Cruciform structure of a DNA motif of parvovirus minute virus of mice (prototype strain) involved in the attenuation of gene expression

Abstract

It has previously been reported that the region between nucleotides 259 and 383 immediately downstream from the P4 early promoter of parvovirus minute virus of mice, prototype strain (MVMp) is responsible for transcriptional attenuation. Attenuation results from the premature pausing of RNA polymerase II within this sequence (designated to as att) and seems to depend on potential RNA secondary structure. To assess the attenuation capacity of att under near physiological conditions, the early transcription unit of MVMp was replaced by the chloramphenicol acetyltransferase reporter gene under control of the early P4 promoter, in the presence or absence of att. The resulting recombinant vectors were encapsidated in parvovirus particles and replicated in cells after co-infection with the wild-type virus. The att fragment reduced the rate of expression of the reporter gene by approximately threefold, confirming previously reported data from transfection experiments performed in the same cellular system. This attenuation factor is unexpectedly high, considering that the 'readthrough' fold of the nascent viral transcript is thermodynamically more stable than the 'attenuation' configuration. In an attempt to elucidate this point, we sought for the presence of secondary structures in the template DNA molecule. In vitro nuclease probing of viral dsDNA revealed that the att fragment had a cruciform configuration with both complementary strands folding into the computer-predicted stem-loop 'attenuation' structure. These observations lead us to propose that the secondary structure of the DNA template may prompt the formation of the 'attenuation' stem-loop in nascent mRNAs by bringing corresponding self-complementary sequences into close proximity.