Dual-function Spot 42 RNA encodes a 15-amino acid protein that regulates the CRP transcription factor

Abstract

The 109-nucleotide Spot 42 RNA whose transcription is repressed by the cyclic adenosine monophosphate receptor protein (CRP) is one of the best-characterized base-pairing small RNAs (sRNAs) in Escherichia coli. Consistent with high levels of Spot 42 in glucose-grown cells, the RNA blocks the expression of transporters and enzymes involved in the utilization of nonpreferred carbon, such as galactose. We now document that Spot 42 also encodes a 15-amino acid protein denoted SpfP. Previous studies showed that overexpression of Spot 42 reduces growth in galactose and other nonpreferred carbon sources. Overexpression of just the small protein from a Spot 42 derivative deficient in base-pairing activity also prevented growth on galactose, revealing that the sRNA and protein impact the same pathway. Copurification experiments showed that SpfP binds CRP. This binding blocks the ability of CRP to activate specific genes, such as the galETKM operon, impacting the kinetics of induction when cells are shifted from glucose to galactose medium. Thus, the small protein reinforces the feedforward loop regulated by the base-pairing activity of the Spot 42 RNA.