Regulation of the Expression of the N Gene of Bacteriophage Lambda

Abstract

A quantitative assay for the N protein of bacteriophage X has been used to study the in vivo regulation of N gene expression. The assay makes use of the observation that in a cell-free protein-synthesizing system from Escherichia coli programmed with XN-DNA the X endolysin is made only if N protein is added to the reaction.The rate of synthesis of N protein in vivo is negatively controlled by the products of the CI and tof genes of the phage. Furthermore, N protein activity is extremely unstable in vivo. During normal cell growth at 350, the halflife of N protein is about 2 min.The protein product of the N gene of phage X is a positive control element required for the expression of almost all other phage genes (1-5). In a gene N-defective phage, or in cells infected in the presence of chloramphenicol-an inhibitor of protein synthesis-phage transcription is almost entirely restricted to the region of nonhomology between phages A and Ximm2' (see Fig. 1) (6-11). The synthesis of an active N protein allows transcription to occur in all other regions of the genome. However, N protein cannot by itself efficiently cause transcription of any region of the genome if initiation of transcription at the early promotors PL and PR (where the subscripts refer to left and right) is prevented by the action of A repressor at the operators . Roberts has suggested that the function of N protein may be to prevent termination of transcription that had originally initiated, independently of N protein action, at the promoters PL and PR (15).I have recently described a direct assay for N protein (16). It is based on the observation that a cell-free protein-synthesizing system programmed with XN-DNA makes the gene R endolysin only if N protein is added to the reaction. The assay is used here to study the in vivo regulation of the expression of the N gene. METHODSMaterials. An Escherichia coli K12 strain JG148 containing lac and ara deletions was used as a source of cell-free extract for in vitro protein synthesis. The DNA used to program the in vitro system in all experiments was derived from the phage XN7N53CI557S7. The methods used for DNA preparation and cell-free protein synthesis were modified only slightly from those of Zubay et al. (17), and have been described (16). In these experiments the source of N protein was an E. coli K12 strain lysogenic for one of the heat-inducible prophages ACIn87X13, XCIs57Ps8c7R6, or ACI867bio11 H1. ACIss7X,3 was obtained from Jan Pero and XCI857bio11 H1 from Helen Greer.AC8I57PsoS7R5 is a recombinant between ACI857P80, obtained from Paul Chadwick, and AS7R5, obtained from Ira Herskowitz. The method used to prepare extracts for N protein assay has been described (16).The Endolysin Assay. The endolysin of phage A can be assayed by measurement of the optical density decrease (lysis) of a suspension of EDTA-sensitized E. coli cells (16,18

Independent Ligand-Induced Folding of the RNA-Binding Domain and Two Functionally Distinct Antitermination Regions in the Phage λ N Protein

“…Historically, N has been difficult to purify because of its folding of N could have an important regulatory role. Partial proteolysis would leave boxB RNA occupied by very short in vivo half-life ‫2ف(‬ min) (Schwartz, 1970;Greenblatt, 1973). Clearly, as a disordered protein, N is an amino-terminal N fragment, preventing the binding of functional N protein and, therefore, blocking antiter-an ideal target for the lon protease, which is the primary protease for misfolded proteins in E. coli (Gottesman, mination.…”

Little Lambda, Who Made Thee?

“…Using an S30 transcription-translation system, Jack Greenblatt (working at Harvard at the time) demonstrated a requirement for N to synthesize endolysin from a DNA template (78). Using this assay, he showed that CI and Cro downregulated N expression (79). Furthermore, N itself was highly unstable (175 21 indicated another remarkable feature of the N reaction.…”

Energy-dependent degradation of lambda O protein in Escherichia coli

“…These proteins are subjected to rapid degradation by various proteases, some of which are energy dependent (12). Among the proteins which are subjected to rapid degradation in E. coli are several proteins of bacteriophage X which regulate the life cycle of the phage (6,13,14,19,25,26). For example, the antitermination protein N of bacteriophage X is degraded by the product of the E. coli lon gene, which is an ATP-dependent protease (22).…”