During the past 15 years, we have carried out several different systematic screens for small regulatory RNA genes in E. coli. These screens, which have included computational screens for conservation of intergenic regions and direct detection after size selection or co-immunoprecipitation with the RNA binding protein Hfq, are all applicable to other organisms. We are now examining small RNA expression using tiled microarrays together with deep sequencing to further extend our identification of small RNAs, particularly antisense RNAs. A large focus of the group has been to elucidate the functions of the small RNAs we and others have identified. Early on we showed that the OxyS RNA, whose expression is induced in response to oxidative stress, acts to repress translation through limited base pairing with target mRNAs. We discovered that the OxyS action is dependent on the Sm-like Hfq protein, which functions as a chaperone to facilitate OxyS RNA base pairing with its target mRNAs. We also found that another abundant and broadly-conserved small RNA mimics the DNA structure of an open promoter and modulates RNA polymerase activity. It is now clear that Hfq-binding small RNAs, which act through limited base pairing are integral to many different stress responses in E. coli. We showed that MicC, whose expression is induced in minimal medium and at low temperature, represses translation of the OmpC outer membrane porin. We also reported that FnrS, whose expression is induced by FNR upon a shift from aerobic to anaerobic conditions, acts to down regulate the levels of a variety of mRNAs encoding metabolic enzymes. In both of these examples, the small RNA represses the synthesis of proteins and enzymes that are not needed under the conditions when the levels of the small RNA are highest. In more recent work, we discovered that the McaS RNA whose levels are elevated in stationary phase or when glucose is limiting regulates mRNA targets involved in various aspects of biofilm formation. McaS represses csgD, the transcription regulator of curli biogenesis and activates flhD, the master transcription regulator of flagella synthesis leading to increased motility, a process not previously reported to be regulated by sRNAs. McaS also regulates pgaA, a porin required for the export of the polysaccharide poly &#946;-1,6-N-acetyl-d-glucosamine. Consequently, high levels of McaS result in increased biofilm formation while a strain lacking mcaS shows reduced biofilm formation. Based on these observations, we propose that McaS modulates steps in the progression to a sessile lifestyle. Other recent studies showed that the Spot 42 RNA, whose levels are highest when glucose is present, plays a broad role in catabolite repression by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse nonpreferred carbon sources. Many of the genes repressed by Spot 42 are transcriptionally activated by the global regulator CRP. Since CRP represses Spot 42, these regulators participate in a specific regulatory circuit called a multioutput feedforward loop. We found that this loop can reduce leaky expression of target genes in the presence of glucose and can maintain repression of target genes under changing nutrient conditions. Our results suggest that base-pairing RNAs in feedforward loops can help shape the steady-state levels and dynamics of gene expression. In addition to small RNAs that act via limited base pairing, we have become interested in small antisense RNAs that have the potential to form extensive base pairing interactions with their mRNA targets encoded on the opposite strand. We previously showed that base pairing between the GadY RNA and the 3-untranslated region of the gadX mRNA encoded opposite GadY leads to increased levels of the gadX mRNA and GadX protein. Recently, we demonstrated that gadX is transcribed in an operon with gadW and that base pairing of GadY with the gadXW mRNA results in processing giving rise to two halves that accumulate to higher levels than the full length mRNA. Multiple enzymes, including the double strand RNA-specific endoribonuclease RNase III, are involved in the GadY-direct cleavage. We have also reported that a large class of antisense RNAs acts to repress the synthesis of small toxic proteins. For example, in characterizing the Sib RNAs, which are encoded by five repeats in E. coli K-12, we observed an overexpression phenotype reminiscent of plasmid addiction. Further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18-19 amino acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed, and this toxicity could be prevented by co-expression of the corresponding Sib RNA. Two other RNAs encoded divergently in another intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Computational screens together with experimental validation have shown that these small hydrophobic protein-antisense RNA gene modules, termed type 1 toxin-antitoxin modules, are much more widely distributed among bacteria than previously appreciated. Studies to further characterize other Hfq-binding RNAs and antisense RNAs and to elucidate the roles of small RNAs that act in ways other than base pairing are ongoing.