N Antiterminator

Background Information

The N antiterminator is one of three critical proteins that we chose to regulate. N is an antiterminator that allows RNA polymerase to code through regions of the genome it would otherwise be unable to transcribe. As depicted in the diagram below, there is a termination sequence early in the λ genome, which causes the mRNA transcript to terminate in the absence of N. However, N binds a specific sequence called Nut and modifies the RNA polymerase. Once modified, the polymerase can ignore the termination sequence downstream of the Nut site, allowing the elongation of the mRNA transcript.

Transcription in the absence of N terminates at Nut

Transcription in the presence of N continues through genes necessary for lysis

Role in Lambda Life Cycle

PL and PR are the first promoters activated during λ infection. N is transcribed from PL, so it acts early in the phage’s developmental cycle. As shown below, N is necessary to transcribe genes necessary for both lysis and lysogeny. In the absence of N, the phage cannot go into either pathway and it will not be infectious.

File:Early events.png

Original Goals

The objective of this subproject project was to determine the necessary concentration of N protein required for lysis. The N gene was placed behind a tetracycline-inducible promoter in a low copy (pSB2K3) plasmid. Addition of anhydrotetracycline (aTc) would induce the production of N. For more details, please see our list of constructs here. This construct was transformed into D1210 E. coli and titered using N amber mutant phage.

Status and Future Plans

Results of titering N amber mutant phage on D1210 containing the tet-regulated N construct.

Relevant Protocols

• Titering
• Liquid Lysis Assay