Mutations in and monoclonal antibody binding to evolutionary hypervariable region of Escherichia coli RNA polymerase beta' subunit inhibit transcript cleavage and transcript elongation.
A 190 amino acid-long region centered around position 1050 of the 1407-amino acid-long beta' subunit of Escherichia coli RNA polymerase (RNAP) is absent from homologues in eukaryotes, archaea and many bacteria. In chloroplasts, the corresponding region can be more than 900 amino acids long. The role of this hypervariable region was studied by deletion mutagenesis of the cloned E. coli rpoC, encoding beta'. Long deletions mimicking beta' from Gram-positive bacteria failed to assemble into RNAP. Mutants with short, 40-60-amino acid-long deletions spanning beta' residues 941-1130 assembled into active RNAP in vitro. These mutant enzymes were defective in the transcript cleavage reaction and had dramatically reduced transcription elongation rates at subsaturating substrate concentrations due to prolonged pausing at sites of transcriptional arrest. Binding of a monoclonal antibody, Pyn1, to the hypervariable region inhibited transcription elongation and intrinsic transcript cleavage and, to a lesser degree, GreB-induced transcript cleavage, but did not interfere with GreB binding to RNAP. We propose that mutations in and antibody binding to the hypervariable, functionally dispensable region of beta' inhibit transcript cleavage and elongation by distorting the flanking conserved segment G in the active center.
Zakharova
N
N
Waksman Institute, Piscataway, New Jersey 08854, USA.
Bass
I
I
Arsenieva
E
E
Nikiforov
V
V
Severinov
K
K
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001419:Bacteria / Q000201:enzymology; D001666:Binding Sites, Antibody; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D019143:Evolution, Molecular; D014644:Genetic Variation; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases