We examine the role of synaptic activity in the development of identified Drosophila embryonic motorneurons. Synaptic activity was blocked by both pan-neuronal expression of tetanus toxin light chain (TeTxLC) and by reduction of acetylcholine (ACh) using a temperature-sensitive allele of choline acetyltransferase (Cha ts2 ). In the absence of synaptic activity, aCC and RP2 motorneurons develop with an apparently normal morphology and retain their capacity to form synapses. However, blockade of synaptic transmission results in significant changes in the electrical phenotype of these neurons. Specifically, increases are seen in both voltage-gated inward Na ϩ and voltage-gated outward K ϩ currents. Voltage-gated Ca 2ϩ currents do not change. The changes in conductances appear to promote neuron excitability. In the absence of synaptic activity, the number of action potentials fired by a depolarizing ramp (Ϫ60 to ϩ60 mV) is increased and, in addition, the amplitude of the initial action potential fired is also significantly larger. Silencing synaptic input to just aCC, without affecting inputs to other neurons, demonstrates that the capability to respond to changing levels of synaptic excitation is intrinsic to these neurons. The alteration to electrical properties are not permanent, being reversed by restoration of normal synaptic function. Whereas our data suggest that synaptic activity makes little or no contribution to the initial formation of embryonic neural circuits, the electrical development of neurons that constitute these circuits seems to depend on a process that requires synaptic activity.

Noncoding, or intergenic, transcription by RNA polymerase II (RNA-PII) is remarkably widespread in eukaryotic organisms, but the effects of such transcription remain poorly understood. Here we show that noncoding transcription plays a role in activation, but not repression, of the Saccharomyces cerevisiae PHO5 gene. Histone eviction from the PHO5 promoter during activation occurs with normal kinetics even in the absence of the PHO5 TATA box, showing that transcription of the gene itself is not required for promoter remodeling. Nevertheless, we find that mutations that impair transcript elongation by RNAPII affect the kinetics of histone eviction from the PHO5 promoter. Most dramatically, inactivation of RNAPII itself abolishes eviction completely. Under repressing conditions, an Ϸ2.4-kb noncoding exosome-degraded transcript is detected that originates near the PHO5 termination site and is transcribed in the antisense direction. Abrogation of this transcript delays chromatin remodeling and subsequent RNAPII recruitment to PHO5 upon activation. We propose that noncoding transcription through positioned nucleosomes can enhance chromatin plasticity so that chromatin remodeling and activation of traversed genes occur in a timely manner.elongation ͉ intergenic transcription ͉ RNA polymerase II I n addition to transcribing all protein-encoding genes, RNA polymerase II (RNAPII) also transcribes a large group of less known and poorly understood untranslated RNAs. Recent genome-wide studies in several species reveal that such noncoding transcription is much more extensive than previously thought and that it occurs across intergenic regions, introns, and exons (see, for example, refs. 1 and 2). Recently, genome-wide studies in yeast have identified many cases of intergenic transcripts associated with promoters (3-5), raising the question of whether and how intergenic transcription across a promoter is used as a means of regulating that gene's transcription.During our studies on elongation and RNA processing factors in yeast, we discovered an intergenic transcript across the PHO5 promoter. This finding led us to investigate whether noncoding transcription might play a role in regulating this gene. PHO5 encodes an acid phosphatase that is regulated by phosphate availability (6). In high phosphate, four positioned nucleosomes are associated with the PHO5 promoter region (7). During phosphate starvation, the Pho4 activator translocates to the nucleus (8) and binds to PHO5 upstream activation sequences (UASp1 and UASp2) along with the Pho2 activator (9-11). This leads to eviction of the four positioned nucleosomes, making a 600-bp region effectively fully accessible (7,(12)(13)(14). Promoter remodeling is facilitated by, although not always absolutely dependent on, several transcription factor complexes including SAGA, Swi/Snf complex, INO80, and the Asf1 chaperone (14-18). High phosphate causes Pho4 accumulation in the cytoplasm, nucleosome reassembly on the promoter, and transcriptional repression of the gene.Here we show tha...

The human mitochondrial transcription machinery generates the primers required for initiation of leading-strand DNA replication. According to one model, the 3′ end of the primer is defined by transcription termination at conserved sequence block II (CSB II) in the mitochondrial DNA control region. We here demonstrate that this site-specific termination event is caused by G-quadruplex structures formed in nascent RNA upon transcription of CSB II. We also demonstrate that a poly-dT stretch downstream of CSB II has a modest stimulatory effect on the termination efficiency. The mechanism is reminiscent of Rho-independent transcription termination in prokaryotes, with the exception that a G-quadruplex structure replaces the hairpin loop formed in bacterial mRNA during transcription of terminator sequences. mitochondrion | RNA polymerase H uman mitochondria contain their own genetic material called mitochondrial DNA (mtDNA), a double-stranded circular molecule encoding 13 subunits of the respiratory chain, 22 tRNAs, and 2 rRNAs. The two strands are referred to as the heavy and light strand based on their buoyant density in cesium chloride gradients (1). Transcription from the light strand promoter (LSP) and heavy strand promoter is polycistronic and produces near genome-length transcripts, which are processed to release the individual RNA molecules (2, 3). MtDNA contains few noncoding sequences, with the exception of the control region, which harbors DNA elements required for initiation of transcription and DNA replication (Fig. 1A).Mitochondrial DNA replication initiates at two major sites, the origins of light and heavy (OriH) strand replication. The mitochondrial RNA polymerase generates the RNA primers required for initiation of DNA synthesis at both these sites (4-7). Primers required for OriH activation are produced by transcription initiated at LSP, and there must thus exist a mechanism that enables the transcription machinery to switch from genomelength transcription to primer formation. One model suggests that the primers are formed by cleavage of the primary LSP transcript by the mitochondrial RNA processing endonuclease (8, 9), but this idea has been questioned, because at least human mitochondrial RNA processing endonuclease is primarily localized to the nucleolus, where it is required for rRNA processing (10). Some years ago, we suggested an alternative model for primer formation at OriH. Using a reconstituted in vitro transcription system containing the mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factors A (TFAM) and B2 (TFB2M), we observed that a large fraction (up to 65%) of the transcription events initiated at LSP were prematurely terminated at a conserved DNA element within the control region, denoted conserved sequence block II (CSB II). The 3′ ends of these terminated transcription products coincided with the RNA-to-DNA transition sites mapped in vivo (5). The mechanism behind the observed transcription termination was not elucidated, but mutational analysis demonstrated ...

In human mitochondria the transcription machinery generates the RNA primers needed for initiation of DNA replication. A critical feature of the leading-strand origin of mitochondrial DNA replication is a CG-rich element denoted conserved sequence block II (CSB II). During transcription of CSB II, a G-quadruplex structure forms in the nascent RNA, which stimulates transcription termination and primer formation. Previous studies have shown that the newly synthesized primers form a stable and persistent RNA–DNA hybrid, a R-loop, near the leading-strand origin of DNA replication. We here demonstrate that the unusual behavior of the RNA primer is explained by the formation of a stable G-quadruplex structure, involving the CSB II region in both the nascent RNA and the non-template DNA strand. Based on our data, we suggest that G-quadruplex formation between nascent RNA and the non-template DNA strand may be a regulated event, which decides the fate of RNA primers and ultimately the rate of initiation of DNA synthesis in human mitochondria.

The lethal mutation l(2)CA4 causes specific defects in local growth of neuronal processes. We uncovered four alleles of l(2)CA4 and mapped it to bands 50A-C on the polytene chromosomes and found it to be allelic to kakapo (Prout et al. 1997. Genetics. 146:275– 285). In embryos carrying our kakapo mutant alleles, motorneurons form correct nerve branches, showing that long distance growth of neuronal processes is unaffected. However, neuromuscular junctions (NMJs) fail to form normal local arbors on their target muscles and are significantly reduced in size. In agreement with this finding, antibodies against kakapo (Gregory and Brown. 1998. J. Cell Biol. 143:1271–1282) detect a specific epitope at all or most Drosophila NMJs. Within the central nervous system of kakapo mutant embryos, neuronal dendrites of the RP3 motorneuron form at correct positions, but are significantly reduced in size. At the subcellular level we demonstrate two phenotypes potentially responsible for the defects in neuronal branching: first, transmembrane proteins, which can play important roles in neuronal growth regulation, are incorrectly localized along neuronal processes. Second, microtubules play an important role in neuronal growth, and kakapo appears to be required for their organization in certain ectodermal cells: On the one hand, kakapo mutant embryos exhibit impaired microtubule organization within epidermal cells leading to detachment of muscles from the cuticle. On the other, a specific type of sensory neuron (scolopidial neurons) shows defects in microtubule organization and detaches from its support cells.