Effects of DNA base analogs on transcription termination at the tryptophan operon attenuator of EScherichia coli.
We have devised a method to specifically incorporate deoxyribonucleotide base analogs in vitro into either strand of the tryptophan (trp) operon attenuator region, using primed synthesis on bacteriophage M13 derivatives carrying cloned trp attenuator DNA. We have employed these techniques to extend previous studies implicating both RNA-RNA and RNA-DNA interactions in transcription termination in an attempt to determine the nature of the contribution from the template DNA molecule in termination regions. In general, we find that the dramatic effects upon transcription termination seen with base analog incorporation into mRNA do not occur when similar analogs are incorporated into the DNA. Only the analog 2,6-diaminopurine deoxyribonucleotide triphosphate (dDapTP), which strengthens A.T or A.U base pairing, elicits a significant response: in the template DNA strand, the presence of this analog increases read-through at the trp attenuator. The analog 5-bromouracil deoxyribonucleoside triphosphate (BrdUTP), which also strengthens pairing with its complementary base, has no detectable effect on termination when it is placed in either strand of the trp attenuator or the mutant attenuator trp a1419. Surprisingly, though the analog 5-iodocytosine deoxyribonucleoside triphosphate (IdCTP) does not affect termination, it has a great effect on initiation of transcription, depressing trp promoter activity as well as stimulating transcription from other regions. These results support the postulated interaction between terminal uridines in mRNA and the template DNA strand in enhancing termination and suggest that there are no significant additional contributions from the DNA. In addition, the novel use of M13 derivatives for incorporating analogs into the DNA on a preparative scale provides a technique for introducing mutations in a general but controlled fashion as a new means for studying other regulatory regions.
D004269:DNA, Bacterial / Q000502:physiology*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D003845:Deoxycytosine Nucleotides / Q000494:pharmacology; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D009876:Operon; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects; D014364:Tryptophan / Q000096:biosynthesis*
D004269:DNA, Bacterial; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003856:Deoxyuracil Nucleotides; C032549:bromodeoxyuridine triphosphate; C033845:5-iodocytosine deoxyribonucleoside triphosphate; C033846:2-amino-2'-deoxyadenosine 5'-triphosphate; D014364:Tryptophan