Transcription at bacteriophage T4 variant late promoters. An application of a newly devised promoter-mapping method involving RNA chain retraction.

Bacteriophage T4 late promoters display a conserved sequence that extends over about 18 base pairs, in which the central 8-base pair sequence (TATAAATA in the nontranscribed strand) is absolutely conserved. Transcription by T4-modified RNA polymerase in vitro nevertheless initiates within a cluster of 6 overlapping variant T4 late promoters that deviate from the absolutely conserved sequence at one or two positions. One of these variant promoters is dominant, and its sequence implies that two of the absolutely conserved nucleotides (underlined above) are not essential. Three other variant promoters that contain only one of the deviations found in the dominant variant promoter are, at best, utilized weakly, suggesting that sequences outside the absolutely conserved segment are important for promoter function. A newly devised method, based on arrest of RNA chain elongation with ribonucleotide analogs and its reversal, has been used to precisely map initiation within the overlapping promoter cluster. Multiple cycles of RNA chain arrest, pyrophosphorolysis, and resynthesis can be executed. This process permits a precise stepwise control of the advance of a transcription complex through a gene.