Effect of bacteriophage infection on the sulfur-labeling of sRNA.

Abstract

Several examples of viral-induced modifications of cellular ribonucleic acids have been reported. After infection of Escherichia coli with T-even bacteriophage, Sueoka and Kano-Sueokal 2 found quantitative as well as qualitative changes in leucyl-tRNA's when compared to uninfected cells by chromatography on methylated albumin-kieselguhr (.IMAE). ]More recently, Waters and Novelli,3 using reverse phase chromatography, were able to confirm the early changes in leucyltRNA after T2 infection of E. coli and, in addition, observed two new leucyl-tRNA peaks which appeared very late after infection. Wainfan, Srinivasan, and Borek4 have reported on the alteration in the relative' activities of the base-specific methylases after T2 infection.The presence of thiolated bases in sRNA and the demonstration that cell-free extracts catalyze the transfer of cysteine-sulfur to sRNA,5' 6 prompted an inquiry as to whether any changes occurred in sulfur-containing RNA's after viral infection. The present communication describes changes in the 1\IAK chromatographic profiles of S15-labeled sRNA after E. coli infection with T-even bacteriophages. Evidence is offered which indicates that these changes are viral-induced, and that they require protein synthesis to be manifest.Experimental.-Growth of cells and viral infection: E. coli B cells were grown in a medium that contained the following constituents per liter: 2 gm of NH4Cl, 6 gm of NaCl, 0.01 gm of MgCl2, 6 gm of Na2HPO4, 3 gm of KH2PO4, 0.026 gm of Na2SO4, 2 gm of glucose, and 10 mM Tris -HCl of pH 7.5. This medium was further supplemented with 0.04 vol of 3XD medium.7 The cell suspension (2%7 inoculum of an overnight culture) was vigorously shaken in a gyratory apparatus at 370 (generation time approximately 50 min) until a density of 7 X 108 cells/ml was reached, at which time L-tryptophan was added to a concentration of 100 ,gg/ml, and then T4 phage at a ratio of 13 plaque-forming units per cell. Under these conditions, cell death was found to be greater than 99% after 2.5 mim, and the number of infective centers was 9.5%/ or more of the bacterial cell count. The infected cell suspension was shaken at 370, and after a given time infection was stopped by the rapid addition of 0.5 Al NaN3 (2 ml per 100 ml of culture) and crushed ice, and rapid cooling in an ice-salt water bath to 4°. The cells were collected by centrifugation and either handled immediately for RNA extraction or stored frozen at -20°. Infection with the coliphages T2, T7, MS2, and OX174p-was carried out in a similar manner except that the infecting ratio of phage/cell was 10, and E. coli K12W1485 and E. coli C were used as the respective hosts for M\S2 and qX174p-phages.S35-labeling of cells: E. coli cells (either infected or uninfected) were labeled with S35 by the addition of radioactive Na2SO4 (New England Nuclear Corp.) to the culture medium. Cells were collected as described above except that nonradioactive 0.5 Al Na2SO4 (2 ml per 100 ml of culture) was added to the suspension at the same time as the azide. "P...

Mechanism of protein biosynthesis.

“…The studies of cysteinedependent thiolation of tRNA (163) provided another tool for testing tRNA after phage infection. In uninfected and phage-infected cells grown on IS-sodium sulfate, all sulfur-containing RNA proved to be 4S RNA, the elution profiles of which on MAK columns were different for tRNA from infected and uninfected cells (163). The material of the major new peak from infected cells does not hybridize with E. coli DNA, but it hybridizes with T4 DNA; T4 mRNA and E. coli tRNA do not compete in this reaction (407).…”

Coding by T4 phage DNA of soluble RNA containing pseudouridylic acid.

“…20 ' rpm in a Spinco rotor SW-39. 10 20 30 Tube number that the T4 P32-sRNA (cpm) and E. coli sRNA (OD) have identical sedimentation profiles and consequently the same sedimentation constants, when centrifuged in a linear sucrose gradient.…”

Transfer RNA coded by the T4 bacteriophage genome.

“…Modifications in transfer RNA (tRNA) chromatographic patterns after virus infection have been described by several laboratories.1-5 In a recent communication, we described distinct alterations in the chromatographic behavior of pulselabeled S35-tRNA after infection of Escherichia coli B with T-even bacteriophage. 6 The experimental data suggested that the observed S35 transition from the "normal" to the "infected" tRNA profile was virus-induced and that these changes were not the result of host tRNA modifications. The "infected" and "uninfected" sulfur-labeled profiles of tRNA as observed by chromatography on methylated albumin kieselguhr (iVIAK) are shown in Figure 1 where we have arbitrarily designated the radioactive regions as peaks 1, 2, and 3.…”