Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, *

The RpoS transcription factor (also called S or 38) is required for the expression of a number of stationary-phase and osmotically inducible genes in Escherichia coli. RpoS is also a virulence factor for several pathogenic bacteria, including Salmonella typhimurium. The activity of RpoS is regulated in response to several different signals, at the transcriptional and translational levels as well as by proteolysis. Here we report that host factor I (HF-I), the product of the hfq gene, is required for efficient expression of rpoS in S. typhimurium. HF-I is a small, heat-stable, site-specific RNA-binding protein originally characterized for its role in replication of the RNA bacteriophage Q␤ of E. coli. Its role in the uninfected bacterial cell has previously been unknown. Assays of ␤-galactosidase in strains with rpoS-lac fusions, Western blot (immunoblot) analysis, and pulse-labeling and immunoprecipitation of both fusion proteins and native RpoS show that an S. typhimurium hfq mutant has a four-to sevenfold reduction in expression of rpoS that is attributable primarily to a defect in translation. These results add a new level of complexity to the regulation of RpoS activity.

, the stationary phase sigma factor of Escherichia coli and Salmonella, is regulated at multiple levels. The s S protein is unstable during exponential growth and is stabilized during stationary phase and after various stress treatments. Degradation requires both the ClpXP protease and the adaptor RssB. The small antiadaptor protein IraP is made in response to phosphate starvation and interacts with RssB, causing s S stabilization under this stress condition. IraP is essential for s S stabilization in some but not all starvation conditions, suggesting the existence of other anti-adaptor proteins. We report here the identification of new regulators of s S stability, important under other stress conditions. IraM (inhibitor of RssB activity during Magnesium starvation) and IraD (inhibitor of RssB activity after DNA damage) inhibit s S proteolysis both in vivo and in vitro. Our results reveal that multiple anti-adaptor proteins allow the regulation of s S stability through the regulation of RssB activity under a variety of stress conditions.

Identifying the proteins synthesized in defined cells at specific times in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chemically modifiable analogs of amino acids at diverse sense codons in cells in rich media. We apply SORT-M to Drosophila melanogaster fed standard food to label and image proteins in specific tissues at precise developmental stages with diverse chemistries, including cyclopropene-tetrazine inverse electron demand Diels-Alder cycloaddition reactions. We also use SORT-M to identify proteins synthesized in germ cells of the fly ovary without dissection. SORT-M will facilitate the definition of proteins synthesized in specific sets of cells to study development, and learning and memory in flies, and may be extended to other animals.Defining the proteins synthesized in specific cells, at specific times within animals will provide molecular insight into diverse biological processes including development, 1 differentiation, 1 circadian oscillations, 2 morphogenesis 3 and learning and memory. 4 Despite the importance of this challenge, there are no general methods for identifying the proteins synthesized in specific cells at specific times within an animal.