Previous work has identified three intergenic regions from the early region of actinophage phi C31 where transcription was either terminated or the mRNA was processed. Here we show using in vivo and in vitro approaches that these regions contain rho-independent terminators designated et(a), et(b) and et(c). Transcripts through et(a-c) would be expected to form stable RNA stem-loops but would lack poly-U tails. Et(a-c) contained part or all of the conserved sequences 5' AGCCCC and 5' GGGGCTT. A Streptomyces 'terminator probe' vector, pUGT1, was constructed and used to assay the efficiency of termination of transcription by et(a-c) from the thiostrepton-inducible tipA promoter by measuring the expression of a downstream reporter gene (aphll). In pUGT1 et(b) was at best a minor terminator in vivo whilst et(a) and et(c) exhibited strong termination activity. In vitro termination was assayed using templates containing a synthetic promoter recognised by E.coli RNA polymerase and fragments containing et(a-c) inserted downstream. All three terminators stimulated the formation of 3' ends in the promoter-distal arm of the inverted repeats with efficiencies et(a) > et(c) > et(b). As all three terminators either overlap with or lie close to sequences which interact with phage repressor proteins (conserved inverted repeats, CIRs) and these can potentially form stem-loop structures in RNA, the effect of CIRs on termination was also investigated. Termination at et(b) was unaffected by the presence or absence on the transcription template of CIR3. CIR4 forms the central 17 bp of et(c) and a 37 nt deletion which eliminated this stem-loop abolished termination in vivo and in vitro. Et(a) was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of et(a) inhibited termination whilst an oligo antisense to CIR5 was ineffective and an oligo targeted further upstream enhanced termination. Taken together these data show that et(a-c) are intrinsic, rho-independent terminators of varying efficiencies despite the absence of a poly-U tail.
|Number of pages||7|
|Journal||Nucleic Acids Research|
|Publication status||Published - 11 Feb 1995|
- TRANSCRIPTION TERMINATION
- CLONING VECTORS
- PHAGE PHI-C31