No abstract

The T box leader sequence is an RNA element that controls gene expression by binding directly to a specific tRNA and sensing its aminoacylation state. This interaction controls expression of amino acid-related genes in a negative feedback loop. The T box RNA structure is highly conserved, but its tRNA binding mechanism is only partially understood. Known sequence elements are the specifier sequence, which recognizes the tRNA anticodon, and the antiterminator bulge, which base pairs with the tRNA acceptor end. Here, we reveal the crucial function of the highly conserved stem I distal region in tRNA recognition and report its 2.65-Å crystal structure. The apex of this region contains an intricately woven looploop interaction between two conserved motifs, the Adenine-guanine (AG) bulge and the distal loop. This loop-loop structure presents a base triple on its surface that is optimally positioned for basestacking interactions. Mutagenesis, cross-linking, and small-angle X-ray scattering data demonstrate that the apical base triple serves as a binding platform to dock the tRNA D-and T-loops. Strikingly, the binding platform strongly resembles the D-and T-loop binding elements from RNase P and the ribosome exit site, suggesting that this loop-loop structure may represent a widespread tRNA recognition platform. We propose a two-checkpoint molecular ruler model for tRNA decoding in which the information content of tRNA is first examined through specifier sequence-anticodon interaction, and the length of the tRNA anticodon arm is then measured by the distal loop-loop platform. When both conditions are met, tRNA is secured, and its aminoacylation state is sensed.riboswitch | RNA-RNA complex C ells must maintain appropriate intracellular pools of aminoacylated transfer RNA (aa-tRNA) to survive. This is usually accomplished by tight regulation of many cellular factors, including amino acid biosynthesis and transporter genes, and tRNA synthetases (1). Most Gram-positive bacteria use the T box regulatory system to control their aa-tRNA levels (2). T box elements are a special family of regulatory RNAs located in the 5′-untranslated region of the mRNA for a regulated gene or operon. Unlike small molecule-sensing riboswitches, T box RNAs recognize tRNAs containing the cognate anticodon and discriminate between uncharged tRNA and aa-tRNA. They usually control gene expression via premature transcription termination, although there are rare examples that are predicted to regulate at the level of translation initiation (2-4). The T box motif was initially identified in the Bacillus subtilis (Bsub) tyrosyl-tRNA synthetase gene (tyrS) and was shown to respond to the aminoacylation state of tyrosine tRNA (5, 6). More than 1,000 T box elements have since been identified (3, 4).Selective tRNA recognition is achieved through a collective effort from several conserved domains in the T box RNA [Geobacillus kaustophilus (Gkau) glyQS T box sequence is shown in Fig. 1]. Stem I is the largest element. It is an ∼100-nucleotide (nt) stem-loop s...

The nucleosome core particle (NCP) is the basic structural unit for genome packaging in eukaryotic cells and consists of DNA wound around a core of eight histone proteins. DNA access is modulated through dynamic processes of NCP disassembly. Partly disassembled structures, such as the hexasome (containing six histones) and the tetrasome (four histones), are important for transcription regulation in vivo. However, the pathways for their formation have been difficult to characterize. We combine time-resolved (TR) small-angle X-ray scattering and TR-FRET to correlate changes in the DNA conformations with composition of the histone core during saltinduced disassembly of canonical NCPs. We find that H2A-H2B histone dimers are released sequentially, with the first dimer being released after the DNA has formed an asymmetrically unwrapped, teardrop-shape DNA structure. This finding suggests that the octasome-to-hexasome transition is guided by the asymmetric unwrapping of the DNA. The link between DNA structure and histone composition suggests a potential mechanism for the action of proteins that alter nucleosome configurations such as histone chaperones and chromatin remodeling complexes.G enome access is highly regulated through the hierarchical organization of proteins and nucleic acids within the cell nucleus. The nucleosome core particle (NCP) is the first level of this hierarchy (1) and contains two dimers of H2A-H2B histones and an (H3-H4) 2 tetramer that is assembled as a dimer of dimers. Around this symmetric octamer core, ∼146 base pairs of dsDNA are wrapped in ∼1.7 superhelical turns (1, 2). The NCP structure physically impedes access to DNA, but is dynamically modulated by numerous mechanisms: posttranslational modification (PTM) of histones, incorporation of histone variants, DNA sequence-dependent effects, and the actions of extrinsic protein factors (e.g., histone chaperones, ATP-dependent remodeling complexes, and histone PTM binding proteins) (3, 4).Studies of the intrinsic properties and dynamics of NCPs are critical for understanding how nuclear machinery gains DNA access in vivo (3,5,6). Insight into the nature of partially unfolded NCP structures has been gleaned from in vitro studies of NCP assembly and disassembly. Intermediate species with partially unwrapped DNA (5, 7), disrupted histone-histone interfaces (8, 9), and dissociation of one (hexasomes) or two (tetrasomes) H2A-H2B dimers have been reported (10-12). Some of these NCP intermediates have been directly connected to chromatin function in vivo. For example, the hexasome is formed by the action of RNA Pol II (13) and the essential histone chaperone FACT (14).In addition to equilibrium studies, the kinetics of nucleosome assembly and disassembly have been characterized by bulk and single-molecule methods, including Förster resonance energy transfer (FRET) (7,8,(15)(16)(17), atomic force microscopy (AFM) (9, 18), force spectroscopy (19-21), and small-angle X-ray scattering (SAXS) (10,22). Many studies focused primarily on specific DNA-histon...

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome.

Zebrafish faithfully regenerate their caudal fin after amputation. During this process, both differentiated cells and resident progenitors migrate to the wound site and undergo lineage-restricted, programmed cellular state transitions to populate the new regenerate. Until now, systematic characterizations of cells comprising the new regenerate and molecular definitions of their state transitions have been lacking. We hereby characterize the dynamics of gene regulatory programs during fin regeneration by creating single-cell transcriptome maps of both preinjury and regenerating fin tissues at 1/2/4 days post-amputation. We consistently identified epithelial, mesenchymal, and hematopoietic populations across all stages. We found common and cell type–specific cell cycle programs associated with proliferation. In addition to defining the processes of epithelial replenishment and mesenchymal differentiation, we also identified molecular signatures that could better distinguish epithelial and mesenchymal subpopulations in fish. The insights for natural cell state transitions during regeneration point to new directions for studying this regeneration model.