Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase.
T7 gene 4, which is required for DNA replication, specifies two proteins whose coding sequences overlap in the same reading frame: the 4A protein, a 566-amino acid primase/helicase, and the 4B protein, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. To study better the individual functions of these two overlapping proteins, we made clones that express both 4A and 4B proteins, only 4B protein, or only what we refer to as the 4A' protein, in which methionine 64 is replaced by leucine, thereby eliminating the 4B initiation codon. These clones provide considerably more gene 4 protein for biochemical analysis than do infected cells. They can also be used to isolate and propagate T7 gene 4 deletion mutants, and we have made T7 mutants which lack all gene 4 coding sequences, or which express 4A' protein but no 4B protein, or 4B protein but no 4A protein. Analysis of these phage mutants shows that 4A' protein without any 4B protein can support essentially normal replication and growth, whereas 4B protein without any 4A protein supports little replication or growth. Apparently, the primase activity of the 4A protein is essential for replication, but the 4B protein is dispensable, presumably because the 4A protein also supplies helicase activity. The mutation at amino acid 64 of 4A' appears to have little effect on 4A function. The rate of replication during normal T7 infection appears to be limited by the amount of gene 4 protein, but too high a level of either 4A or 4B protein is inhibitory to growth.
Rosenberg
A H
AH
Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
Patel
S S
SS
Johnson
K A
KA
Studier
F W
FW
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005814:Genes, Viral; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D015183:Restriction Mapping; D013604:T-Phages / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication
D007527:Isoenzymes; D014764:Viral Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases