Radiolysis of water with a synchrotron x-ray beam permits the hydroxyl radical-accessible surface of an RNA to be mapped with nucleotide resolution in 10 milliseconds. Application of this method to folding of the Tetrahymena ribozyme revealed that the most stable domain of the tertiary structure, P4-P6, formed cooperatively within 3 seconds. Exterior helices became protected from hydroxyl radicals in 10 seconds, whereas the catalytic center required minutes to be completely folded. The results show that rapid collapse to a partially disordered state is followed by a slow search for the active structure.

Monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags and components of biosensors for deep-tissue imaging and multicolour microscopy. We report three bright and spectrally distinct monomeric NIR FPs, termed miRFPs, engineered from bacterial phytochrome, which can be used as easily as GFP-like FPs. miRFPs are 2–5-fold brighter in mammalian cells than other monomeric NIR FPs and perform well in protein fusions, allowing multicolour structured illumination microscopy. miRFPs enable development of several types of NIR biosensors, such as for protein–protein interactions, RNA detection, signalling cascades and cell fate. We demonstrate this by engineering the monomeric fluorescence complementation reporters, the IκBα reporter for NF-κB pathway and the cell cycle biosensor for detection of proliferation status of cells in culture and in animals. miRFPs allow non-invasive visualization and detection of biological processes at different scales, from super-resolution microscopy to in vivo imaging, using the same probes.

Radiolysis of peptide and protein solutions with high-energy X-ray beams induces stable, covalent modifications of amino acid residues that are useful for synchrotron protein footprinting. A series of 5-14 amino acid residue peptides of varied sequences were selected to study their synchrotron radiolysis chemistry. Radiolyzed peptide products were detected within 10 ms of exposure to a white light synchrotron X-ray beam. Mass spectrometry techniques were used to characterize radiolytic modification to amino acids cysteine (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), proline (Pro), histidine (His), and leucine (Leu). A reactivity order of Cys, Met >> Phe, Tyr, > Trp > Pro > His, Leu was determined under aerobic reaction conditions from MS/MS analysis of the radiolyzed peptide products. Radiolysis of peptides in 18O-labeled water under aerobic conditions revealed that oxygenated radical species from air and water both contribute to the modification of amino acid side chains. Cysteine and methionine side chains reacted with hydroxyl radicals generated from radiolysis of water as well as molecular oxygen. Phenylalanine and tyrosine residues were modified predominantly by hydroxyl radicals, and the source of modification of proline was exclusively through molecular oxygen.

No abstract

No abstract

How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend primarily on sequence information. In this study, we demonstrate that specific recognition of messenger RNAs (mRNAs) by RNA-binding proteins requires the correct spatial positioning of these sequences. We characterized both the cis-acting sequence elements and the spatial restraints that define the mode of RNA binding of the zipcode-binding protein 1 (ZBP1/IMP1/IGF2BP1) to the b-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element comprised of a 59 element (CGGAC) followed by a variable 39 element (C/A-CA-C/U) that must be appropriately spaced. Remarkably, the orientation of these elements is interchangeable within target transcripts bound by ZBP1. The spatial relationship of this consensus binding site identified conserved transcripts that were verified to associate with ZBP1 in vivo. The dendritic localization of one of these transcripts, spinophilin, was found to be dependent on both ZBP1 and the RNA elements recognized by ZBP1 KH34.

The TATA-binding protein (TBP) initiates assembly of transcription preinitiation complexes on eukaryotic class II promoters, binding to and restructuring consensus and variant "TATA box" sequences. The sequence dependence of the DNA structure in TBP⅐TATA complexes has been investigated in solution using fluorescence resonance energy transfer. The mean 5dye-3dye distance varies significantly among oligomers bearing the adenovirus major late promoter sequence (AdMLP) and five single-site variants bound to Saccharomyces cerevisiae TBP, consistent with solution bend angles for AdMLP of 76°and for the variants ranging from 30°to 62°. These solution bends contrast sharply with the corresponding co-crystal structures, which show ϳ80°b ends for all sequences. Transcription activities for these TATA sequences are strongly correlated with the solution bend angles but not with TBP⅐DNA binding affinities. Our results support a model in which transcription efficiency derives primarily from the sequence-dependent structure of the TBP⅐TATA binary complex. Specifically, the distance distribution for the average solution structure of the TBP⅐TATA complex may reflect the sequence-dependent probability for the complex to assume a conformation in which the TATA box DNA is severely bent. Upon assumption of this geometry, the binary complex becomes a target for binding and correctly orienting the other components of the preinitiation complex.The TATA-binding protein (TBP) 1 binds to eukaryotic class II promoters at specific sequences of DNA of the consensus sequence TATA(a/t)A(a/t)N, nucleating assembly of the proteins required for transcription. Atomic resolution co-crystal structures of complexes of DNA bearing consensus strong promoter sequences bound to Saccharomyces cerevisiae (1), Arabidopsis thaliana (2), and human (3, 4) TBPs are extremely similar, characterized by a TBP-induced ϳ80°bend in the DNA helix. TBP also binds to numerous variant TATA sequences, many of which occur naturally in promoters (5, 6). For 21 such single-point mutants of the adenovirus major late promoter (AdMLP) TATA box sequence, in vitro transcription activity was found to range from Ͻ1% to 107% of that of the reference AdMLP TATA sequence (6).The wide range of observed transcription activities suggested that TBP does not bind similarly to all TATA elements. Gel electrophoresis circular permutation analysis of TBP⅐DNA complexes shows that the electrophoretic mobility of the complexes is TATA sequence-dependent, with bend angles from Ͻ34°to 106°inferred from the gel mobility patterns (7). In contrast, the co-crystal structures of 11 TATA sequence variants of varying affinity bound to A. thaliana TBP are all very similar, with the DNA helix bent as in the strong promoters (3,8).The present study was undertaken to further explore the TATA box sequence dependence of TBP binding and DNA structure using native, full-length S. cerevisiae TBP together with the AdMLP TATA sequence and five single-base-pair variant sequences. End-to-end distance distributions for...

No abstract