Selection and characterization of randomly produced mutants of gene V protein of bacteriophage M13.
Gene V protein of bacteriophage Ff (M13, f1, fd) is a master regulator of phage DNA replication and phage mRNA translation. It exerts these two functions by binding to single-stranded viral DNA or to specific sequences in the 5' ends of its target mRNAs, respectively. To study the structure/function relationship of gene V protein, M13 gene V was inserted in a phagemid expression vector and a library of missense and nonsense mutants was constructed by random chemical mutagenesis. Phagemids encoding gene V proteins with decreased biological activities were selected and the nucleotide sequences of their gene V fragments were determined. Furthermore, the mutant proteins were characterized both with respect to their ability to inhibit the production of phagemid DNA transducing particles and their ability to repress the translation of a chimeric lacZ reporter gene whose expression is controlled by the promoter and translational initiation signals of M13 gene II. From the data obtained, it can be deduced that the mechanism by which gene V protein binds to single-stranded DNA differs from the mechanism by which it binds to its target sequence in the gene II mRNA.
Stassen
A P
AP
Laboratory of Molecular Biology, University of Nijmegen, The Netherlands.
Zaman
G J
GJ
van Deursen
J M
JM
Schoenmakers
J G
JG
Konings
R N
RN
D001435:Bacteriophages / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011897:Random Allocation; D012641:Selection, Genetic; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D014764:Viral Proteins; C057816:gene 5 protein, Enterobacteria phage M13