Characterization of the prohead-pRNA interaction of bacteriophage phi 29.
The small prohead RNA (pRNA) of the Bacillus subtilis bacteriophage phi 29 is essential for ATP-dependent packaging of viral DNA. The 174-, 124-, and 120-residue forms of pRNA produced in vitro using T7 RNA polymerase were equivalent in prohead binding and DNA packaging activity to pRNAs produced in phi 29-infected cells. pRNA binding to proheads, characterized by the use of Northern hybridization and filter binding assays, was specific, rapid, and irreversible in the presence of 10 mM Mg2+. Proheads produced in phage-infected cells carried 5.8 +/- 2.7 copies of pRNA, and proheads assembled in Escherichia coli in the absence of pRNA bound 6.0 +/- 3.5 copies of pRNA. Footprints of proheads on pRNA generated with the ribonucleases A, T1, and V1 showed that nucleotides 22-84, 5' to 3', were protected from ribonuclease attack. Enhanced cleavage at nucleotides 37-40 with ribonuclease V1 suggested a conformational change of pRNA upon prohead binding.
Reid
R J
RJ
Department of Genetics and Cell Biology, University of Minnesota, Minneapolis 55455.
Bodley
J W
JW
Anderson
D
D
D017103:Bacillus Phages / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002213:Capsid / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral