Sequence-specific interaction of nascent antiterminator RNA with the zinc-finger motif of Escherichia coli RNA polymerase

Abstract

The N-terminal Zn-finger motif of the β′ subunit of RNA polymerase contains two pairs of invariant cysteines flanking a moderately well-conserved segment of 13 amino acids that is rich in basic residues. Previous work showed that replacement of certain Zn-finger residues prevented transcription antitermination in response to phage HK022 put sites. Nascent put RNA binds to and modifies transcribing polymerase, so that it becomes resistant to termination. To characterize the Zn finger further, we replaced each of the basic residues with alanine and determined the effects of the substitutions on termination, antitermination and cell viability. All the mutants were defective in put-mediated antitermination. The severity of the defect depended on the mutant and on the sequence of the upstream stem-loop of put RNA. Some, but not all, mutants distinguished between put variants that differed in this region. This suggests that the Zn-finger motif interacts directly and specifically with put RNA. All the mutants in the basic residues complemented a temperature-sensitive β′ mutant for cell growth at a non-permissive temperature, and those mutant enzymes that were tested transcribed and terminated normally in vitro on a template that lacked a put site.