The group currently has two main interests: (1) the identification and characterization of small, noncoding RNAs and (2) the identification and characterization of small ORFs. Both of these classes of genes are frequently overlooked because they are missed by most genome annotation, are poor targets for genetic approaches and are not detected in biochemical assays. [unreadable] [unreadable] Identification of small, noncoding RNAs[unreadable] We have been carrying out several different systematic screens for small, noncoding RNA genes in E. coli. These screens are all applicable to other organisms. One approach based on computer searches of intergenic regions for extended regions of conservation among closely related species led to the identification of 17 conserved small RNAs. Another screen for small RNAs that coimmunoprecipitate with the RNA binding protein Hfq allowed us to detect six less well conserved RNAs. A third approach of size fraction of total RNA followed by linker ligation and cDNA synthesis resulted in the identification of still other small RNAs. In collaboration with Dr. Rosner and Dr. Martin, we also carried out an expression-based screen to detect small RNAs encoded within or on the opposite strand of protein coding genes. Chromosomal fragments of approximately 160 bp were fused to a promoterless lacZ reporter gene on a multi-copy plasmid and 105 clones were selected at random. Among the randomly-selected clones, 56 had significantly elevated activity. Of these, 49 had inserts which mapped within or downstream of ORFs. For six of the nine most active sequences with orientations opposite to that of the ORF, chromosomal expression was detected by RT-PCR, but defined transcripts were not detected by northern analysis. Although this approach did not result in the identification of new small RNAs, the results did show that the E. coli chromosome carries numerous -35 and -10 sequences with weak promoter activity. [unreadable] [unreadable] Development of general approaches for the characterization of small, noncoding RNAs[unreadable] We also have been developing tools for the characterization of small RNA regulators. Detection of RNAs on microarrays has become a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. In collaboration with Dr. Gottesman and Dr. Leppla, we developed an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of E. coli much more efficiently than the commonly-used cDNA-based method. [unreadable] [unreadable] Many bacterial sRNAs act as post-transcriptional regulators by basepairing with target mRNAs. While the number of characterized small RNAs has steadily increased, only a limited number of the corresponding mRNA targets have been identified. In collaboration with Dr. Gottesman and Dr. Tjaden, we developed and tested a program, TargetRNA, that predicts the targets of these small RNA regulators. The program was evaluated by assessing whether previously known targets could be identified. The program was then used to predict targets for the E. coli RNAs RyhB, OmrA, OmrB and OxyS, and the predictions were compared with changes in whole genome expression patterns observed upon expression of the small RNAs. [unreadable] [unreadable] Characterization of specific small, noncoding RNAs[unreadable] A growing focus of the group has been to elucidate the functions of the small RNAs in E. coli. We previously showed that OxyS RNA, whose expression is induced by OxyR in response to oxidative stress, acts to repress translation by basepairing with target mRNAs. OxyS RNA action is dependent on the Sm-like Hfq protein, which functions as a chaperone to facilitate OxyS RNA basepairing with its target mRNAs. We also discovered that the abundant 6S RNA binds and modifies RNA polymerase. Recently, we elucidated the functions of two other small RNAs that bind Hfq and act by basepairing: the 109 nucleotide MicC RNA and the 105 nucleotide GadY RNA. We found that the MicC RNA represses translation of the OmpC outer membrane porin. Interestingly, under most conditions, the MicC RNA shows expression opposite that of the MicF RNA, which represses expression of the OmpF porin. Thus we suggest that the MicF and MicC RNAs act to control the OmpF:OmpC protein ratio in response to a variety of environmental stimuli. In contrast, basepairing between the GadY RNA and the 3?-untranslated region (3? UTR) of the gadX mRNA encoded opposite gadY leads to increased levels of the gadX mRNA and GadX protein. Increased GadX levels in turn result in increased expression of the acid-response genes controlled by the GadX transcription factor. Studies to further characterize the GadY RNA and the roles of other newly-discovered small RNAs are ongoing.[unreadable] [unreadable] Characterization of small ORFs[unreadable] In our genome-wide screens for small RNAs, we found that a number of short RNAs actually encode small proteins. Although small proteins have largely been missed, the few small proteins that have been studied in detail in bacterial and mammalian cells have been shown to have important functions in signaling and in cellular defenses. Thus we established a project to elucidate the functions of E. coli proteins of less than 50 amino acids using many of the approaches the group has used to characterize the functions of small, noncoding RNAs.