Dissection of the beta subunit in the Escherichia coli RNA polymerase into domains by proteolytic cleavage.
The 1342 amino acid long beta subunit of Escherichia coli RNA polymerase includes a dispensable region (residues 940-1040) that is absent in homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria (Borukhov, S., Severinov, K., Kashlev, M., Lebedev, A., Bass, I., Rowland, G. C., Lim, P.-P., Glass, R. E., Nikiforov, V., and Goldfarb, A. (1991) J. Biol. Chem. 266, 23921-23926). Genetic disruption of this region by in-frame deletion or insertion sensitizes the beta subunit in assembled RNA polymerase molecules to attack by trypsin. We demonstrate that RNA polymerase with the beta polypeptide cleaved in the dispensable region retains normal in vitro activity. Moreover, the RNA polymerase activity is completely restored after denaturation and reconstitution of the enzyme carrying cleaved beta subunit indicating that its carboxyl- and amino-terminal parts fold and assemble into RNA polymerase as separate entities.
Severinov
K
K
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Mustaev
A
A
Kashlev
M
M
Borukhov
S
S
Nikiforov
V
V
Goldfarb
A
A
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004251:DNA Transposable Elements; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014357:Trypsin
D004251:DNA Transposable Elements; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin