RHO-INDEPENDENT TERMINATORS WITHOUT 3' POLY-U TAILS FROM THE EARLY REGION OF ACTINOPHAGE-PHI-C31

Previous work has identified three intergenic regions from the early r egion of actinophage phi C31 where transcription was either terminated or the mRNA was processed. Here we show using in vivo and in vitro ap proaches that these regions contain rho-independent terminators design ated et(a), et(b) and et(c). Transcripts through et(a-c) would be expe cted 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' GG GGCTT. A Streptomyces 'terminator probe' vector, pUGT1, was constructe d 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 st rong termination activity. In vitro termination was assayed using temp lates containing a synthetic promoter recognised by E.coli RNA polymer ase and fragments containing et(a-c) inserted downstream. All three te rminators stimulated the formation of 3' ends in the promoter-distal a rm of the inverted repeats with efficiencies et(a) > et(c) > et(b). As all three terminators either overlap with or lie close to sequences w hich interact with phage repressor proteins (conserved inverted repeat s, CIRs) and these can potentially form stem-loop structures in RNA, t he 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 de letion which eliminated this stem-loop abolished termination in vivo a nd in vitro. Et(a) was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of et(a) inh ibited termination whilst an oligo antisense to CIR5 was ineffective a nd an oligo targeted further upstream enhanced termination. Taken toge ther these data show that et(a-c) are intrinsic, rho-independent termi nators of varying efficiencies despite the absence of a poly-U tail.