Dissection of the beta subunit in the Escherichia coli RNA polymerase into domains by proteolytic cleavage

• Published in: The Journal of biological chemistry 1992-06, Vol.267 (18), p.12813-12819
• Main Authors: SEVERINOV, K, MUSTAEV, A, KASHLEV, M, BORUKHOV, S, NIKIFOROV, V, GOLDFARB, A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Blotting, Western; DNA Transposable Elements; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Enzymes and enzyme inhibitors; Escherichia coli; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Kinetics; Molecular Sequence Data; Promoter Regions, Genetic; Sequence Alignment; Structure-Activity Relationship; Transcription, Genetic; Transferases; Trypsin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)42348-4

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.