Recognition mechanisms of the minus-strand origin of phage f1 by Escherichia coli RNA polymerase.
BACKGROUND
The primer RNA for the synthesis of the minus strand of filamentous coliphages is produced by host RNA polymerase at a specific site on the plus strand template. The mechanism used by the enzyme in recognizing the origin is unknown, but minus strand replication requires the holoenzyme form of RNA polymerase. The origin contains two inverted repeats which can form hairpins.


RESULTS
When the origin sequence is drawn arranging the two hairpins horizontally, it resembles a stretch of a mostly double-stranded molecule. The nucleotides protected from nucleases by the holoenzyme were found to be located in two regions on this drawing: one around the 35th nucleotide upstream of the RNA start site, and the other ranging from the 10th nucleotide upstream to the 10th downstream, of the start site. The core enzyme did not show any protection. Mutational analyses of the origin indicated that the base-paired structure in the former region was important for origin activity. In the region around the 10th nucleotide upstream of the start site, specific base(s) in the non-template strand were required for origin activity, while the base sequence of the template strand was irrelevant.


CONCLUSIONS
It is likely that the recognition mechanism of the origin by RNA polymerase shares common features with that of transcriptional promoters.
Higashitani
N
N
Division of Microbial Genetics, National Institute of Genetics, Mishima, Shizuoka-ken, Japan.
Higashitani
A
A
Guan
Z W
ZW
Horiuchi
K
K
D001483:Base Sequence; D003090:Coliphages / Q000235:genetics*; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012313:RNA / Q000096:biosynthesis; D018741:Replication Origin / Q000235:genetics*
D004268:DNA-Binding Proteins; C034477:RNA primers; D012313:RNA; D012321:DNA-Directed RNA Polymerases