Transcription and Translation of Prereplicative Bacteriophage T4 Genes in Vitro

• Published in: The Journal of biological chemistry 1973-08, Vol.248 (15), p.5512-5519
• Main Authors: O'Farrell, Patricia Z., Gold, Lawrence M.
• Format: Article
• Language: English
• Subjects: Amino Acids; Carbon Radioisotopes; Cell-Free System; Chromatography, DEAE-Cellulose; Coliphages - drug effects; Coliphages - metabolism; DNA, Viral - metabolism; DNA-Directed RNA Polymerases - metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli - cytology; Escherichia coli - drug effects; Escherichia coli - enzymology; Kinetics; Protein Biosynthesis; Rifampin - pharmacology; RNA, Viral - biosynthesis; Sodium Dodecyl Sulfate; Subcellular Fractions - metabolism; Templates, Genetic; Time Factors; Transcription, Genetic; Viral Proteins - biosynthesis; Virus Replication
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)43632-6

Bacteriophage T4 DNA was used to direct transcription and translation in vitro in extracts prepared from uninfected Escherichia coli. The radioactive protein products of the cell-free reactions were examined on sodium dodecyl sulfate acrylamide gels. We conclude that the cell-free system prepared from uninfected E. coli has the capacity to synthesize most T4 prereplicative RNAs and proteins. Furthermore, synthesis of those RNAs and proteins occurs in the same temporal order as occurs in vivo during the first minutes after T4 infection. The relative molar yields of early proteins in vitro are similar to the relative yields obtained in vivo. We found no evidence in support of the subdivision of early RNAs into immediate early and delayed early species; promoter recognition in vitro is followed by the elaboration of polycistronic mRNAs with no constraints against promoter distal transcription. A small class of prereplicative proteins was not synthesized efficiently in vitro. Those proteins are derived from the special class of prereplicative cistrons called quasi-lates; these genes in vivo are under the control of promoters first recognized after a delay of about 1½ min. The components required for quasi-late promoter recognition in vivo appear to be missing or nonfunctional in cell-free extracts of uninfected E. coli.