A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of

A complete transcript of the Bacillus subtilis pyr operon contains the following elements in 5' to 3' order: a 151-nucleotide (nt) untranslated leader; pyrR, encoding a 20-kDa protein; a 173-nt intercistronic region; pyrP, encoding a 46-kDa protein; a 145-nt intercistronic region; and eight overlapping cistrons encoding all of the six enzymes for de novo pyrimidine biosynthesis. Transcription is controlled by the availability of pyrimidines via an attenuation mechanism. There are three transcription terminators within the operon, each of which is preceded by another stem-loop structure, the antiterminator, whose formation would prevent formation of the terminator stem-loop. These are located in the leader, the pyrR-pyrP intercistronic region, and the pyrP-pyrB intercistronic region. Northern (RNA) blot analysis has identified transcripts of lengths which coincide with termination at these proposed attenuation sites and whose relative abundances vary in the expected pyrimidine-dependent manner. Each antiterminator contains a 50-base conserved sequence in its promoter-proximal half. Various transcriptional fusions of the pyr promoter and surrounding sequences to promoterless reporter genes support an attenuation mechanism whereby when pyrimidines are abundant, the PyrR protein binds to the conserved sequence in the pyr mRNA and disrupts the antiterminator, permitting terminator hairpin formation and promoting transcription termination. Deletion of pyrR from the chromosome resulted in the constitutive, elevated expression of aspartate transcarbamylase, which is encoded by pyrB, the third gene in the operon. Complementation of an E. coli upp mutant, as well as direct enzymatic assay, has demonstrated that pyrR also confers uracil phosphoribosyltransferase activity. Analysis of pyrR and upp deletion mutants demonstrated that upp, not pyrR, encodes the quantitatively important uracil phosphoribosyltransferase activity. The pyrP gene probably encodes an integral membrane uracil permease.

Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway. Mutations that disrupt Ca2+-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca2+-sensor for replenishment. The Ca2+-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca2+-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals.DOI: http://dx.doi.org/10.7554/eLife.01524.001

Lignin is highly branched phenolic polymer and accounts 15-30% by weight of lignocellulosic biomass (LCBM). The acceptable molecular structure of lignin is composed with three main constituents linked by different linkages. However, the structure of lignin varies significantly according to the type of LCBM, and the composition of lignin strongly depends on the degradation process. Thus, the elucidation of structural features of lignin is important for the utilization of lignin in high efficient ways. Up to date, degradation of lignin with destructive methods is the main path for the analysis of molecular structure of lignin. Spectroscopic techniques can provide qualitative and quantitative information on functional groups and linkages of constituents in lignin as well as the degradation products. In this review, recent progresses on lignin degradation were presented and compared. Various spectroscopic methods, such as ultraviolet spectroscopy, Fourier-transformed infrared spectroscopy, Raman spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy, for the characterization of structural and compositional features of lignin were summarized. Various NMR techniques, such as 1 H, 13 C, 19 F, and 31 P, as well as 2D NMR, were highlighted for the comprehensive investigation of lignin structure. Quantitative 13 C NMR and various 2D NMR techniques provide both qualitative and quantitative results on the detailed lignin structure and composition produced from various processes which proved to be ideal methods in practice.

Expression of the Bacillus subtilis pyr operon is regulated by exogenous pyrimidines and the protein product of the first gene of the operon, PyrR. It has been proposed that PyrR mediates transcriptional attenuation at three untranslated segments of the operon (R. J. Turner, Y. Lu, and R. L. Switzer, J. Bacteriol., 176:3708-3722, 1994). In this study, transcriptional fusions of the pyr promoter followed by the pyr attenuation sequences, either individually or in tandem to a lacZ reporter gene, were used to examine the physiological functions of all three attenuators through their ability to affect ␤-galactosidase expression. These fusions were studied as chromosomal integrants in various B. subtilis strains to examine the entire range of control by pyrimidines, PyrR dependence, and developmental control of pyr gene expression. The nutritional regulation of each attenuator separately was roughly equivalent to that of the other two and was totally dependent upon PyrR, and that of tandem attenuators was cumulative. The regulation of a fusion of the spac promoter followed by the pyrP:pyrB intercistronic region to lacZ produced results similar to those obtained with the corresponding fusion containing the pyr promoter, demonstrating that attenuator-dependent regulation is independent of the promoter. Extreme pyrimidine starvation gave rise to two-to threefold-higher levels of expression of a pyr-lacZ fusion that lacked attenuators, independent of PyrR, than were obtained with cells that were not starved. Increased expression of a similar spac-lacZ fusion during pyrimidine starvation was also observed, however, indicating that attenuator-independent regulation is not a specific property of the pyr operon. Conversion of the initiator AUG codon in a small open reading frame in the pyrP:pyrB intercistronic region to UAG reduced expression by about half but did not alter regulation by pyrimidines, which excludes the possibility of a coupled transcription-translation attenuation mechanism. Developmental regulation of pyr expression during early stationary phase was found to be dependent upon the attenuators and PyrR, and the participation of Spo0A was excluded.The genes encoding the enzymes of de novo pyrimidine nucleotide biosynthesis in Bacillus subtilis (29,30,35,44) and Bacillus caldolyticus (15, 16) are clustered into a 10-cistron operon, which appears to be expressed as a single transcriptional unit. The coding sequences of the pyr cluster all overlap by 1 to 32 nucleotides (nt) except at the 5Ј end, where three untranslated regions are found (Fig. 1). These regions comprise a 150-nt 5Ј leader sequence, a 173-nt intercistronic region between pyrR and pyrP (pyrR:pyrP), and a 145-nt intercistronic region between pyrP and pyrB (pyrP:pyrB). Examination of the sequences of each of these untranslated regions revealed striking similarities among them (44). Each of them encodes transcripts that are capable of forming stem-loop structures characteristic of transcription terminators. Northern (RNA) hybridization analysis of py...

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Transcriptionally active chromosome (TAC) is a fraction of protein/DNA complexes with RNA polymerase activity in the plastid. However, the function of most TAC proteins remains unknown. Here, we isolated two allelic mutants of the gene for a TAC component, TAC7, and performed functional analysis in plastid gene expression and chloroplast development in Arabidopsis. tac7-1 is a mutant with a premature translation termination isolated from a population treated with ethyl methane sulfonate, and tac7-2 is a transfer-DNA tagging mutant. Both of them showed an albino phenotype when grown under normal light conditions, and a few appressed membranes were observed inside the defective chloroplasts. These data indicate that TAC7 is important for thylakoid biogenesis. The TAC7 gene encodes an uncharacterized 161 amino acids polypeptide localized in chloroplast. The transcriptional levels of plastid-encoded polymerase (PEP)-dependent genes were downregulated in tac7-2, suggesting that PEP activity was decreased in the mutant. Yeast two-hybrid assay shows that TAC7 can interact with the four TAC components including FLN1, TAC10, TAC12 and TAC14 which are involved in redox state changes, phosphorylation processes and phytochrome-dependent light signaling, respectively, These data indicate that TAC7 plays an important role for TAC to regulate PEP-dependent chloroplast gene expression and chloroplast development.

The Bacillus subtilis pyr operon is regulated by exogenous pyrimidines by a transcriptional attenuation mechanism. Transcription in vitro from pyr DNA templates specifying attenuation regions yielded terminated and read-through transcripts of the expected lengths. Addition of the PyrR regulatory protein plus UMP led to greatly increased termination. Synthetic antisense deoxyoligonucleotides were used to probe possible secondary structures in the pyr mRNA that were proposed to play roles in controlling attenuation. Oligonucleotides predicted to disrupt terminator structures suppressed termination, whereas oligonucleotides predicted to disrupt the stem of antiterminator stem-loops strongly promoted termination at the usual termination site. Oligonucleotides that disrupt a previously unrecognized stem-loop structure, called the antiantiterminator, the formation of which interferes with formation of the downstream antiterminator, suppressed termination. We propose that transcriptional attenuation of the pyr operon is governed by switching between alternative antiterminator versus anti-antiterminator plus terminator structures, and that PyrR acts by UMP-dependent binding to and stabilization of the anti-antiterminator.Transcription of the Bacillus subtilis pyrimidine biosynthetic (pyr) operon is regulated by an attenuation mechanism that involves a regulatory protein, PyrR, which is encoded by the first gene of the operon (1, 2). PyrR is thought to act by modulating the function of three attenuators in the operon. These attenuators are located in the pyr 5Ј leader, between the first and second genes, and between the second and third genes of the operon. Each attenuation region is predicted to specify mRNA that is capable of folding into a factor-independent transcription terminator (T) stem-loop, the formation of which can be prevented by the formation of a competing and more stable upstream stem-loop, called the antiterminator (AT). The balance between read-through, with consequent expression of downstream genes, versus termination and reduced expression of pyr genes has been proposed to be determined by the interconversion between AT and T structures in the mRNA, mediated in a UMP-dependent manner by the binding of PyrR to a conserved sequence in the 5Ј strand of each AT stem. PyrR binding would destabilize the AT secondary structure and favor the formation of the alternative T secondary structure. This model was supported by the analysis of expression of pyr-lacZ fusions in which portions of the 5Ј leader specifying the proposed AT and T structures were deleted (1).Further examination of the conserved putative PyrR binding sequences in the three B. subtilis pyr attenuation regions, together with corresponding sequences from the B. caldolyticus pyr operon (3), revealed that each sequence is capable of folding into a stem-loop in which the most highly conserved region (UCCAGAGAGG in B. subtilis) is located at the top of the structure. The formation of such a stem-loop would in each case disrupt the base pair...