Overexpression of N antitermination proteins of bacteriophages lambda, 21, and P22: loss of N protein specificity.
The N protein of bacteriophage lambda (N lambda) modifies Escherichia coli RNA polymerase in such a way that it transcribes through termination signals, a process called antitermination. N antitermination normally occurs only if the template contains a specific utilization or nut site upstream of the terminators and only in the presence of host-encoded Nus proteins. The lambda-related phages 21 and P22 produce N analogs, N21 and N22, but these require different nut sites and show a different pattern of functional interaction with one of the Nus factors, NusA, according to whether this protein is of E. coli or Salmonella origin (NusAEc or NusASal). We report the overproduction of N lambda, N21, or N22, each of which was induced by isopropyl-beta-D-thiogalactopyranoside at 37 degrees C from its cloned position downstream from ptac on a high-expression plasmid, each in a host that provided NusAEc or NusASal. Overproduction of each of these N proteins resulted in relaxed specificity for nut, which was shown by the ability to complement N mutants of heterologous phages; NusA specificity was determined by the N type that was present in these complementation tests. We also observed that excess N was able to suppress transcriptional polarity in the particular case of cloned 'trpA, the last gene of the tryptophan operon, although there was no effect on polarity within chromosomal trpE. Such polarity is attributed to the presence of cryptic intragenic terminators that become exposed in the absence of translation. Because there is no known nut site cis to 'trpA, we suggest that the 'trpA segment itself fortuitously contains a nut sequence that is able to function with excess N of any of the types tested and with either NusAEc or NusASal. We also found that excess N of any specificity, or even inactive N with missense mutation, could cause an increase in the level of NusAEc or NusASal, possibly because interaction between N and NusA, but independent of nut, whether functional or not, interferes with the autoregulation of NusA synthesis. These observations highlight the importance of protein concentration for the specificity of interactions both with other proteins and with nucleic acids. They also indicate that the interaction between N and NusA requires nut participation both for specificity and functionality.
Franklin
N C
NC
Department of Biology, University of Utah, Salt Lake City 84112.
Doelling
J H
JH
D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012483:Salmonella Phages / Q000235:genetics*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins
C059417:N protein, Bacteriophage lambda; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins