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged-protein dynamics and fluorescence-activated cell sorting, but not for monitoring single-protein trafficking over extended periods of time.

The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein–protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe‐64→Leu, Ser‐65→Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single‐protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher‐intensity emission after blue‐light excitation with respect to wild‐type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged‐protein dynamics and fluorescence‐activated cell sorting, but not for monitoring single‐protein trafficking over extended periods of time.