Rho-independent terminators without 3' poly-U tails from the early region of actinophage øC31.
Previous work has identified three intergenic regions from the early region of actinophage øC31 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 eta, etb and etc. Transcripts through eta-c would be expected to form stable RNA stem-loops but would lack poly-U tails. Eta-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 eta-c from the thiostrepton-inducible tipA promoter by measuring the expression of a downstream reporter gene (aphII). In pUGT1 etb was at best a minor terminator in vivo whilst eta and etc exhibited strong termination activity. In vitro termination was assayed using templates containing a synthetic promoter recognised by E.coli RNA polymerase and fragments containing eta-c inserted downstream. All three terminators stimulated the formation of 3' ends in the promoter-distal arm of the inverted repeats with efficiencies eta > etc > etb. 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 etb was unaffected by the presence or absence on the transcription template of CIR3. CIR4 forms the central 17 bp of etc and a 37 nt deletion which eliminated this stem-loop abolished termination in vivo and in vitro. Eta was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of eta inhibited termination whilst an oligo antisense to CIR5 was ineffective and an oligo targeted further upstream enhanced termination. Taken together these data show that eta-c are intrinsic, rho-independent terminators of varying efficiencies despite the absence of a poly-U tail.
Ingham
C J
CJ
Robertson Laboratory of Biotechnology, University of Glasgow, UK.
Hunter
I S
IS
Smith
M C
MC
D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D017124:Conserved Sequence / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012317:RNA Phages; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics / Q000821:virology*; D013728:Terminator Regions, Genetic / Q000235:genetics*; D013883:Thiostrepton / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics
D012333:RNA, Messenger; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases; D013883:Thiostrepton