Effects of DNA base analogs on transcription termination at the tryptophan operon attenuator of Escherichia coli

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Abstract
A method was devised to specifically incorporate deoxyribonucleotide base analogs in vitro into either strand of the tryptophan (trp) operon attenuator region. The method uses primed synthesis on bacteriophage M13 derivatives carrying cloned trp attenuator DNA. These techniques were used 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, the dramatic effects on 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.cntdot.T or A.cntdot.U base pairing, elicits a significant response: in the template DNA strand, the presence of this analog increases readthrough at the trp attenuator. The analog 5-bromouracil deoxyribonucleotide 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 deoxyribonucleotide 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. The postulated interaction between terminal uridines in mRNA and the template DNA strand in enhancing termination is supported and it is further suggested 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.

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