Bacteriophage-Specific Proteins in E. Coli Infected With an Rna Bacteriophage

Abstract

The f2 group of RNA coliphages (f2, MS2, R17, fr), first isolated by Loeb and Zinder,1 are closely related, small viruses with single-stranded RNA of about one million molecular weight (for a review of RNA phages, see ref.2). The number of proteins encoded in the viral RNA is therefore small. To date, two phage-specified proteins have been identified chemically or enzymatically in infected cells, the phage coat subunit and an RNA synthetase,3-5 and the presence of a third protein has been inferred from genetic experiments.6' 7 By analysis of radioactive proteins present in phage-infected cells, Haywood and Sinsheimer have provided evidence for the presence of four phage-specific, noncoat proteins in cells infected with coliphage MS2.8We have recently described a method for labeling phage-specific proteins in E. coli infected with MS2 under conditions where host protein synthesis is virtually eliminated by actinomycin.9 Such cells synthesize predominantly phage coat protein, the bulk of which is made later in the infectious cycle than is noncoat protein. The identification of noncoat protein in those studies depended on the incorporation of radioactive histidine, which is not present in the virus coat. In this report we describe the fractionation of phage-specific proteins by polyacrylamide gel electrophoresis. The results show that in addition to phage coat, three discrete proteins are present in infected cells. One of these proteins is missing in cells infected with an amber mutant with a defect in phage maturation. In confirmation of our earlier results,9 it was found that coat protein constitutes about 80 per cent of the total phage-specific protein synthesized in the infected cell. In addition, preliminary findings are presented on the identification of noncoat proteins made in cell extracts under the direction of MS2 RNA and on the presence of minor proteins in the phage particle itself.Materials and Methods.-The source of most of the bacterial strains and of bacteriophage MS2 has been given previously.' 10 Bacteriophage f2 and an amber mutant of f2 (sus-11) were kindly supplied by N. D. Zinder, as were E. coli K38, a nonpermissive host, and E. coli K37, an ambersuppressing strain.'The procedures for sensitization of E. coli C3000 to actinomycin and for incorporation of amino acids into protein after infection with MS2 have been described.9To prepare the radioactive protein for electrophoresis, the labeled cells were centrifuged at 10,000 g for 5 min, washed once with cold saline containing nonlabeled amino acids, and the cell pellet was resuspended in a small volume of water. The cells were broken by sonication for 3 min and all labeled protein was solubilized by addition of 1/lo vol of glacial acetic acid, freshly deionized urea to a concentration of 0.5 M, sodium dodecyl sulfate (SDS) to a concentration of 1.0%, and mercaptoethanol to a concentration of 0.01 M.12 After incubation at 370 for 60 mi, the solution was centrifuged at 30,000 g for 10 min, and the supernate, which contained all the radioactive p...

Translational activation in coliphage qβ: on a polycistronic messenger RNA, repression of one gene can activate translation of another 1 1Edited by M. Gottesman

“…Expression of maturation/lysis protein in RNA coliphage Qb is tightly regulated, and is primarily required late in the infectious cycle for both capsid formation and host cell lysis (Nathans et al, 1966;Karnik & Billeter, 1983;Winter & Gold, 1983; for a review, see van Duin, 1988). Translational downregulation of the Qb maturation gene (nucleotides 61 through 1320) has been shown to involve an extremely stable, long-range RNA loop structure comprising nearly 470 nucleotides at the 5 H end of the genome (de Smit & van Duin, 1990, 1994.…”

Structure of the Poly(G) Polymerase Component of the Bacteriophage f2 Replicase

“…and electrophoretic behavior of the f2 and MS2 proteins has received much attention (19,25,26). Of the three known viral proteins, the largest has been identified as the product of the replicase cistron and is known to be hyperproduced by coat mutants of MS2 and f2 (25,27).…”

“…cells, but not in uninfected cells after actinomycin (25) or rifampicin treatment (P. Model, N. Fedoroff, unpublished observations). Fig.…”

Cell-free protein synthesis in a microfabricated reactor

“…The detailed mechanism of the translation of MS2 RNA has been well investigated both in vivo [11] and in vitro [12] . Nathans [13] has shown that the ®ngerprint of the product in E. coli cell-free system shows the same pattern of that of the coat protein expressed in E. coli cell in which the coat protein can be measured as a major spot.…”