An RNA polymerase α subunit mutant impairs N‐dependent transcriptional antiterminationin Escherichia coli

Abstract

SummaryWe show that the rpoA341 mutation in the gene encoding the ␣ subunit of Escherichia coli RNA polymerase results in a decreased level of transcripts originating from the lytic promoters p L and p R of infecting phage. However, using lacZ fusions we demonstrate that initiation of transcription from both p L and p R is not impaired in the rpoA341 host. Rather, it is the level of the longer, antiterminated p L -and p R -derived transcripts which is altered: the activity of ␤-galactosidase in bacteria harbouring a source of N and a p L -nutL-t L1 -t I -lacZ or p R -nutR-t R1 -lacZ fusion is considerably lower in the rpoA341 mutant relative to the rpoA + strain. In the absence of the antiterminator protein N no difference is observed in the level of longer p R -derived transcripts between wild-type (rpoA + ) and mutant (rpoA341) hosts. Although synthesis of N appears to be similar in both phageinfected rpoA + and rpoA341 cells, overexpression of the N gene leads to restoration of wild-type levels of the longer p L -and p R -derived transcripts in the mutant host. While this mutation does not appear to affect vegetative phage growth in nus + backgrounds, in combination with certain nus mutations it retards lytic development. Therefore, we conclude that the rpoA341 mutation specifically interferes with the function of the N-antitermination complex, suggesting that the C-terminal domain of the RNA polymerase ␣ subunit may play an important role in N-dependent transcriptional antitermination.

ClpP/ClpX-mediated degradation of the bacteriophage λ O protein and regulation of λ phage and λ plasmid replication

“…1) or both λ plasmid and a plasmid overexpressing the N gene. As a control strain, we used the rpoA341 mutant, which was previously shown to be defective in N-dependent anti-termination (Obuchowski et al 1997). As expected, we did not observe any significant effect of the provision of the N protein in this mutant (Table 4).…”

“…The single copy p R -lacZ and p L -lacZ fusions have been described previously (Obuchowski et al 1997).…”

Escherichia coli RNA Polymerase Mutations Located Near the Upstream Edge of an RNA:DNA Hybrid and the Beginning of the RNA-exit Channel are Defective for Transcription Antitermination by the N Protein from Lambdoid Phage H-19B

“…RNA polymerase mutations in b [19][20][21][22] and a subunits, [23][24][25] defective in supporting the growth of l phage as well as impaired for N-mediated antitermination, have been reported. Here, we describe the isolation and characterization of RNAP mutants near the upstream edge of an RNA:DNA hybrid and at the beginning of the RNA-exit channel of the EC that are defective specifically for transcription antitermination mediated by the N protein of a lambdoid phage, H-19B.…”

“…11,12 N interacts with nut RNA through its N-terminal ARM sequences and with the EC through its C-terminal domain. 18 Although RNAP mutants defective in l phage growth have been reported, [19][20][21][22][23][24][25] the interacting surface on the EC for N has remained elusive, as has the mechanism by which N imparts the antitermination property to RNAP.…”

Regulation of replication of λ phage and λ plasmid DNAs at low temperature

“…6). Despite impaired N-dependent antitermination (Obuchowski et al 1997c) phage k lytic development is normal in the rpoA341 mutant (Szalewska-Palasz et al 1996); we therefore decided to use this strain as a host in experiments on the k lytic development in cells that overproduce CII and CIII at 30°C. We found that overexpression of cII and cIII causes a dramatic inhibition of phage kcIb2 lytic growth (Fig.…”