Eleven mutations have been identified in 23S rRNA that make Haloarcula marismortui resistant to anisomycin, an antibiotic that competes with the amino acid side chains of aminoacyl tRNAs for binding to the A-site cleft of the large ribosomal unit. The correlation observed between the sensitivity of H. marismortui to anisomycin and the affinity of its large ribosomal subunits for the drug indicates that its response to anisomycin is determined primarily by binding of the drug to its large ribosomal subunit. The structures of large ribosomal subunits containing resistance mutations show that these mutations can be divided into two classes: (1) those that interfere with specific drug-ribosome interactions, and (2) those that stabilize the apo-conformation of the A-site cleft of the ribosome relative to its drug-bound conformation. The conformational effects of some mutations of the second kind propagate through the ribosome for considerable distances, and are reversed when A-site substrates bind to the ribosome.

A better understanding of the molecular mechanisms of signaling by the neurotransmitter serotonin is required to assess the hypothesis that defects in serotonin signaling underlie depression in humans. Caenorhabditis elegans uses serotonin as a neurotransmitter to regulate locomotion, providing a genetic system to analyze serotonin signaling. From large-scale genetic screens we identified 36 mutants of C. elegans in which serotonin fails to have its normal effect of slowing locomotion, and we molecularly identified eight genes affected by 19 of the mutations. Two of the genes encode the serotonin-gated ion channel MOD-1 and the G-protein-coupled serotonin receptor SER-4. mod-1 is expressed in the neurons and muscles that directly control locomotion, while ser-4 is expressed in an almost entirely non-overlapping set of sensory and interneurons. The cells expressing the two receptors are largely not direct postsynaptic targets of serotonergic neurons. We analyzed animals lacking or overexpressing the receptors in various combinations using several assays for serotonin response. We found that the two receptors act in parallel to affect locomotion. Our results show that serotonin functions as an extrasynaptic signal that independently activates multiple receptors at a distance from its release sites and identify at least six additional proteins that appear to act with serotonin receptors to mediate serotonin response. DEPRESSION is hypothesized to involve dysfunction of the neurotransmitter serotonin (Cowen 2008). Understanding the molecular mechanism of serotonin signaling is complicated by the fact that the human brain expresses 14 types of serotonin receptors, one of which is a serotonin-gated ion channel, and the rest of which are G-protein-coupled receptors (Millan et al. 2008). An additional challenge to understanding serotonin signaling is the fact that serotonin can act locally at synapses where it is released or diffuse away and act at distant receptors. While classical neurotransmitters such as GABA and glutamate appear to function mainly locally at synapses, serotonin can diffuse several microns from its release sites at concentrations sufficient to activate its receptors (Bunin and Wightman 1998). Furthermore, serotonin receptors are often localized at nonsynaptic sites (Kia et al. 1996). These observations suggest that serotonin might act predominantly as an extrasynaptic signal to activate several receptor types on cells distant from its release sites and that the combined action of these several receptors somehow coordinates appropriate responses to serotonin. The details of how such action might occur remain unclear.Caenorhabditis elegans uses serotonin as a neurotransmitter (Horvitz et al. 1982;Chase and Koelle 2007) and provides a model system with the potential to make important contributions to the study of serotonin signaling. First, C. elegans allows the use of forward genetic screens to identify the proteins beyond serotonin receptors that mediate serotonin response. Second, the known...

Structures have been obtained for the complexes tiamulin, homoharringtonine and bruceatin form with the large ribosomal subunit of Haloarcula marismortui at resolutions ranging from 2.8 to 3.2 Å. They show that these inhibitors all block protein synthesis by competing with the amino acid side chains of incoming aminoacyl-tRNAs for binding in the A-site cleft in the peptidyl transferase center, which is universally conserved. In addition these structures support the hypothesis that the speciesspecificity exhibited by the A-site cleft inhibitors is determined by the interactions they make, or fail to make, with a single nucleotide, U2504 (E. coli). In the ribosome, the position of U2504 is controlled by its interactions with neighboring nucleotides, whose identities vary among kingdoms.

Structures have been obtained for the complexes that triacetyloleandomycin and mycalamide A form with the large ribosomal subunit of Haloarcula marismortui. Triacetyloleandomycin binds in the nascent peptide tunnel and inhibits the activity of ribosomes by blocking the growth of the nascent peptide chain. Mycalamide A binds to the E site and inhibits protein synthesis by occupying the space normally occupied by the CCA end of E-site-bound tRNAs.

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