The C-terminal 12 amino acids of sigma(N) are required for structure and function.
The sigma(N) protein is an alternative sigma subunit of bacterial RNA polymerase. We investigated the role of a 12-amino-acid "tail" at the C-terminus of Klebsiella pneumoniae sigma(N), which was predicted to be largely surface-exposed and to be mostly loop (that is not alpha-helical or beta-strand). Deletion of this tail from N-terminal hexahistidine-tagged sigma(N) led to loss of sigma(N)-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 sigma(N).
Studholme
D J
DJ
Department of Biology, Imperial College of Science Technology and Medicine, Sir Alexander Fleming Building, London, SW7 2AZ, United Kingdom.
Finn
R D
RD
Chaney
M K
MK
Buck
M
M
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D051877:RNA Polymerase Sigma 54; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017384:Sequence Deletion; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54