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Showing papers on "Small hairpin RNA published in 1995"


Journal ArticleDOI
TL;DR: It is documented that leaky expression of the T7 RNA polymerase is responsible for the frequently observed loss of the culture's ability to express genes of interest, and it is shown that the inability to achieve detectable expression levels can be overcome by using a tightly repressed expression system.
Abstract: One of the more efficient systems for high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulatable transcription unit supplying the specific T7 RNA polymerase. Using various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase, we document that leaky expression of the latter is responsible for the frequently observed loss of the culture's ability to express genes of interest. We further show that the inability to achieve detectable expression levels can be overcome by using a tightly repressed expression system. We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is attenuated by a series of tandemly arranged transcription terminators. The plasmids also incorporate the phage lambda-derived nutL/N protein antitermination function, allowing conditional reversion of attenuation upon induction. The applicability of the system is illustrated by the strictly regulatable, high-level production of several cytokines of human and murine origin.

118 citations


Journal ArticleDOI
TL;DR: A model for the loop-loop interaction is incorporated which accounts for the importance of positions one and seven and the first two nucleotides of the stem, while providing potentially unique structures for recognition by the RNA one modulator protein.

82 citations


Journal ArticleDOI
TL;DR: Analysis of ancestral sequences of the primitive tRNA molecule seems to indicate that self-alignment of these sequences makes it possible for the 3′-half of the reconstructed molecule to display a significant similarity to the 5′- half, indicating that the two halves of tRNA might be paralogous.

59 citations


Journal ArticleDOI
TL;DR: It is found that the sequences bounded by the hairpins as well as those 3' to them are excluded from particles in packaging reactions, implying that packaging occurs from the 5' end and that the explanation for the facilitation of recombination by thehairpin structures is the lack of entry of the 3' ends rather than a difficulty of progressing through the hairpin by the phage polymerase.
Abstract: Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a procapsid composed of four different proteins. The preformed procapsid is capable of packaging plus-strand transcripts of the genomic segments in an in vitro reaction. Minus-strand synthesis within the procapsid then results in the production of the double-stranded RNA genome. When plus-strand transcripts contain strong hairpin structures near the 3' ends, they are subject to heterologous recombination to remove the hairpins. We now find that the sequences bounded by the hairpins as well as those 3' to them are excluded from particles in packaging reactions. This finding implies that packaging occurs from the 5' end and that the explanation for the facilitation of recombination by the hairpin structures is the lack of entry of the 3' ends rather than a difficulty of progressing through the hairpin by the phage polymerase. Packaging of segment M is dependent on the packaging of segment S. An S segment containing a strong hairpin is able to facilitate the packaging of segment M. This result implies that there is more than one entry pore into the procapsid.

27 citations


Journal ArticleDOI
TL;DR: The results suggest that there are three distinct pausing activities by RNA polymerase during its transcription of the S10 leader, with three corresponding signals in the S 10 leader.

23 citations