The Escherichia coli sRNA GcvB regulates several genes involved in transport of amino acids and peptides (sstT, oppA, dppA, and cycA). Two regions of GcvB from nt +124 to +161 and from nt +73 to +82 are complementary with essentially the same region of the cycA mRNA. Transcriptional fusions of cycA to lacZ showed the region of cycA mRNA that can pair with either region of GcvB is necessary for regulation by GcvB. However, mutations in either region of gcvB predicted to disrupt pairing between cycA mRNA and GcvB did not alter expression of a cycA-lacZ translational fusion. A genetic analysis identified nts in GcvB necessary for regulation of the cycA-lacZ fusion. The results show that either region of GcvB complementary to cycA mRNA can basepair with and independently repress cycA-lacZ and both regions need to be changed to cause a significant loss of repression. 1. Introduction The E. coli gcvB gene encodes a sRNA of 206 nts [1]. Transcription of gcvB is activated by GcvA when cellular glycine is high and repressed by GcvA when glycine is limiting; repression by GcvA requires the accessory GcvR protein [1]. GcvB regulates cycA, encoding the glycine transport protein [2]. Thus, GcvB regulates its own synthesis by controlling the level of glycine transported into the cell. A ΔgcvB strain shows constitutive synthesis of OppA and DppA, the periplasmic binding protein components of the two major peptide transport systems, SstT, a serine transport system, and CycA, a glycine transport system [1–4]. The Salmonella enterica serovar Typhimurium GcvB also regulates OppA and DppA levels and several other genes involved in transport of polar and branched amino acids and general amino acid metabolism [5, 6]. Evidence suggests GcvB regulates its target mRNAs by an antisense mechanism, basepairing with the mRNAs to prevent translation initiation [3–6]. Although it is unclear how extensive pairing between a sRNA and a mRNA must be, research indicates one or two regions of 8-9 basepairs is sufficient for regulation [7]. In cases where basepairing interactions occur, the RNA chaperone Hfq is required, likely to alter RNA secondary structures or to bring together sRNAs and target mRNAs, increasing local RNA concentrations [8–11]. Hfq binds GcvB [11, 12], stabilizing the RNA [5, 13], and loss of Hfq results in the loss of repression of GcvB target mRNAs [2, 4, 5, 13]. For sRNAs studied in detail that regulate by an antisense mechanism, often a single basepair change in the sRNA or its target mRNA results in a loss of regulation by the sRNA (e.g., the sRNA SgrS and its
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