The C-Terminal 12 Amino Acids of ςN Are Required for Structure and Function

The ςN protein is an alternative ς subunit of bacterial RNA polymerase. We investigated the role of a 12-amino-acid “tail” at the C-terminus of Klebsiella pneumoniae ςN, which was predicted to be largely surface-exposed and to be mostly loop (that is not α-helical or β-strand). Deletion of this tail from N-terminal hexahistidine-tagged ςN led to loss of ςN-dependent transcription activity in vivo. We overexpressed and purified this deletion-mutant protein for in vitro characterization. The purified deleted protein showed decreased RNA polymerase core- and DNA-binding activities compared to the full-length protein and transcription activity was greatly impaired. Furthermore, evidence from circular dichroism and protease digestion experiments together suggested that deletion of the C-terminus tail resulted in a loss of conformational constraint in the protein. We discuss a possible structural role for the 12 amino acids at the C-terminus of ςN.