Functional complementation between mutations at two distant positions in Escherichia coli RNA polymerase as revealed by second-site suppression.
Subunit-subunit interactions are critical for the assembly of the core of Escherichia coli RNA polymerase. The mutant alpha-subunit C131A is unable to complement the temperature-sensitive alpha-R45C mutant strain, which is defective for binding of the beta-subunit. In vitro reconstitution experiments, however, indicate that the alpha-C131A variant is able to form the intermediate alpha2beta, but is defective in contacting the beta'-subunit. We used this alpha-C131A mutant to isolate a suppressor mutation in the beta'-subunit. Genetic and biochemical characterization of the beta' suppressor indicates the allele-specific nature of its effect. Sequence analysis of the suppressor revealed a single substitution of Gly at position 333, an evolutionarily conserved position in the conserved region C of the beta'-subunit, by Asp. However, the crystal structure of the bacterial RNA polymerase indicates that the primary mutation (alpha-C131A) and its suppressor lie far apart. Thus, we propose that long-range interactions, as in this case, may play an important role in the functional assembly of E. coli RNA polymerase.
Sujatha
S
S
Centre for Cellular and Molecular Biology, Hyderabad, India.
Ishihama
A
A
Chatterji
D
D
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D005998:Glycine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic; D013696:Temperature
D001224:Aspartic Acid; D012321:DNA-Directed RNA Polymerases; D005998:Glycine